Sample records for early hematopoietic progenitor

  1. Circulating hematopoietic progenitor cells in patients affected by Chornobyl accident.

    PubMed

    Bilko, N M; Dyagil, I S; Russu, I Z; Bilko, D I

    2016-12-01

    High radiation sensitivity of stem cells and their ability to accumulate sublethal radiation damage provides the basis for investigation of hematopoietic progenitors using in vivo culture methodology. Unique samples of peripheral blood and bone marrow were derived from the patients affected by Chornobyl accident during liquidation campaign. To investigate functional activity of circulating hematopoietic progenitor cells from peripheral blood and bone marrow of cleanup workers in early and remote periods after the accident at Chornobyl nuclear power plant (CNPP). The assessment of the functional activity of circulating hematopoietic progenitor cells was performed in samples of peripheral blood and bone marrow of 46 cleanup workers, who were treated in the National Scientific Center for Radiation Medicine of the Academy of Medical Sciences of Ukraine alongside with 35 non radiated patients, who served as a control. Work was performed by culturing peripheral blood and bone marrow mononuclear cells in the original gel diffusion capsules, implanted into the peritoneal cavity of CBA mice. It was shown that hematopoietic progenitor cells could be identified in the peripheral blood of liquidators of CNPP accident. At the same time the number of functionally active progenitor cells of the bone marrow was significantly decreased and during the next 10 years after the accident, counts of circulating progenitor cells in the peripheral blood as well as functionally active hematopoietic cells in bone marrow returned to normal levels. It was shown that hematopoietic progenitor cells are detected not only in the bone marrow but also in the peripheral blood of liquidators as a consequence of radiation exposure associated with CNPP accident. This article is a part of a Special Issue entitled "The Chornobyl Nuclear Accident: Thirty Years After".

  2. Biology and flow cytometry of proangiogenic hematopoietic progenitors cells.

    PubMed

    Rose, Jonathan A; Erzurum, Serpil; Asosingh, Kewal

    2015-01-01

    During development, hematopoiesis and neovascularization are closely linked to each other via a common bipotent stem cell called the hemangioblast that gives rise to both hematopoietic cells and endothelial cells. In postnatal life, this functional connection between the vasculature and hematopoiesis is maintained by a subset of hematopoietic progenitor cells endowed with the capacity to differentiate into potent proangiogenic cells. These proangiogenic hematopoietic progenitors comprise a specific subset of bone marrow (BM)-derived cells that homes to sites of neovascularization and possess potent paracrine angiogenic activity. There is emerging evidence that this subpopulation of hematopoietic progenitors plays a critical role in vascular health and disease. Their angiogenic activity is distinct from putative "endothelial progenitor cells" that become structural cells of the endothelium by differentiation into endothelial cells. Proangiogenic hematopoietic progenitor cell research requires multidisciplinary expertise in flow cytometry, hematology, and vascular biology. This review provides a comprehensive overview of proangiogenic hematopoietic progenitor cell biology and flow cytometric methods to detect these cells in the peripheral blood circulation and BM. © 2014 International Society for Advancement of Cytometry.

  3. Hematopoietic progenitor migration to the adult thymus

    PubMed Central

    Zlotoff, Daniel A.; Bhandoola, Avinash

    2010-01-01

    While most hematopoietic lineages develop in the bone marrow (BM), T cells uniquely complete their development in the specialized environment of the thymus. Hematopoietic stem cells with long-term self-renewal capacity are not present in the thymus. As a result, continuous T cell development requires that BM-derived progenitors be imported into the thymus throughout adult life. The process of thymic homing begins with the mobilization of progenitors out of the bone marrow, continues with their circulation in the bloodstream, and concludes with their settling in the thymus. This review will discuss each of these steps as they occur in the unirradiated and post-irradiation scenarios, focusing on the molecular mechanisms of regulation. Improved knowledge about these early steps in T cell generation may accelerate the development of new therapeutic options in patients with impaired T cell number or function. PMID:21251013

  4. Nutraceutical augmentation of circulating endothelial progenitor cells and hematopoietic stem cells in human subjects.

    PubMed

    Mikirova, Nina A; Jackson, James A; Hunninghake, Ron; Kenyon, Julian; Chan, Kyle W H; Swindlehurst, Cathy A; Minev, Boris; Patel, Amit N; Murphy, Michael P; Smith, Leonard; Ramos, Famela; Ichim, Thomas E; Riordan, Neil H

    2010-04-08

    The medical significance of circulating endothelial or hematopoietic progenitors is becoming increasing recognized. While therapeutic augmentation of circulating progenitor cells using G-CSF has resulted in promising preclinical and early clinical data for several degenerative conditions, this approach is limited by cost and inability to perform chronic administration. Stem-Kine is a food supplement that was previously reported to augment circulating EPC in a pilot study. Here we report a trial in 18 healthy volunteers administered Stem-Kine twice daily for a 2 week period. Significant increases in circulating CD133 and CD34 cells were observed at days 1, 2, 7, and 14 subsequent to initiation of administration, which correlated with increased hematopoietic progenitors as detected by the HALO assay. Augmentation of EPC numbers in circulation was detected by KDR-1/CD34 staining and colony forming assays. These data suggest Stem-Kine supplementation may be useful as a stimulator of reparative processes associated with mobilization of hematopoietic and endothelial progenitors.

  5. CD44-mediated hyaluronan binding marks proliferating hematopoietic progenitor cells and promotes bone marrow engraftment

    PubMed Central

    Lee-Sayer, Sally S. M.; Dougan, Meghan N.; Cooper, Jesse; Sanderson, Leslie; Dosanjh, Manisha; Maxwell, Christopher A.

    2018-01-01

    CD44 is a widely expressed cell adhesion molecule that binds to the extracellular matrix component, hyaluronan. However, this interaction is not constitutive in most immune cells at steady state, as the ability of CD44 to engage hyaluronan is highly regulated. While activated T cells and macrophages gain the ability to bind hyaluronan by CD44, the status in other immune cells is less studied. Here we found a percentage of murine eosinophils, natural killer and natural killer T cells were capable of interacting with hyaluronan at steady state. To further investigate the consequences of hyaluronan binding by CD44 in the hematopoietic system, point mutations of CD44 that either cannot bind hyaluronan (LOF-CD44) or have an increased affinity for hyaluronan (GOF-CD44) were expressed in CD44-deficient bone marrow. Competitive bone marrow reconstitution of irradiated mice revealed an early preference for GOF-CD44 over WT-CD44 expressing cells, and for WT-CD44 over LOF-CD44 expressing cells, in the hematopoietic progenitor cell compartment. The advantage of the hyaluronan-binding cells was observed in the hematopoietic stem and progenitor populations, and was maintained throughout the immune system. Hematopoietic stem cells bound minimal hyaluronan at steady state, and this was increased when the cells were induced to proliferate whereas multipotent progenitors had an increased ability to bind hyaluronan at steady state. In vitro, the addition of hyaluronan promoted their proliferation. Thus, proliferating hematopoietic progenitors bind hyaluronan, and hyaluronan binding cells have a striking competitive advantage in bone marrow engraftment. PMID:29684048

  6. Human hematopoietic progenitors express erythropoietin.

    PubMed

    Stopka, T; Zivny, J H; Stopkova, P; Prchal, J F; Prchal, J T

    1998-05-15

    Erythropoietin (EPO) is a factor essential for erythroid cell proliferation, differentiation, and survival. The production of EPO by the kidneys in response to hypoxia and anemia is well documented. To determine whether EPO is also produced by hematopoietic cells, we analyzed the expression of EPO in normal human hematopoietic progenitors and in their progeny. Undifferentiated CD34(+)lin- hematopoietic progenitors do not have detectable EPO mRNA. Differentiating CD34(+) cells that are stimulated with recombinant human EPO in serum-free liquid cultures express both EPO and EPO receptor (EPOR). Because CD34(+) cells represent a heterogeneous cell population, we analyzed individual burst-forming units-erythroid (BFU-E) and nonerythroid colony-forming unit-granulocyte-macrophage colonies for EPO mRNA. Only BFU-E colonies were positive for EPO mRNA. Lysates from pooled BFU-E colonies stained positively for EPO by immunoblotting. To further confirm the intrinsic nature of erythroid EPO, we replaced extrinsic EPO in erythroid colony cultures with EPO-mimicking peptide (EMP). We show EPO expression in the EMP-stimulated BFU-Es at both mRNA and protein levels. Stimulation of bone marrow mononuclear cells (BMMCs) with EMP upregulated EPO expression. Furthermore, we found EPO and EPOR mRNAs as well as EPO protein in K562 cells, a human erythroleukemia cell line. Stimulation of K562 cells with EMP upregulated EPO expression. We suggest that EPO of erythroid origin may have a role in the regulation of erythropoiesis.

  7. Pluripotent cell models of fanconi anemia identify the early pathological defect in human hemoangiogenic progenitors.

    PubMed

    Suzuki, Naoya M; Niwa, Akira; Yabe, Miharu; Hira, Asuka; Okada, Chihiro; Amano, Naoki; Watanabe, Akira; Watanabe, Ken-Ichiro; Heike, Toshio; Takata, Minoru; Nakahata, Tatsutoshi; Saito, Megumu K

    2015-04-01

    Fanconi anemia (FA) is a disorder of genomic instability characterized by progressive bone marrow failure (BMF), developmental abnormalities, and an increased susceptibility to cancer. Although various consequences in hematopoietic stem/progenitor cells have been attributed to FA-BMF, the quest to identify the initial pathological event is still ongoing. To address this issue, we established induced pluripotent stem cells (iPSCs) from fibroblasts of six patients with FA and FANCA mutations. An improved reprogramming method yielded iPSC-like colonies from all patients, and iPSC clones were propagated from two patients. Quantitative evaluation of the differentiation ability demonstrated that the differentiation propensity toward the hematopoietic and endothelial lineages is already defective in early hemoangiogenic progenitors. The expression levels of critical transcription factors were significantly downregulated in these progenitors. These data indicate that the hematopoietic consequences in FA patients originate from the early hematopoietic stage and highlight the potential usefulness of iPSC technology for elucidating the pathogenesis of FA-BMF. ©AlphaMed Press.

  8. Menin regulates the function of hematopoietic stem cells and lymphoid progenitors

    PubMed Central

    Chen, Ya-Xiong; Friedman, Ann; Yang, Yuqing; Tubbs, Anthony T.; Shestova, Olga; Pear, Warren S.

    2009-01-01

    Men1 is a tumor suppressor gene mutated in endocrine neoplasms. Besides its endocrine role, the Men1 gene product menin interacts with the mixed lineage leukemia (MLL) protein, a histone H3 lysine 4 methyltransferase. Although menin and MLL fusion proteins cooperate to activate Homeobox (Hox) gene expression during transformation, little is known about the normal hematopoietic functions of menin. Here, we studied hematopoiesis after Men1 ablation. Menin loss modestly impaired blood neutrophil, lymphocyte, and platelet counts. Without hematopoietic stress, multilineage and myelo-erythroid bone marrow progenitor numbers were preserved, while B lymphoid progenitors were decreased. In contrast, competitive transplantation revealed a marked functional defect of long-term hematopoietic stem cells (HSC) in the absence of menin, despite normal initial homing of progenitors to the bone marrow. HoxA9 gene expression was only modestly decreased in menin-deficient HSCs. These observations reveal a novel and essential role for menin in HSC homeostasis that was most apparent during situations of hematopoietic recovery, suggesting that menin regulates molecular pathways that are essential during the adaptive HSC response to stress. PMID:19228930

  9. Adenosine signaling promotes hematopoietic stem and progenitor cell emergence

    PubMed Central

    Jing, Lili; Tamplin, Owen J.; Chen, Michael J.; Deng, Qing; Patterson, Shenia; Kim, Peter G.; Durand, Ellen M.; McNeil, Ashley; Green, Julie M.; Matsuura, Shinobu; Ablain, Julien; Brandt, Margot K.; Schlaeger, Thorsten M.; Huttenlocher, Anna; Daley, George Q.; Ravid, Katya

    2015-01-01

    Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1+/cmyb+ HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl+ hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP–protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates. PMID:25870200

  10. Protein Tyrosine Phosphatase PRL2 Mediates Notch and Kit Signals in Early T Cell Progenitors.

    PubMed

    Kobayashi, Michihiro; Nabinger, Sarah C; Bai, Yunpeng; Yoshimoto, Momoko; Gao, Rui; Chen, Sisi; Yao, Chonghua; Dong, Yuanshu; Zhang, Lujuan; Rodriguez, Sonia; Yashiro-Ohtani, Yumi; Pear, Warren S; Carlesso, Nadia; Yoder, Mervin C; Kapur, Reuben; Kaplan, Mark H; Daniel Lacorazza, Hugo; Zhang, Zhong-Yin; Liu, Yan

    2017-04-01

    The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow hematopoietic stem and progenitor cells (HSPCs) that continuously feed thymic progenitors remain largely unknown. While Notch signal is indispensable for T cell specification and differentiation, the downstream effectors are not well understood. PRL2, a protein tyrosine phosphatase that regulates hematopoietic stem cell proliferation and self-renewal, is highly expressed in murine thymocyte progenitors. Here we demonstrate that protein tyrosine phosphatase PRL2 and receptor tyrosine kinase c-Kit are critical downstream targets and effectors of the canonical Notch/RBPJ pathway in early T cell progenitors. While PRL2 deficiency resulted in moderate defects of thymopoiesis in the steady state, de novo generation of T cells from Prl2 null hematopoietic stem cells was significantly reduced following transplantation. Prl2 null HSPCs also showed impaired T cell differentiation in vitro. We found that Notch/RBPJ signaling upregulated PRL2 as well as c-Kit expression in T cell progenitors. Further, PRL2 sustains Notch-mediated c-Kit expression and enhances stem cell factor/c-Kit signaling in T cell progenitors, promoting effective DN1-DN2 transition. Thus, we have identified a critical role for PRL2 phosphatase in mediating Notch and c-Kit signals in early T cell progenitors. Stem Cells 2017;35:1053-1064. © 2016 AlphaMed Press.

  11. Zinc finger protein 521 antagonizes early B-cell factor 1 and modulates the B-lymphoid differentiation of primary hematopoietic progenitors.

    PubMed

    Mega, Tiziana; Lupia, Michela; Amodio, Nicola; Horton, Sarah J; Mesuraca, Maria; Pelaggi, Daniela; Agosti, Valter; Grieco, Michele; Chiarella, Emanuela; Spina, Raffaella; Moore, Malcolm A S; Schuringa, Jan Jacob; Bond, Heather M; Morrone, Giovanni

    2011-07-01

    Zinc finger protein 521 (EHZF/ZNF521) is a multi-functional transcription co-factor containing 30 zinc fingers and an amino-terminal motif that binds to the nucleosome remodelling and histone deacetylase (NuRD) complex. ZNF521 is believed to be a relevant player in the regulation of the homeostasis of the hematopoietic stem/progenitor cell compartment, however the underlying molecular mechanisms are still largely unknown. Here, we show that this protein plays an important role in the control of B-cell development by inhibiting the activity of early B-cell factor-1 (EBF1), a master factor in B-lineage specification. In particular, our data demonstrate that: (1) ZNF521 binds to EBF1 via its carboxyl-terminal portion and this interaction is required for EBF1 inhibition; (2) NuRD complex recruitment by ZNF521 is not essential for the inhibition of transactivation of EBF1-dependent promoters; (3) ZNF521 represses EBF1 target genes in a human B-lymphoid molecular context; and (4) RNAi-mediated silencing of ZNF521/Zfp521 in primary human and murine hematopoietic progenitors strongly enhances the generation of B-lymphocytes in vitro. Taken together, our data indicate that ZNF521 can antagonize B-cell development and lend support to the notion that it may contribute to conserve the multipotency of primitive lympho-myeloid progenitors by preventing or delaying their EBF1-driven commitment toward the B-cell lineage.

  12. GATA-3 is required for early T lineage progenitor development

    PubMed Central

    Hosoya, Tomonori; Kuroha, Takashi; Moriguchi, Takashi; Cummings, Dustin; Maillard, Ivan; Lim, Kim-Chew

    2009-01-01

    Most T lymphocytes appear to arise from very rare early T lineage progenitors (ETPs) in the thymus, but the transcriptional programs that specify ETP generation are not completely known. The transcription factor GATA-3 is required for the development of T lymphocytes at multiple late differentiation steps as well as for the development of thymic natural killer cells. However, a role for GATA-3 before the double-negative (DN) 3 stage of T cell development has to date been obscured both by the developmental heterogeneity of DN1 thymocytes and the paucity of ETPs. We provide multiple lines of in vivo evidence through the analysis of T cell development in Gata3 hypomorphic mutant embryos, in irradiated mice reconstituted with Gata3 mutant hematopoietic cells, and in mice conditionally ablated for the Gata3 gene to show that GATA-3 is required for ETP generation. We further show that Gata3 loss does not affect hematopoietic stem cells or multipotent hematopoietic progenitors. Finally, we demonstrate that Gata3 mutant lymphoid progenitors exhibit neither increased apoptosis nor diminished cell-cycle progression. Thus, GATA-3 is required for the cell-autonomous development of the earliest characterized thymic T cell progenitors. PMID:19934022

  13. Development of hematopoietic stem and progenitor cells from human pluripotent stem cells.

    PubMed

    Chen, Tong; Wang, Fen; Wu, Mengyao; Wang, Zack Z

    2015-07-01

    Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), provide a new cell source for regenerative medicine, disease modeling, drug discovery, and preclinical toxicity screening. Understanding of the onset and the sequential process of hematopoietic cells from differentiated hPSCs will enable the achievement of personalized medicine and provide an in vitro platform for studying of human hematopoietic development and disease. During embryogenesis, hemogenic endothelial cells, a specified subset of endothelial cells in embryonic endothelium, are the primary source of multipotent hematopoietic stem cells. In this review, we discuss current status in the generation of multipotent hematopoietic stem and progenitor cells from hPSCs via hemogenic endothelial cells. We also review the achievements in direct reprogramming from non-hematopoietic cells to hematopoietic stem and progenitor cells. Further characterization of hematopoietic differentiation in hPSCs will improve our understanding of blood development and expedite the development of hPSC-derived blood products for therapeutic purpose. © 2015 Wiley Periodicals, Inc.

  14. Collection and use of circulating hematopoietic progenitor cells.

    PubMed

    Lee, J H; Klein, H G

    1995-02-01

    Although lymphocytes and monocytes are becoming increasingly important in transfusion therapy, peripheral stem cells have been responsible for the recent explosive interest in harvesting mononuclear cells from the peripheral circulation. Despite their low concentration in peripheral blood and the consequent difficulty in cell collection, circulating hematopoietic progenitor cells are collected and used almost routinely. These mononuclear cells, possessing the capacity for hematopoietic reconstitution and the potential for definitive therapy of a variety of disorders, have been the focus of recent intense interest in transfusion medicine.

  15. Direct Reprogramming of Murine Fibroblasts to Hematopoietic Progenitor Cells

    PubMed Central

    Batta, Kiran; Florkowska, Magdalena; Kouskoff, Valerie; Lacaud, Georges

    2014-01-01

    Summary Recent reports have shown that somatic cells, under appropriate culture conditions, could be directly reprogrammed to cardiac, hepatic, or neuronal phenotype by lineage-specific transcription factors. In this study, we demonstrate that both embryonic and adult somatic fibroblasts can be efficiently reprogrammed to clonal multilineage hematopoietic progenitors by the ectopic expression of the transcription factors ERG, GATA2, LMO2, RUNX1c, and SCL. These reprogrammed cells were stably expanded on stromal cells and possessed short-term reconstitution ability in vivo. Loss of p53 function facilitated reprogramming to blood, and p53−/− reprogrammed cells efficiently generated erythroid, megakaryocytic, myeloid, and lymphoid lineages. Genome-wide analyses revealed that generation of hematopoietic progenitors was preceded by the appearance of hemogenic endothelial cells expressing endothelial and hematopoietic genes. Altogether, our findings suggest that direct reprogramming could represent a valid alternative approach to the differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) for disease modeling and autologous blood cell therapies. PMID:25466247

  16. Erythro-Myeloid Progenitors: “definitive” hematopoiesis in the conceptus prior to the emergence of hematopoietic stem cells

    PubMed Central

    Frame, Jenna M.; McGrath, Kathleen E.; Palis, James

    2013-01-01

    Erythro-myeloid progenitors (EMP) serve as a major source of hematopoiesis in the developing conceptus prior to the formation of a permanent blood system. In this review, we summarize the current knowledge regarding the emergence, fate, and potential of this hematopoietic stem cell (HSC)-independent wave of hematopoietic progenitors, focusing on the murine embryo as a model system. A better understanding of the temporal and spatial control of hematopoietic emergence in the embryo will ultimately improve our ability to derive hematopoietic stem and progenitor cells from embryonic stem cells and induced pluripotent stem cells to serve therapeutic purposes. PMID:24095199

  17. ADAM17 limits the expression of CSF1R on murine hematopoietic progenitors

    PubMed Central

    Becker, Amy M.; Walcheck, Bruce; Bhattacharya, Deepta

    2014-01-01

    All-lymphoid progenitors (ALPs) yield few myeloid cells in vivo, but readily generate such cells in vitro. The basis for this difference remains unknown. We hypothesized that ALPs limit responsiveness to in vivo concentrations of myeloid-promoting cytokines by reducing expression of the corresponding receptors, potentially through post-transcriptional mechanisms. Consistent with such a mechanism, ALPs express higher levels of Csf1r transcripts than their upstream precursors, yet show limited cell surface protein expression of CSF1R. ALPs and other hematopoietic progenitors deficient in ADAM17, a metalloprotease that can cleave CSF1R, display elevated cell surface CSF1R expression. Adam17−/− ALPs, however, fail to yield myeloid cells upon transplantation into irradiated recipients. Moreover, Adam17−/− ALPs yield fewer macrophages in vitro than control ALPs at high concentrations of M-CSF. Mice with hematopoietic-specific deletion of Adam17 have grossly normal numbers of myeloid and lymphoid progenitors and mature cells in vivo. These data demonstrate that ADAM17 limits CSF1R protein expression on hematopoietic progenitors, but that compensatory mechanisms prevent elevated CSF1R levels from altering lymphoid progenitor potential. PMID:25308957

  18. Extended flow cytometry characterization of normal bone marrow progenitor cells by simultaneous detection of aldehyde dehydrogenase and early hematopoietic antigens: implication for erythroid differentiation studies

    PubMed Central

    Mirabelli, Peppino; Di Noto, Rosa; Lo Pardo, Catia; Morabito, Paolo; Abate, Giovanna; Gorrese, Marisa; Raia, Maddalena; Pascariello, Caterina; Scalia, Giulia; Gemei, Marica; Mariotti, Elisabetta; Del Vecchio, Luigi

    2008-01-01

    Background Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. Results In normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA). Conclusion Our study, comparing surface antigen expression of ALDH+/CD34+, ALDH

  19. HIV-1 infection depletes human CD34+CD38- hematopoietic progenitor cells via pDC-dependent mechanisms.

    PubMed

    Li, Guangming; Zhao, Juanjuan; Cheng, Liang; Jiang, Qi; Kan, Sheng; Qin, Enqiang; Tu, Bo; Zhang, Xin; Zhang, Liguo; Su, Lishan; Zhang, Zheng

    2017-07-01

    Chronic human immunodeficiency virus-1 (HIV-1) infection in patients leads to multi-lineage hematopoietic abnormalities or pancytopenia. The deficiency in hematopoietic progenitor cells (HPCs) induced by HIV-1 infection has been proposed, but the relevant mechanisms are poorly understood. We report here that both human CD34+CD38- early and CD34+CD38+ intermediate HPCs were maintained in the bone marrow (BM) of humanized mice. Chronic HIV-1 infection preferentially depleted CD34+CD38- early HPCs in the BM and reduced their proliferation potential in vivo in both HIV-1-infected patients and humanized mice, while CD34+CD38+ intermediate HSCs were relatively unaffected. Strikingly, depletion of plasmacytoid dendritic cells (pDCs) prevented human CD34+CD38- early HPCs from HIV-1 infection-induced depletion and functional impairment and restored the gene expression profile of purified CD34+ HPCs in humanized mice. These findings suggest that pDCs contribute to the early hematopoietic suppression induced by chronic HIV-1 infection and provide a novel therapeutic target for the hematopoiesis suppression in HIV-1 patients.

  20. Acquired initiating mutations in early hematopoietic cells of CLL patients.

    PubMed

    Damm, Frederik; Mylonas, Elena; Cosson, Adrien; Yoshida, Kenichi; Della Valle, Véronique; Mouly, Enguerran; Diop, M'boyba; Scourzic, Laurianne; Shiraishi, Yuichi; Chiba, Kenichi; Tanaka, Hiroko; Miyano, Satoru; Kikushige, Yoshikane; Davi, Frederick; Lambert, Jérôme; Gautheret, Daniel; Merle-Béral, Hélène; Sutton, Laurent; Dessen, Philippe; Solary, Eric; Akashi, Koichi; Vainchenker, William; Mercher, Thomas; Droin, Nathalie; Ogawa, Seishi; Nguyen-Khac, Florence; Bernard, Olivier A

    2014-09-01

    Appropriate cancer care requires a thorough understanding of the natural history of the disease, including the cell of origin, the pattern of clonal evolution, and the functional consequences of the mutations. Using deep sequencing of flow-sorted cell populations from patients with chronic lymphocytic leukemia (CLL), we established the presence of acquired mutations in multipotent hematopoietic progenitors. Mutations affected known lymphoid oncogenes, including BRAF, NOTCH1, and SF3B1. NFKBIE and EGR2 mutations were observed at unexpectedly high frequencies, 10.7% and 8.3% of 168 advanced-stage patients, respectively. EGR2 mutations were associated with a shorter time to treatment and poor overall survival. Analyses of BRAF and EGR2 mutations suggest that they result in deregulation of B-cell receptor (BCR) intracellular signaling. Our data propose disruption of hematopoietic and early B-cell differentiation through the deregulation of pre-BCR signaling as a phenotypic outcome of CLL mutations and show that CLL develops from a pre-leukemic phase. The origin and pathogenic mechanisms of CLL are not fully understood. The current work indicates that CLL develops from pre-leukemic multipotent hematopoietic progenitors carrying somatic mutations. It advocates for abnormalities in early B-cell differentiation as a phenotypic convergence of the diverse acquired mutations observed in CLL. ©2014 American Association for Cancer Research.

  1. Estrogen signaling selectively induces apoptosis of hematopoietic progenitors and myeloid neoplasms without harming steady-state hematopoiesis.

    PubMed

    Sánchez-Aguilera, Abel; Arranz, Lorena; Martín-Pérez, Daniel; García-García, Andrés; Stavropoulou, Vaia; Kubovcakova, Lucia; Isern, Joan; Martín-Salamanca, Sandra; Langa, Xavier; Skoda, Radek C; Schwaller, Jürg; Méndez-Ferrer, Simón

    2014-12-04

    Estrogens are potent regulators of mature hematopoietic cells; however, their effects on primitive and malignant hematopoietic cells remain unclear. Using genetic and pharmacological approaches, we observed differential expression and function of estrogen receptors (ERs) in hematopoietic stem cell (HSC) and progenitor subsets. ERα activation with the selective ER modulator (SERM) tamoxifen induced apoptosis in short-term HSCs and multipotent progenitors. In contrast, tamoxifen induced proliferation of quiescent long-term HSCs, altered the expression of self-renewal genes, and compromised hematopoietic reconstitution after myelotoxic stress, which was reversible. In mice, tamoxifen treatment blocked development of JAK2(V617F)-induced myeloproliferative neoplasm in vivo, induced apoptosis of human JAK2(V617F+) HSPCs in a xenograft model, and sensitized MLL-AF9(+) leukemias to chemotherapy. Apoptosis was selectively observed in mutant cells, and tamoxifen treatment only had a minor impact on steady-state hematopoiesis in disease-free animals. Together, these results uncover specific regulation of hematopoietic progenitors by estrogens and potential antileukemic properties of SERMs. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Hematopoietic progenitors express neural genes

    PubMed Central

    Goolsby, James; Marty, Marie C.; Heletz, Dafna; Chiappelli, Joshua; Tashko, Gerti; Yarnell, Deborah; Fishman, Paul S.; Dhib-Jalbut, Suhayl; Bever, Christopher T.; Pessac, Bernard; Trisler, David

    2003-01-01

    Bone marrow, or cells selected from bone marrow, were reported recently to give rise to cells with a neural phenotype after in vitro treatment with neural-inducing factors or after delivery into the brain. However, we showed previously that untreated bone marrow cells express products of the neural myelin basic protein gene, and we demonstrate here that a subset of ex vivo bone marrow cells expresses the neurogenic transcription factor Pax-6 as well as neuronal genes encoding neurofilament H, NeuN (neuronal nuclear protein), HuC/HuD (Hu-antigen C/Hu-antigen D), and GAD65 (glutamic acid decarboxylase 65), as well as the oligodendroglial gene encoding CNPase (2′,3′ cyclic nucleotide 3′-phosphohydrolase). In contrast, astroglial glial fibrillary acidic protein (GFAP) was not detected. These cells also were CD34+, a marker of hematopoietic stem cells. Cultures of these highly proliferative CD34+ cells, derived from adult mouse bone marrow, uniformly displayed a phenotype comparable with that of hematopoietic progenitor cells (CD45+, CD34+, Sca-1+, AA4.1+, cKit+, GATA-2+, and LMO-2+). The neuronal and oligodendroglial genes expressed in ex vivo bone marrow also were expressed in all cultured CD34+ cells, and GFAP was not observed. After CD34+ cell transplantation into adult brain, neuronal or oligodendroglial markers segregated into distinct nonoverlapping cell populations, whereas astroglial GFAP appeared, in the absence of other neural markers, in a separate set of implanted cells. Thus, neuronal and oligodendroglial gene products are present in a subset of bone marrow cells, and the expression of these genes can be regulated in brain. The fact that these CD34+ cells also express transcription factors (Rex-1 and Oct-4) that are found in early development elicits the hypothesis that they may be pluripotent embryonic-like stem cells. PMID:14634211

  3. Sensitivity and dose dependency of radiation-induced injury in hematopoietic stem/progenitor cells in mice

    PubMed Central

    Guo, Chang-Ying; Luo, Lan; Urata, Yoshishige; Goto, Shinji; Huang, Wen-Jing; Takamura, Syu; Hayashi, Fumiko; Doi, Hanako; Kitajima, Yuriko; Ono, Yusuke; Ogi, Tomoo; Li, Tao-Sheng

    2015-01-01

    We evaluated the sensitivity and dose dependency of radiation-induced injury in hematopoietic stem/progenitor cells. Adult C57BL/6 mice were daily exposed to 0, 2, 10, 50, and 250 mGy γ-ray for 1 month in succession, respectively. The damage of hematopoietic stem/progenitor cells in bone marrow were investigated within 2 hours (acute phase) or at 3 months (chronic phase) after the last exposure. Daily exposure to over 10 mGy γ-ray significantly decreased the number and colony-forming capacity of hematopoietic stem/progenitor cells at acute phase, and did not completely recover at chronic phase with 250 mGy exposure. Interestingly, the daily exposure to 10 or 50 mGy γ-ray decreased the formation of mixed types of colonies at chronic phase, but the total number of colonies was comparable to control. Immunostaining analysis showed that the formation of 53BP1 foci in c-kit+ stem/progenitor cells was significantly increased with daily exposure to 50 and 250 mGy at acute phase, and 250 mGy at chronic phase. Many genes involved in toxicity responses were up- or down-regulated with the exposures to all doses. Our data have clearly shown the sensitivity and dose dependency of radiation-induced injury in hematopoietic stem/progenitor cells of mice with daily exposures to 2 ~ 250 mGy γ-ray. PMID:25623887

  4. Hematopoietic stem and progenitor cells regulate the regeneration of their niche by secreting Angiopoietin-1

    PubMed Central

    Zhou, Bo O; Ding, Lei; Morrison, Sean J

    2015-01-01

    Hematopoietic stem cells (HSCs) are maintained by a perivascular niche in bone marrow but it is unclear whether the niche is reciprocally regulated by HSCs. Here, we systematically assessed the expression and function of Angiopoietin-1 (Angpt1) in bone marrow. Angpt1 was not expressed by osteoblasts. Angpt1 was most highly expressed by HSCs, and at lower levels by c-kit+ hematopoietic progenitors, megakaryocytes, and Leptin Receptor+ (LepR+) stromal cells. Global conditional deletion of Angpt1, or deletion from osteoblasts, LepR+ cells, Nes-cre-expressing cells, megakaryocytes, endothelial cells or hematopoietic cells in normal mice did not affect hematopoiesis, HSC maintenance, or HSC quiescence. Deletion of Angpt1 from hematopoietic cells and LepR+ cells had little effect on vasculature or HSC frequency under steady-state conditions but accelerated vascular and hematopoietic recovery after irradiation while increasing vascular leakiness. Hematopoietic stem/progenitor cells and LepR+ stromal cells regulate niche regeneration by secreting Angpt1, reducing vascular leakiness but slowing niche recovery. DOI: http://dx.doi.org/10.7554/eLife.05521.001 PMID:25821987

  5. Progressive alterations in multipotent hematopoietic progenitors underlie lymphoid cell loss in aging.

    PubMed

    Young, Kira; Borikar, Sneha; Bell, Rebecca; Kuffler, Lauren; Philip, Vivek; Trowbridge, Jennifer J

    2016-10-17

    Declining immune function with age is associated with reduced lymphoid output of hematopoietic stem cells (HSCs). Currently, there is poor understanding of changes with age in the heterogeneous multipotent progenitor (MPP) cell compartment, which is long lived and responsible for dynamically regulating output of mature hematopoietic cells. In this study, we observe an early and progressive loss of lymphoid-primed MPP cells (LMPP/MPP4) with aging, concomitant with expansion of HSCs. Transcriptome and in vitro functional analyses at the single-cell level reveal a concurrent increase in cycling of aging LMPP/MPP4 with loss of lymphoid priming and differentiation potential. Impaired lymphoid differentiation potential of aged LMPP/MPP4 is not rescued by transplantation into a young bone marrow microenvironment, demonstrating cell-autonomous changes in the MPP compartment with aging. These results pinpoint an age and cellular compartment to focus further interrogation of the drivers of lymphoid cell loss with aging. © 2016 Young et al.

  6. Cadmium modulates hematopoietic stem and progenitor cells and skews toward myelopoiesis in mice

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Yandong; Yu, Xinchun

    The heavy metal cadmium (Cd) is known to modulate immunity and cause osteoporosis. However, how Cd influences on hematopoiesis remain largely unknown. Herein, we show that wild-type C57BL/6 (B6) mice exposed to Cd for 3 months had expanded bone marrow (BM) populations of long-term hematopoietic stem cells (LT-HSCs), common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs), while having reduced populations of multipotent progenitors (MPPs) and common lymphoid progenitors (CLPs). A competitive mixed BM transplantation assay indicates that BM from Cd-treated mice had impaired LT-HSC ability to differentiate into mature cells. In accordance with increased myeloid progenitors and decreased lymphoid progenitors,more » the BM and spleens of Cd-treated mice had more monocytes and/or neutrophils and fewer B cells and T cells. Cd impaired the ability of the non-hematopoietic system to support LT-HSCs, in that lethally irradiated Cd-treated recipients transplanted with normal BM cells had reduced LT-HSCs after the hematopoietic system was fully reconstituted. This is consistent with reduced osteoblasts, a known critical component for HSC niche, observed in Cd-treated mice. Conversely, lethally irradiated control recipients transplanted with BM cells from Cd-treated mice had normal LT-HSC reconstitution. Furthermore, both control mice and Cd-treated mice that received Alendronate, a clinical drug used for treating osteoporosis, had BM increases of LT-HSCs. Thus, the results suggest Cd increase of LT-HSCs is due to effects on HSCs and not on osteoblasts, although, Cd causes osteoblast reduction and impaired niche function for maintaining HSCs. Furthermore, Cd skews HSCs toward myelopoiesis. - Highlights: • Cd increases the number of LT-HSCs but impairs their development. • Cd-treated hosts have compromised ability to support LT-HSCs. • Cd promotes myelopoiesis at the expense of lymphopoiesis at the MPP level.« less

  7. Hematopoietic stem cells can differentiate into restricted myeloid progenitors before cell division in mice.

    PubMed

    Grinenko, Tatyana; Eugster, Anne; Thielecke, Lars; Ramasz, Beáta; Krüger, Anja; Dietz, Sevina; Glauche, Ingmar; Gerbaulet, Alexander; von Bonin, Malte; Basak, Onur; Clevers, Hans; Chavakis, Triantafyllos; Wielockx, Ben

    2018-05-15

    Hematopoietic stem cells (HSCs) continuously replenish all blood cell types through a series of differentiation steps and repeated cell divisions that involve the generation of lineage-committed progenitors. However, whether cell division in HSCs precedes differentiation is unclear. To this end, we used an HSC cell-tracing approach and Ki67 RFP knock-in mice, in a non-conditioned transplantation model, to assess divisional history, cell cycle progression, and differentiation of adult HSCs. Our results reveal that HSCs are able to differentiate into restricted progenitors, especially common myeloid, megakaryocyte-erythroid and pre-megakaryocyte progenitors, without undergoing cell division and even before entering the S phase of the cell cycle. Additionally, the phenotype of the undivided but differentiated progenitors correlated with the expression of lineage-specific genes and loss of multipotency. Thus HSC fate decisions can be uncoupled from physical cell division. These results facilitate a better understanding of the mechanisms that control fate decisions in hematopoietic cells.

  8. EVI2B is a C/EBPα target gene required for granulocytic differentiation and functionality of hematopoietic progenitors.

    PubMed

    Zjablovskaja, Polina; Kardosova, Miroslava; Danek, Petr; Angelisova, Pavla; Benoukraf, Touati; Wurm, Alexander A; Kalina, Tomas; Sian, Stephanie; Balastik, Martin; Delwel, Ruud; Brdicka, Tomas; Tenen, Daniel G; Behre, Gerhard; Fiore, Fréderic; Malissen, Bernard; Horejsi, Vaclav; Alberich-Jorda, Meritxell

    2017-04-01

    Development of hematopoietic populations through the process of differentiation is critical for proper hematopoiesis. The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) is a master regulator of myeloid differentiation, and the identification of C/EBPα target genes is key to understand this process. Here we identified the Ecotropic Viral Integration Site 2B (EVI2B) gene as a direct target of C/EBPα. We showed that the product of the gene, the transmembrane glycoprotein EVI2B (CD361), is abundantly expressed on the surface of primary hematopoietic cells, the highest levels of expression being reached in mature granulocytes. Using shRNA-mediated downregulation of EVI2B in human and murine cell lines and in primary hematopoietic stem and progenitor cells, we demonstrated impaired myeloid lineage development and altered progenitor functions in EVI2B-silenced cells. We showed that the compromised progenitor functionality in Evi2b-depleted cells can be in part explained by deregulation of cell proliferation and apoptosis. In addition, we generated an Evi2b knockout murine model and demonstrated altered properties of hematopoietic progenitors, as well as impaired G-CSF dependent myeloid colony formation in the knockout cells. Remarkably, we found that EVI2B is significantly downregulated in human acute myeloid leukemia samples characterized by defects in CEBPA. Altogether, our data demonstrate that EVI2B is a downstream target of C/EBPα, which regulates myeloid differentiation and functionality of hematopoietic progenitors.

  9. Interleukin-7-induced Stat-5 acts in synergy with Flt-3 signaling to stimulate expansion of hematopoietic progenitor cells.

    PubMed

    Åhsberg, Josefine; Tsapogas, Panagiotis; Qian, Hong; Zetterblad, Jenny; Zandi, Sasan; Månsson, Robert; Jönsson, Jan-Ingvar; Sigvardsson, Mikael

    2010-11-19

    The development of lymphoid cells from bone marrow progenitors is dictated by interplay between internal cues such as transcription factors and external signals like the cytokines Flt-3 ligand and Il-7. These proteins are both of large importance for normal lymphoid development; however, it is unclear if they act in direct synergy to expand a transient Il-7R(+)Flt-3(+) population or if the collaboration is created through sequential activities. We report here that Flt-3L and Il-7 synergistically stimulated the expansion of primary Il-7R(+)Flt-3(+) progenitor cells and a hematopoietic progenitor cell line ectopically expressing the receptors. The stimulation resulted in a reduced expression of pro-apoptotic genes and also mediated survival of primary progenitor cells in vitro. However, functional analysis of single cells suggested that the anti-apoptotic effect was additive indicating that the synergy observed mainly depends on stimulation of proliferation. Analysis of downstream signaling events suggested that although Il-7 induced Stat-5 phosphorylation, Flt-3L caused activation of the ERK and AKT signaling pathways. Flt-3L could also drive proliferation in synergy with ectopically expressed constitutively active Stat-5. This synergy could be inhibited with either receptor tyrosine kinase or MAPK inhibitors suggesting that Flt-3L and Il-7 act in synergy by activation of independent signaling pathways to expand early hematopoietic progenitors.

  10. bantam miRNA is important for Drosophila blood cell homeostasis and a regulator of proliferation in the hematopoietic progenitor niche

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lam, Victoria; Tokusumi, Tsuyoshi; Tokusumi, Yumiko

    Highlights: • bantam miRNA is endogenously expressed in the hematopoietic progenitor niche. • bantam is necessary and sufficient to induce cellular proliferation in the PSC. • bantam is upstream of the Insulin Receptor signaling pathway. • A model for positive regulation of hematopoietic niche growth is proposed. - Abstract: The Drosophila hematopoietic system is utilized in this study to gain novel insights into the process of growth control of the hematopoietic progenitor niche in blood development. The niche microenvironment is an essential component controlling the balance between progenitor populations and differentiated, mature blood cells and has been shown to leadmore » to hematopoietic malignancies in humans when misregulated. MicroRNAs are one class of regulators associated with blood malignancies; however, there remains a relative paucity of information about the role of miRNAs in the niche. Here we demonstrate that bantam miRNA is endogenously active in the Drosophila hematopoietic progenitor niche, the posterior signaling center (PSC), and functions in the primary hematopoietic organ, the lymph gland, as a positive regulator of growth. Loss of bantam leads to a significant reduction in the PSC and overall lymph gland size, as well as a loss of the progenitor population and correlative premature differentiation of mature hemocytes. Interestingly, in addition to being essential for proper lymph gland development, we have determined bantam to be a novel upstream component of the insulin signaling cascade in the PSC and have unveiled dMyc as one factor central to bantam activity. These important findings identify bantam as a new hematopoietic regulator, place it in an evolutionarily conserved signaling pathway, present one way in which it is regulated, and provide a mechanism through which it facilitates cellular proliferation in the hematopoietic niche.« less

  11. Reduced Erg Dosage Impairs Survival of Hematopoietic Stem and Progenitor Cells.

    PubMed

    Xie, Ying; Koch, Mia Lee; Zhang, Xin; Hamblen, Melanie J; Godinho, Frank J; Fujiwara, Yuko; Xie, Huafeng; Klusmann, Jan-Henning; Orkin, Stuart H; Li, Zhe

    2017-07-01

    ERG, an ETS family transcription factor frequently overexpressed in human leukemia, has been implicated as a key regulator of hematopoietic stem cells. However, how ERG controls normal hematopoiesis, particularly at the stem and progenitor cell level, and how it contributes to leukemogenesis remain incompletely understood. Using homologous recombination, we generated an Erg knockdown allele (Erg kd ) in which Erg expression can be conditionally restored by Cre recombinase. Erg kd/kd animals die at E10.5-E11.5 due to defects in endothelial and hematopoietic cells, but can be completely rescued by Tie2-Cre-mediated restoration of Erg in these cells. In Erg kd/+ mice, ∼40% reduction in Erg dosage perturbs both fetal liver and bone marrow hematopoiesis by reducing the numbers of Lin - Sca-1 + c-Kit + (LSK) hematopoietic stem and progenitor cells (HSPCs) and megakaryocytic progenitors. By genetic mosaic analysis, we find that Erg-restored HSPCs outcompete Erg kd/+ HSPCs for contribution to adult hematopoiesis in vivo. This defect is in part due to increased apoptosis of HSPCs with reduced Erg dosage, a phenotype that becomes more drastic during 5-FU-induced stress hematopoiesis. Expression analysis reveals that reduced Erg expression leads to changes in expression of a subset of ERG target genes involved in regulating survival of HSPCs, including increased expression of a pro-apoptotic regulator Bcl2l11 (Bim) and reduced expression of Jun. Collectively, our data demonstrate that ERG controls survival of HSPCs, a property that may be used by leukemic cells. Stem Cells 2017;35:1773-1785. © 2017 AlphaMed Press.

  12. CXCR2 and CXCL4 regulate survival and self-renewal of hematopoietic stem/progenitor cells.

    PubMed

    Sinclair, Amy; Park, Laura; Shah, Mansi; Drotar, Mark; Calaminus, Simon; Hopcroft, Lisa E M; Kinstrie, Ross; Guitart, Amelie V; Dunn, Karen; Abraham, Sheela A; Sansom, Owen; Michie, Alison M; Machesky, Laura; Kranc, Kamil R; Graham, Gerard J; Pellicano, Francesca; Holyoake, Tessa L

    2016-07-21

    The regulation of hematopoietic stem cell (HSC) survival and self-renewal within the bone marrow (BM) niche is not well understood. We therefore investigated global transcriptomic profiling of normal human HSC/hematopoietic progenitor cells [HPCs], revealing that several chemokine ligands (CXCL1-4, CXCL6, CXCL10, CXCL11, and CXCL13) were upregulated in human quiescent CD34(+)Hoescht(-)Pyronin Y(-) and primitive CD34(+)38(-), as compared with proliferating CD34(+)Hoechst(+)Pyronin Y(+) and CD34(+)38(+) stem/progenitor cells. This suggested that chemokines might play an important role in the homeostasis of HSCs. In human CD34(+) hematopoietic cells, knockdown of CXCL4 or pharmacologic inhibition of the chemokine receptor CXCR2, significantly decreased cell viability and colony forming cell (CFC) potential. Studies on Cxcr2(-/-) mice demonstrated enhanced BM and spleen cellularity, with significantly increased numbers of HSCs, hematopoietic progenitor cell-1 (HPC-1), HPC-2, and Lin(-)Sca-1(+)c-Kit(+) subpopulations. Cxcr2(-/-) stem/progenitor cells showed reduced self-renewal capacity as measured in serial transplantation assays. Parallel studies on Cxcl4 demonstrated reduced numbers of CFC in primary and secondary assays following knockdown in murine c-Kit(+) cells, and Cxcl4(-/-) mice showed a decrease in HSC and reduced self-renewal capacity after secondary transplantation. These data demonstrate that the CXCR2 network and CXCL4 play a role in the maintenance of normal HSC/HPC cell fates, including survival and self-renewal. © 2016 by The American Society of Hematology.

  13. CXCR2 and CXCL4 regulate survival and self-renewal of hematopoietic stem/progenitor cells

    PubMed Central

    Sinclair, Amy; Park, Laura; Shah, Mansi; Drotar, Mark; Calaminus, Simon; Hopcroft, Lisa E. M.; Kinstrie, Ross; Guitart, Amelie V.; Dunn, Karen; Abraham, Sheela A.; Sansom, Owen; Michie, Alison M.; Machesky, Laura; Kranc, Kamil R.; Graham, Gerard J.; Pellicano, Francesca

    2016-01-01

    The regulation of hematopoietic stem cell (HSC) survival and self-renewal within the bone marrow (BM) niche is not well understood. We therefore investigated global transcriptomic profiling of normal human HSC/hematopoietic progenitor cells [HPCs], revealing that several chemokine ligands (CXCL1-4, CXCL6, CXCL10, CXCL11, and CXCL13) were upregulated in human quiescent CD34+Hoescht−Pyronin Y− and primitive CD34+38−, as compared with proliferating CD34+Hoechst+Pyronin Y+ and CD34+38+ stem/progenitor cells. This suggested that chemokines might play an important role in the homeostasis of HSCs. In human CD34+ hematopoietic cells, knockdown of CXCL4 or pharmacologic inhibition of the chemokine receptor CXCR2, significantly decreased cell viability and colony forming cell (CFC) potential. Studies on Cxcr2−/− mice demonstrated enhanced BM and spleen cellularity, with significantly increased numbers of HSCs, hematopoietic progenitor cell-1 (HPC-1), HPC-2, and Lin−Sca-1+c-Kit+ subpopulations. Cxcr2−/− stem/progenitor cells showed reduced self-renewal capacity as measured in serial transplantation assays. Parallel studies on Cxcl4 demonstrated reduced numbers of CFC in primary and secondary assays following knockdown in murine c-Kit+ cells, and Cxcl4−/− mice showed a decrease in HSC and reduced self-renewal capacity after secondary transplantation. These data demonstrate that the CXCR2 network and CXCL4 play a role in the maintenance of normal HSC/HPC cell fates, including survival and self-renewal. PMID:27222476

  14. Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors

    PubMed Central

    Doulatov, Sergei; Vo, Linda T.; Macari, Elizabeth R.; Wahlster, Lara; Kinney, Melissa A.; Taylor, Alison M.; Barragan, Jessica; Gupta, Manav; McGrath, Katherine; Lee, Hsiang-Ying; Humphries, Jessica M.; DeVine, Alex; Narla, Anupama; Alter, Blanche P.; Beggs, Alan H.; Agarwal, Suneet; Ebert, Benjamin L.; Gazda, Hanna T.; Lodish, Harvey F.; Sieff, Colin A.; Schlaeger, Thorsten M.; Zon, Leonard I.; Daley, George Q.

    2017-01-01

    Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. Identifying therapeutics for DBA requires circumventing the paucity of primary patient blood stem and progenitor cells. To this end, we adopted a reprogramming strategy to generate expandable hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs) from DBA patients. Reprogrammed DBA progenitors recapitulate defects in erythroid differentiation, which were rescued by gene complementation. Unbiased chemical screens identified SMER28, a small-molecule inducer of autophagy, which enhanced erythropoiesis in a range of in vitro and in vivo models of DBA. SMER28 acted through autophagy factor ATG5 to stimulate erythropoiesis and up-regulate expression of globin genes. These findings present an unbiased drug screen for hematological disease using iPSCs and identify autophagy as a therapeutic pathway in DBA. PMID:28179501

  15. Comparative study of hematopoietic stem and progenitor cells between sexes in mice under physiological conditions along time.

    PubMed

    Gasco, Samanta; Rando, Amaya; Zaragoza, Pilar; García-Redondo, Alberto; Calvo, Ana Cristina; Osta, Rosario

    2017-12-01

    Hematopoietic stem and progenitor cells (HSPCs) are attractive targets in regenerative medicine, although the differences in their homeostatic maintenance between sexes along time are still under debate. We accurately monitored hematopoietic stem cells (HSCs), common lymphoid progenitors (CLPs), and common myeloid progenitors (CMPs) frequencies by flow cytometry, by performing serial peripheral blood extractions from male and female B6SJL wild-type mice and found no significant differences. Only modest differences were found in the gene expression profile of Slamf1 and Gata2. Our findings suggest that both sexes could be used indistinctly to perform descriptive studies in the murine hematopoietic system, especially for flow cytometry studies in peripheral blood. This would allow diminishing the number of animals needed for the experimental procedures. In addition, the use of serial extractions in the same animals drastically decreases the number of animals needed. © 2017 International Federation for Cell Biology.

  16. Culture materials affect ex vivo expansion of hematopoietic progenitor cells.

    PubMed

    LaIuppa, J A; McAdams, T A; Papoutsakis, E T; Miller, W M

    1997-09-05

    Ex vivo expansion of hematopoietic cells is important for applications such as cancer treatment, gene therapy, and transfusion medicine. While cell culture systems are widely used to evaluate the biocompatibility of materials for implantation, the ability of materials to support proliferation of primary human cells in cultures for reinfusion into patients has not been addressed. We screened a variety of commercially available polymer (15 types), metal (four types), and glass substrates for their ability to support expansion of hematopoietic cells when cultured under conditions that would be encountered in a clinical setting. Cultures of peripheral blood (PB) CD34+ cells and mononuclear cells (MNC) were evaluated for expansion of total cells and colony-forming unit-granulocyte monocyte (CFU-GM; progenitors committed to the granulocyte and/or monocyte lineage). Human hematopoietic cultures in serum-free medium were found to be extremely sensitive to the substrate material. The only materials tested that supported expansion at or near the levels of polystyrene were tissue culture polystyrene, Teflon perfluoroalkoxy, Teflon fluorinated ethylene propylene, cellulose acetate, titanium, new polycarbonate, and new polymethylpentene. MNC were less sensitive to the substrate materials than the primitive CD34+ progenitors, although similar trends were seen for expansion of the two cell populations on the substrates tested. CFU-GM expansion was more sensitive to substrate materials than was total cell expansion. The detrimental effects of a number of the materials on hematopoietic cultures appear to be caused by protein adsorption and/or leaching of toxins. Factors such as cleaning, sterilization, and reuse significantly affected the performance of some materials as culture substrates. We also used PB CD34+ cell cultures to examine the biocompatibility of gas-permeable cell culture and blood storage bags and several types of tubing commonly used with biomedical equipment

  17. Defective Hematopoietic Stem Cell and Lymphoid Progenitor Development in the Ts65Dn Mouse Model of Down Syndrome: Potential Role of Oxidative Stress

    PubMed Central

    Lorenzo, Laureanne Pilar E.; Chen, Haiyan; Shatynski, Kristen E.; Clark, Sarah; Yuan, Rong; Harrison, David E.; Yarowsky, Paul J.

    2011-01-01

    Abstract Aims Down Syndrome (DS), a genetic disease caused by a triplication of chromosome 21, is characterized by increased markers of oxidative stress. In addition to cognitive defects, patients with DS also display hematologic disorders and increased incidence of infections and leukemia. Using the Ts65Dn mouse model of DS, the goal of this study was to examine hematopoietic stem and lymphoid progenitor cell function in DS. Results Analysis of hematopoietic progenitor populations showed that Ts65Dn mice possessed fewer functional hematopoietic stem cells and a significantly decreased percentage of bone marrow lymphoid progenitors. Increased reactive oxygen species and markers of oxidative stress were detected in hematopoietic stem cell populations and were associated with a loss of quiescence. Bone marrow progenitor populations expressed diminished levels of the IL-7Rα chain, which was associated with decreased proliferation and increased apoptosis. Modulating oxidative stress in vitro suggested that oxidative stress selectively leads to decreased IL-7Rα expression, and inhibits the survival of IL-7Rα-expressing hematopoietic progenitors, potentially linking increased reactive oxygen species and immunopathology. Innovation The study results identify a link between oxidative stress and diminished IL-7Rα expression and function. Further, the data suggest that this decrease in IL-7Rα is associated with defective hematopoietic development in Down Syndrome. Conclusion The data suggest that hematopoietic stem and lymphoid progenitor cell defects underlie immune dysfunction in DS and that increased oxidative stress and reduced cytokine signaling may alter hematologic development in Ts65Dn mice. Antioxid. Redox Signal. 15, 2083–2094. PMID:21504363

  18. Shear Stress Regulates Adhesion and Rolling of CD44+ Leukemic and Hematopoietic Progenitor Cells on Hyaluronan

    PubMed Central

    Christophis, Christof; Taubert, Isabel; Meseck, Georg R.; Schubert, Mario; Grunze, Michael; Ho, Anthony D.; Rosenhahn, Axel

    2011-01-01

    Leukemic cells and human hematopoietic progenitor cells expressing CD44 receptors have the ability to attach and roll on hyaluronan. We investigated quantitatively the adhesion behavior of leukemic cell lines and hematopoietic progenitor cells on thin films of the polysaccharides hyaluronan and alginate in a microfluidic system. An applied flow enhances the interaction between CD44-positive cells and hyaluronan if a threshold shear stress of 0.2 dyn/cm2 is exceeded. At shear stress ∼1 dyn/cm2, the cell rolling speed reaches a maximum of 15 μm/s. Leukemic Jurkat and Kasumi-1 cells lacking CD44-expression showed no adhesion or rolling on the polysaccharides whereas the CD44-expressing leukemic cells KG-1a, HL-60, K-562, and hematopoietic progenitor cells attached and rolled on hyaluronan. Interestingly, the observations of flow-induced cell rolling are related to those found in the recruitment of leukocytes to inflammatory sites and the mechanisms of stem-cell homing into the bone marrow. PMID:21806926

  19. Quantitative stability of hematopoietic stem and progenitor cell clonal output in rhesus macaques receiving transplants

    PubMed Central

    Koelle, Samson J.

    2017-01-01

    Autologous transplantation of hematopoietic stem and progenitor cells lentivirally labeled with unique oligonucleotide barcodes flanked by sequencing primer targets enables quantitative assessment of the self-renewal and differentiation patterns of these cells in a myeloablative rhesus macaque model. Compared with other approaches to clonal tracking, this approach is highly quantitative and reproducible. We documented stable multipotent long-term hematopoietic clonal output of monocytes, granulocytes, B cells, and T cells from a polyclonal pool of hematopoietic stem and progenitor cells in 4 macaques observed for up to 49 months posttransplantation. A broad range of clonal behaviors characterized by contribution level and biases toward certain cell types were extremely stable over time. Correlations between granulocyte and monocyte clonalities were greatest, followed by correlations between these cell types and B cells. We also detected quantitative expansion of T cell–biased clones consistent with an adaptive immune response. In contrast to recent data from a nonquantitative murine model, there was little evidence for clonal succession after initial hematopoietic reconstitution. These findings have important implications for human hematopoiesis, given the similarities between macaque and human physiologies. PMID:28087539

  20. In vivo imaging of transplanted hematopoietic stem and progenitor cells in mouse calvarium bone marrow

    PubMed Central

    Lo Celso, Cristina; Lin, Charles P; Scadden, David T

    2011-01-01

    In vivo imaging of transplanted hematopoietic stem and progenitor cells (HSPCs) was developed to investigate the relationship between HSPCs and components of their microenvironment in the bone marrow. In particular, it allows a direct observation of the behavior of hematopoietic cells during the first few days after transplantation, when the critical events in homing and early engraftment are occurring. By directly imaging these events in living animals, this method permits a detailed assessment of functions previously evaluated by crude assessments of cell counts (homing) or after prolonged periods (engraftment). This protocol offers a new means of investigating the role of cell-intrinsic and cell-extrinsic molecular regulators of hematopoiesis during the early stages of transplantation, and it is the first to allow the study of cell-cell interactions within the bone marrow in three dimensions and in real time. In this paper, we describe how to isolate, label and inject HSPCs, as well as how to perform calvarium intravital microscopy and analyze the resulting images. A typical experiment can be performed and analyzed in ~1 week. PMID:21212779

  1. Dpp dependent Hematopoietic stem cells give rise to Hh dependent blood progenitors in larval lymph gland of Drosophila.

    PubMed

    Dey, Nidhi Sharma; Ramesh, Parvathy; Chugh, Mayank; Mandal, Sudip; Mandal, Lolitika

    2016-10-26

    Drosophila hematopoiesis bears striking resemblance with that of vertebrates, both in the context of distinct phases and the signaling molecules. Even though, there has been no evidence of Hematopoietic stem cells (HSCs) in Drosophila , the larval lymph gland with its Hedgehog dependent progenitors served as an invertebrate model of progenitor biology. Employing lineage-tracing analyses, we have now identified Notch expressing HSCs in the first instar larval lymph gland. Our studies clearly establish the hierarchical relationship between Notch expressing HSCs and the previously described Domeless expressing progenitors. These HSCs require Decapentapelagic (Dpp) signal from the hematopoietic niche for their maintenance in an identical manner to vertebrate aorta-gonadal-mesonephros (AGM) HSCs. Thus, this study not only extends the conservation across these divergent taxa, but also provides a new model that can be exploited to gain better insight into the AGM related Hematopoietic stem cells (HSCs).

  2. UMG Lenti: Novel Lentiviral Vectors for Efficient Transgene- and Reporter Gene Expression in Human Early Hematopoietic Progenitors

    PubMed Central

    Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G.; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B.; Grillone, Teresa; Giovannone, Emilia D.; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A. S.; Bond, Heather M.; Mesuraca, Maria; Morrone, Giovanni

    2014-01-01

    Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and –LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG–LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and

  3. UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

    PubMed

    Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B; Grillone, Teresa; Giovannone, Emilia D; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A S; Bond, Heather M; Mesuraca, Maria; Morrone, Giovanni

    2014-01-01

    Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and

  4. Expression of Coxsackievirus and Adenovirus Receptor Separates Hematopoietic and Cardiac Progenitor Cells in Fetal Liver Kinase 1-Expressing Mesoderm

    PubMed Central

    Tashiro, Katsuhisa; Hirata, Nobue; Okada, Atsumasa; Yamaguchi, Tomoko; Takayama, Kazuo; Mizuguchi, Hiroyuki

    2015-01-01

    In developing embryos or in vitro differentiation cultures using pluripotent stem cells (PSCs), such as embryonic stem cells and induced pluripotent stem cells, fetal liver kinase 1 (Flk1)-expressing mesodermal cells are thought to be a heterogeneous population that includes hematopoietic progenitors, endothelial progenitors, and cardiac progenitors. However, information on cell surface markers for separating these progenitors in Flk1+ cells is currently limited. In the present study, we show that distinct types of progenitor cells in Flk1+ cells could be separated according to the expression of coxsackievirus and adenovirus receptor (CAR, also known as CXADR), a tight junction component molecule. We found that mouse and human PSC- and mouse embryo-derived Flk1+ cells could be subdivided into Flk1+CAR+ cells and Flk1+CAR− cells. The progenitor cells with cardiac potential were almost entirely restricted to Flk1+CAR+ cells, and Flk1+CAR− cells efficiently differentiated into hematopoietic cells. Endothelial differentiation potential was observed in both populations. Furthermore, from the expression of CAR, Flk1, and platelet-derived growth factor receptor-α (PDGFRα), Flk1+ cells could be separated into three populations (Flk1+PDGFRα−CAR− cells, Flk1+PDGFRα−CAR+ cells, and Flk1+PDGFRα+CAR+ cells). Flk1+PDGFRα+ cells and Flk1+PDGFRα− cells have been reported as cardiac and hematopoietic progenitor cells, respectively. We identified a novel population (Flk1+PDGFRα−CAR+ cells) with the potential to differentiate into not only hematopoietic cells and endothelial cells but also cardiomyocytes. Our findings indicate that CAR would be a novel and prominent marker for separating PSC- and embryo-derived Flk1+ mesodermal cells with distinct differentiation potentials. PMID:25762001

  5. Downstream targets of HOXB4 in a cell line model of primitive hematopoietic progenitor cells.

    PubMed

    Lee, Han M; Zhang, Hui; Schulz, Vincent; Tuck, David P; Forget, Bernard G

    2010-08-05

    Enforced expression of the homeobox transcription factor HOXB4 has been shown to enhance hematopoietic stem cell self-renewal and expansion ex vivo and in vivo. To investigate the downstream targets of HOXB4 in hematopoietic progenitor cells, HOXB4 was constitutively overexpressed in the primitive hematopoietic progenitor cell line EML. Two genome-wide analytical techniques were used: RNA expression profiling using microarrays and chromatin immunoprecipitation (ChIP)-chip. RNA expression profiling revealed that 465 gene transcripts were differentially expressed in KLS (c-Kit(+), Lin(-), Sca-1(+))-EML cells that overexpressed HOXB4 (KLS-EML-HOXB4) compared with control KLS-EML cells that were transduced with vector alone. In particular, erythroid-specific gene transcripts were observed to be highly down-regulated in KLS-EML-HOXB4 cells. ChIP-chip analysis revealed that the promoter region for 1910 genes, such as CD34, Sox4, and B220, were occupied by HOXB4 in KLS-EML-HOXB4 cells. Side-by-side comparison of the ChIP-chip and RNA expression profiling datasets provided correlative information and identified Gp49a and Laptm4b as candidate "stemness-related" genes. Both genes were highly ranked in both dataset lists and have been previously shown to be preferentially expressed in hematopoietic stem cells and down-regulated in mature hematopoietic cells, thus making them attractive candidates for future functional studies in hematopoietic cells.

  6. Intrathymic injection of hematopoietic progenitor cells establishes functional T cell development in a mouse model of severe combined immunodeficiency.

    PubMed

    Tuckett, Andrea Z; Thornton, Raymond H; O'Reilly, Richard J; van den Brink, Marcel R M; Zakrzewski, Johannes L

    2017-05-16

    Even though hematopoietic stem cell transplantation can be curative in patients with severe combined immunodeficiency, there is a need for additional strategies boosting T cell immunity in individuals suffering from genetic disorders of lymphoid development. Here we show that image-guided intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγ null mice is feasible and facilitates the generation of functional T cells conferring protective immunity. Hematopoietic stem and progenitor cells were isolated from the bone marrow of healthy C57BL/6 mice (wild-type, Luciferase + , CD45.1 + ) and injected intravenously or intrathymically into both male and female, young or aged NOD-scid IL2rγ null recipients. The in vivo fate of injected cells was analyzed by bioluminescence imaging and flow cytometry of thymus- and spleen-derived T cell populations. In addition to T cell reconstitution, we evaluated mice for evidence of immune dysregulation based on diabetes development and graft-versus-host disease. T cell immunity following intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγ null mice was assessed in a B cell lymphoma model. Despite the small size of the thymic remnant in NOD-scid IL2rγ null mice, we were able to accomplish precise intrathymic delivery of hematopoietic stem and progenitor cells by ultrasound-guided injection. Thymic reconstitution following intrathymic injection of healthy allogeneic hematopoietic cells was most effective in young male recipients, indicating that even in the setting of severe immunodeficiency, sex and age are important variables for thymic function. Allogeneic T cells generated in intrathymically injected NOD-scid IL2rγ null mice displayed anti-lymphoma activity in vivo, but we found no evidence for severe auto/alloreactivity in T cell-producing NOD-scid IL2rγ null mice, suggesting that immune dysregulation is not a major concern. Our findings suggest that intrathymic

  7. Proteomic Cornerstones of Hematopoietic Stem Cell Differentiation: Distinct Signatures of Multipotent Progenitors and Myeloid Committed Cells*

    PubMed Central

    Klimmeck, Daniel; Hansson, Jenny; Raffel, Simon; Vakhrushev, Sergey Y.; Trumpp, Andreas; Krijgsveld, Jeroen

    2012-01-01

    Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors which differentiate into lineage committed progenitors and subsequently mature cells. In this report, we applied an in-depth quantitative proteomic approach to analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations highly enriched for either multipotent hematopoietic stem/progenitor cells (HSPCs, LinnegSca-1+c-Kit+) or myeloid committed precursors (LinnegSca-1−c-Kit+). By employing stable isotope dimethyl labeling and high-resolution mass spectrometry, more than 5000 proteins were quantified. From biological triplicate experiments subjected to rigorous statistical evaluation, 893 proteins were found differentially expressed between multipotent and myeloid committed cells. The differential protein content in these cell populations points to a distinct structural organization of the cytoskeleton including remodeling activity. In addition, we found a marked difference in the expression of metabolic enzymes, including a clear shift of specific protein isoforms of the glycolytic pathway. Proteins involved in translation showed a collective higher expression in myeloid progenitors, indicating an increased translational activity. Strikingly, the data uncover a unique signature related to immune defense mechanisms, centering on the RIG-I and type-1 interferon response systems, which are installed in multipotent progenitors but not evident in myeloid committed cells. This suggests that specific, and so far unrecognized, mechanisms protect these immature cells before they mature. In conclusion, this study indicates that the transition of hematopoietic stem/progenitors toward myeloid commitment is accompanied by a profound change in processing of

  8. The potential benefits of nicaraven to protect against radiation-induced injury in hematopoietic stem/progenitor cells with relative low dose exposures

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ali, Haytham; Department of Medical Physiology and Cell Biology, Qena Faculty of Medicine, South Valley University; Galal, Omima

    Highlights: • Nicaraven mitigated the radiation-induced reduction of c-kit{sup +} stem cells. • Nicaraven enhanced the function of hematopoietic stem/progenitor cells. • Complex mechanisms involved in the protection of nicaraven to radiation injury. - Abstract: Nicaraven, a hydroxyl radical-specific scavenger has been demonstrated to attenuate radiation injury in hematopoietic stem cells with 5 Gy γ-ray exposures. We explored the effect and related mechanisms of nicaraven for protecting radiation injury induced by sequential exposures to a relatively lower dose γ-ray. C57BL/6 mice were given nicaraven or placebo within 30 min before exposure to 50 mGy γ-ray daily for 30 days inmore » sequences (cumulative dose of 1.5 Gy). Mice were victimized 24 h after the last radiation exposure, and the number, function and oxidative stress of hematopoietic stem cells were quantitatively estimated. We also compared the gene expression in these purified stem cells from mice received nicaraven and placebo treatment. Nicaraven increased the number of c-kit{sup +} stem/progenitor cells in bone marrow and peripheral blood, with a recovery rate around 60–90% of age-matched non-irradiated healthy mice. The potency of colony forming from hematopoietic stem/progenitor cells as indicator of function was completely protected with nicaraven treatment. Furthermore, nicaraven treatment changed the expression of many genes associated to DNA repair, inflammatory response, and immunomodulation in c-kit{sup +} stem/progenitor cells. Nicaraven effectively protected against damages of hematopoietic stem/progenitor cells induced by sequential exposures to a relatively low dose radiation, via complex mechanisms.« less

  9. Correlated miR-mRNA expression signatures of mouse hematopoietic stem and progenitor cell subsets predict "Stemness" and "Myeloid" interaction networks.

    PubMed

    Heiser, Diane; Tan, Yee Sun; Kaplan, Ian; Godsey, Brian; Morisot, Sebastien; Cheng, Wen-Chih; Small, Donald; Civin, Curt I

    2014-01-01

    Several individual miRNAs (miRs) have been implicated as potent regulators of important processes during normal and malignant hematopoiesis. In addition, many miRs have been shown to fine-tune intricate molecular networks, in concert with other regulatory elements. In order to study hematopoietic networks as a whole, we first created a map of global miR expression during early murine hematopoiesis. Next, we determined the copy number per cell for each miR in each of the examined stem and progenitor cell types. As data is emerging indicating that miRs function robustly mainly when they are expressed above a certain threshold (∼100 copies per cell), our database provides a resource for determining which miRs are expressed at a potentially functional level in each cell type. Finally, we combine our miR expression map with matched mRNA expression data and external prediction algorithms, using a Bayesian modeling approach to create a global landscape of predicted miR-mRNA interactions within each of these hematopoietic stem and progenitor cell subsets. This approach implicates several interaction networks comprising a "stemness" signature in the most primitive hematopoietic stem cell (HSC) populations, as well as "myeloid" patterns associated with two branches of myeloid development.

  10. Pre-malignant lymphoid cells arise from hematopoietic stem/progenitor cells in chronic lymphocytic leukemia.

    PubMed

    Kikushige, Yoshikane; Miyamoto, Toshihiro

    2015-11-01

    Human malignancies progress through a multistep process that includes the development of critical somatic mutations over the clinical course. Recent novel findings have indicated that hematopoietic stem cells (HSCs), which have the potential to self-renew and differentiate into multilineage hematopoietic cells, are an important cellular target for the accumulation of critical somatic mutations in hematological malignancies and play a central role in myeloid malignancy development. In contrast to myeloid malignancies, mature lymphoid malignancies, such as chronic lymphocytic leukemia (CLL), are thought to originate directly from differentiated mature lymphocytes; however, recent compelling data have shown that primitive HSCs and hematopoietic progenitor cells contribute to the pathogenesis of mature lymphoid malignancies. Several representative mutations of hematological malignancies have been identified within the HSCs of CLL and lymphoma patients, indicating that the self-renewing long-lived fraction of HSCs can serve as a reservoir for the development of oncogenic events. Novel mice models have been established as human mature lymphoma models, in which specific oncogenic events target the HSCs and immature progenitor cells. These data collectively suggest that HSCs can be the cellular target involved in the accumulation of oncogenic events in the pathogenesis of mature lymphoid and myeloid malignancies.

  11. Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

    PubMed Central

    Gori, Jennifer L.; Butler, Jason M.; Chan, Yan-Yi; Chandrasekaran, Devikha; Poulos, Michael G.; Ginsberg, Michael; Nolan, Daniel J.; Elemento, Olivier; Wood, Brent L.; Adair, Jennifer E.; Rafii, Shahin; Kiem, Hans-Peter

    2015-01-01

    Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC–derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina–induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34+CD45+ hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor γ chain–null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood–derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34+ cells, suggesting these ligands are critical for HSC emergence. PMID:25664855

  12. Parathyroid Hormone Regulates the Distribution and Osteoclastogenic Potential of Hematopoietic Progenitors in the Bone Marrow

    PubMed Central

    Jacome-Galarza, Christian E; Lee, Sun-Kyeong; Lorenzo, Joseph A; Aguila, Hector Leonardo

    2011-01-01

    Parathyroid hormone (PTH) increases both the number of osteoclast in bone and the number of early hematopoietic stem cells (HSCs) in bone marrow. We previously characterized the phenotype of multiple populations of bone marrow cells with in vitro osteoclastogenic potential in mice. Here we examined whether intermittent administration of PTH influences these osteoclast progenitor (OCP) populations. C57BL/6 mice were treated with daily injections of bPTH(1–34) (80 μg/kg/day) for 7 or 14 days. We found that PTH caused a significant increase in the percentage of TN/CD115+CD117high and TN/CD115+CD117int cells ( p <.05) in bone marrow on day 7. In contrast, PTH decreased the absolute number of TN/CD115+CD117low cells by 39% on day 7 ( p <.05). On day 14, there was no effect of PTH on osteoclast progenitor distribution in vivo. However, PTH treatment for 7 and 14 days did increase receptor activator of NF-κB ligand (RANKL)– and macrophage colony-stimulating factor (M-CSF)–stimulated in vitro osteoclastogenesis and bone resorption in TN/CD115+ cells. In the periphery, 14 days of treatment increased the percentage and absolute numbers of HSCs (Lin−CD117+Sca-1+) in the spleen ( p <.05). These data correlated with an increase in the percent and absolute numbers of HSCs in bone marrow on day 14 ( p <.05). Interestingly, the effects on hematopoietic progenitors do not depend on osteoclast resorption activity. These results suggest that in vivo PTH treatment increased in vitro osteoclastogenesis and resorption without altering the number of osteoclast precursors. This implies that in vivo PTH induces sustained changes, possibly through an epigenetic mechanism, in the in vitro responsiveness of the cells to M-CSF and RANKL. PMID:21611963

  13. Myeloid-derived suppressor cells can be efficiently generated from human hematopoietic progenitors and peripheral blood monocytes.

    PubMed

    Casacuberta-Serra, Sílvia; Parés, Marta; Golbano, Arantxa; Coves, Elisabet; Espejo, Carmen; Barquinero, Jordi

    2017-07-01

    Myeloid-derived suppressor cells (MDSCs) have an important role in controlling inflammation. As such, they are both a therapeutic target and, based on the administration of ex vivo-generated MDSCs, a therapeutic tool. However, there are relatively few reports describing methods to generate human MDSCs, and most of them rely on cells obtained from peripheral blood monocytes. We investigated alternative approaches to the generation of MDSCs from hematopoietic progenitors and monocytes. Purified CD34 + hematopoietic progenitors from apheresis products and CD14 + cells isolated from buffy coats were cultured in the presence of different combinations of cytokines. The resulting myeloid cell populations were then characterized phenotypically and functionally. Progenitor cells cultured in the presence of SCF+TPO+FLT3-L+GM-CSF+IL-6 gave rise to both monocytic (M)- and granulocytic (G)-MDSCs but production of the latter was partially inhibited by IL-3. M-MDSCs but not G-MDSCs were obtained by culturing peripheral blood monocytes with GM-CSF+IL-6 or GM-CSF+TGF-β1 for 6 days. CD14 expression was downregulated in the cultured cells. PD-L1 expression at baseline was lower in hematopoietic progenitor cell-derived than in monocyte-derived MDSCs, but was markedly increased in response to stimulation with LPS+IFN-γ. The functionality of the two MDSC subtypes was confirmed in studies of the suppression of allogeneic and mitogen-induced proliferation and by cytokine profiling. Here we describe both the culture conditions that allow the generation of MDSCs and the phenotypical and functional characterization of these cell populations.

  14. Differential Reponses of Hematopoietic Stem and Progenitor Cells to mTOR Inhibition

    PubMed Central

    Yang, Aimin; Xiao, Xia; Zhao, Mingfeng; LaRue, Amanda C.; Schulte, Bradley A.; Wang, Gavin Y.

    2015-01-01

    Abnormal activation of the mammalian target of rapamycin (mTOR) signaling pathway has been observed in a variety of human cancers. Therefore, targeting of the mTOR pathway is an attractive strategy for cancer treatment and several mTOR inhibitors, including AZD8055 (AZD), a novel dual mTORC1/2 inhibitor, are currently in clinical trials. Although bone marrow (BM) suppression is one of the primary side effects of anticancer drugs, it is not known if pharmacological inhibition of dual mTORC1/2 affects BM hematopoietic stem and progenitor cells (HSPCs) function and plasticity. Here we report that dual inhibition of mTORC1/2 by AZD or its analogue (KU-63794) depletes mouse BM Lin−Sca-1+c-Kit+ cells in cultures via the induction of apoptotic cell death. Subsequent colony-forming unit (CFU) assays revealed that inhibition of mTORC1/2 suppresses the clonogenic function of hematopoietic progenitor cells (HPCs) in a dose-dependent manner. Surprisingly, we found that dual inhibition of mTORC1/2 markedly inhibits the growth of day-14 cobblestone area-forming cells (CAFCs) but enhances the generation of day-35 CAFCs. Given the fact that day-14 and day-35 CAFCs are functional surrogates of HPCs and hematopoietic stem cells (HSCs), respectively, these results suggest that dual inhibition of mTORC1/2 may have distinct effects on HPCs versus HSCs. PMID:26221145

  15. Lineage-related cytotoxicity and clonogenic profile of 1,4-benzoquinone-exposed hematopoietic stem and progenitor cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chow, Paik Wah; Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur; Abdul Hamid, Zariyantey, E-mail: zyantey@ukm.edu.my

    Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are sensitive targets for benzene-induced hematotoxicity and leukemogenesis. The impact of benzene exposure on the complex microenvironment of HSCs and HPCs remains elusive. This study aims to investigate the mechanism linking benzene exposure to targeting HSCs and HPCs using phenotypic and clonogenic analyses. Mouse bone marrow (BM) cells were exposed ex vivo to the benzene metabolite, 1,4-benzoquinone (1,4-BQ), for 24 h. Expression of cellular surface antigens for HSC (Sca-1), myeloid (Gr-1, CD11b), and lymphoid (CD45, CD3e) populations were confirmed by flow cytometry. The clonogenicity of cells was studied using the colony-formingmore » unit (CFU) assay for multilineage (CFU-GM and CFU-GEMM) and single-lineage (CFU-E, BFU-E, CFU-G, and CFU-M) progenitors. 1,4-BQ demonstrated concentration-dependent cytotoxicity in mouse BM cells. The percentage of apoptotic cells increased (p < 0.05) following 1,4-BQ exposure. Exposure to 1,4-BQ showed no significant effect on CD3e{sup +} cells but reduced the total counts of Sca-1{sup +}, CD11b{sup +}, Gr-1{sup +}, and CD45{sup +} cells at 7 and 12 μM (p < 0.05). Furthermore, the CFU assay showed reduced (p < 0.05) clonogenicity in 1,4-BQ-treated cells. 1,4-BQ induced CFU-dependent cytotoxicity by significantly inhibiting colony growth for CFU-E, BFU-E, CFU-G, and CFU-M starting at a low concentration of exposure (5 μM); whereas for the CFU-GM and CFU-GEMM, the inhibition of colony growth was remarkable only at 7 and 12 μM of 1,4-BQ, respectively. Taken together, 1,4-BQ caused lineage-related cytotoxicity in mouse HPCs, demonstrating greater toxicity in single-lineage progenitors than in those of multi-lineage. - Highlights: • We examine 1,4-BQ toxicity targeting mouse hematopoietic cell lineages. • 1,4-BQ induces concentration-dependent cytotoxicity in bone marrow (BM) cells. • 1,4-BQ shows lineage-related toxicity on hematopoietic stem and

  16. Correction of glucocerebrosidase deficiency after retroviral-mediated gene transfer into hematopoietic progenitor cells from patients with Gaucher disease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fink, J.K.; Correll, P.H.; Perry, L.K.

    1990-03-01

    Retroviral gene transfer has been used successfully to correct the glucocerebrosidase (GCase) deficiency in primary hematopoietic cells from patients with Gaucher disease. For this model of somatic gene therapy, the authors developed a high-titer, amphotropic retroviral vector designated NTG in which the human GCase gene was driven by the mutant polyoma virus enhancer/herpesvirus thymidine kinase gene (tk) promoter (Py{sup +}/Htk). NTG normalized GCase activity in transduced Gaucher fibroblasts and efficiently infected human monocytic and erythroleukemic cell lines. RNA blot-hybridization (Northern blot) analysis of these hemaptopoietic cell lines showed unexpectedly high-level expression from the Moloney murine leukemia virus long terminal repeatmore » (Mo-MLV LTR) and levels of Py{sup +}/Htk enhancer/promoter-initiated human GCase RNA that approximated endogenous GCase RNA levels. Furthermore, NTG efficiently infected human hematopoietic progenitor cells. Detection of the provirus in approximately one-third of NTG-infected progenitor colonies that had not been selected in G418-containing medium indicates that relative resistance to G418 underestimated the actual gene transfer efficiency. Northern blot analysis of NTG-infected, progenitor-derived cells showed expression from both the Mo-MLV LTR and the Py{sup +}/Htk enhancer/promoter. NTG-transduced hematopoietic progenitor cells from patients with Gaucher disease generated progeny in which GCase activity has been normalized.« less

  17. Widespread Non-Hematopoietic Tissue Distribution by Transplanted Human Progenitor Cells with High Aldehyde Dehydrogenase Activity

    PubMed Central

    Hess, David A.; Craft, Timothy P.; Wirthlin, Louisa; Hohm, Sarah; Zhou, Ping; Eades, William C.; Creer, Michael H.; Sands, Mark S.; Nolta, Jan A.

    2011-01-01

    Transplanted adult progenitor cells distribute to peripheral organs and can promote endogenous cellular repair in damaged tissues. However, development of cell-based regenerative therapies has been hindered by the lack of pre-clinical models to efficiently assess multiple organ distribution and difficulty defining human cells with regenerative function. After transplantation into beta-glucuronidase (GUSB)-deficient NOD/SCID/MPSVII mice, we characterized the distribution of lineage depleted human umbilical cord blood-derived cells purified by selection using high aldehyde dehydrogenase activity (ALDH) with CD133 co-expression. ALDHhi or ALDHhiCD133+ cells produced robust hematopoietic reconstitution, and variable levels of tissue distribution in multiple organs. GUSB+ donor cells that co-expressed human (HLA-A,B,C) and hematopoietic (CD45+) cell surface markers were the primary cell phenotype found adjacent to the vascular beds of several tissues, including islet and ductal regions of mouse pancreata. In contrast, variable phenotypes were detected in the chimeric liver, with HLA+/CD45+ cells demonstrating robust GUSB expression adjacent to blood vessels, and CD45−/HLA− cells with diluted GUSB expression predominant in the liver parenchyma. However, true non-hematopoietic human (HLA+/CD45−) cells were rarely detected in other peripheral tissues, suggesting that these GUSB+/HLA−/CD45− cells in the liver were a result of downregulated human surface marker expression in vivo, not widespread seeding of non-hematopoietic cells. However, relying solely on continued expression of cell surface markers, as employed in traditional xenotransplantation models, may underestimate true tissue distribution. ALDH-expressing progenitor cells demonstrated widespread and tissue-specific distribution of variable cellular phenotypes, indicating that these adult progenitor cells should be explored in transplantation models of tissue damage. PMID:18055447

  18. Failure of hepatocyte marker-expressing hematopoietic progenitor cells to efficiently convert into hepatocytes in vitro.

    PubMed

    Lian, Gewei; Wang, Chengyan; Teng, Chunbo; Zhang, Cong; Du, Liying; Zhong, Qian; Miao, Chenglin; Ding, Mingxiao; Deng, Hongkui

    2006-03-01

    Whether bone marrow (BM) hematopoietic stem/progenitor cells can directly differentiate into nonhematopoietic cells remains controversial. The aim of this study is to further investigate the potentiality of BM hematopoietic progenitor cells to convert into hepatocytes in vitro. Different subsets of BM cells from C57/BL6 mice were isolated using markers of hematopoietic stem cells by magnetic cell sorting and by flow cytometry. These cells were induced to transdifferentiate to hepatocytes in vitro in the presence of various cytokines or of hepatocytes (or tissue) from damaged liver, which have been reported to stimulate the conversion. Hepatic gene markers in freshly isolated or cultured BM cells were determined by reverse transcriptase polymerase chain reaction and immunofluorescence. Freshly isolated hematopoietic progenitor cells (HPC) expressed a low level of messenger RNAs of hepatic cell-specific markers including albumin and alpha-fetoprotein (AFP), but did not significantly upregulate expression of these markers, even in the presence of cytokines or cocultured hepatocytes (or tissue). HPCs induced in vitro did not express the message of alpha-anti-trypsin-a mature hepatocyte marker. At protein level, the specific staining of AFP was not detected in the HPCs, either freshly isolated or in vitro induced. Albumin protein was detected in freshly isolated albumin mRNA-positive and -negative BM cell subpopulations. Albumin-stained BM cells disappeared after being induced for 5 days, but restained if mouse serum was supplemented in medium for a 24-hour extended culture, suggesting that albumin was absorbed by BM cells instead of de novo expression. HPCs expressed mRNAs of hepatic cell markers, but could not efficiently convert into hepatocytes in vitro under our experimental conditions. Our observation raises a cautionary note in determining whether in vitro transdifferentiation of BM cells to hepatocytes can actually take place.

  19. The lung is a site of platelet biogenesis and a reservoir for hematopoietic progenitors

    PubMed Central

    Lefrançais, Emma; Ortiz-Muñoz, Guadalupe; Caudrillier, Axelle; Mallavia, Beñat; Liu, Fengchun; Sayah, David M.; Thornton, Emily E.; Headley, Mark B.; David, Tovo; Coughlin, Shaun R.; Krummel, Matthew F.; Leavitt, Andrew D.; Passegué, Emmanuelle; Looney, Mark R.

    2017-01-01

    Platelets are critical for hemostasis, thrombosis, and inflammatory responses1,2, yet the events leading to mature platelet production remain incompletely understood3. The bone marrow (BM) is proposed to be a major site of platelet production although indirect evidence points towards a potential pulmonary contribution to platelet biogenesis4-7. By directly imaging the lung microcirculation in mice8, we discovered that a large number of megakaryocytes (MKs) circulate through the lungs where they dynamically release platelets. MKs releasing platelets in the lung are of extrapulmonary origin, such as the BM, where we observed large MKs migrating out of the BM space. The lung contribution to platelet biogenesis is substantial with approximately 50% of total platelet production or 10 million platelets per hour. Furthermore, we identified populations of mature and immature MKs along with hematopoietic progenitors that reside in the extravascular spaces of the lung. Under conditions of thrombocytopenia and relative stem cell deficiency in the BM9, these progenitors can migrate out of the lung, repopulate the BM, completely reconstitute blood platelet counts, and contribute to multiple hematopoietic lineages. These results position the lung as a primary site of terminal platelet production and an organ with considerable hematopoietic potential. PMID:28329764

  20. Functional evaluation of circulating hematopoietic progenitors in Noonan syndrome

    PubMed Central

    TIMEUS, FABIO; CRESCENZIO, NICOLETTA; BALDASSARRE, GIUSEPPINA; DORIA, ALESSANDRA; VALLERO, STEFANO; FOGLIA, LUISELDA; PAGLIANO, SARA; ROSSI, CESARE; SILENGO, MARGHERITA CIRILLO; RAMENGHI, UGO; FAGIOLI, FRANCA; DI MONTEZEMOLO, LUCA CORDERO; FERRERO, GIOVANNI BATTISTA

    2013-01-01

    Noonan syndrome (NS) is an autosomal dominant disorder, characterized by short stature, multiple dysmorphisms and congenital heart defects. A myeloproliferative disorder (NS/MPD), resembling juvenile myelomonocytic leukemia (JMML), is occasionally diagnosed in infants with NS. In the present study, we performed a functional evaluation of the circulating hematopoietic progenitors in a series of NS, NS/MPD and JMML patients. The different functional patterns were compared with the aim to identify a possible NS subgroup worthy of stringent hematological follow-up for an increased risk of MPD development. We studied 27 NS and 5 JMML patients fulfilling EWOG-MDS criteria. The more frequent molecular defects observed in NS were mutations in the PTPN11 and SOS genes. The absolute count of monocytes, circulating CD34+ hematopoietic progenitors, their apoptotic rate and the number of circulating CFU-GMs cultured in the presence of decreasing concentrations or in the absence of granulocyte-macrophage colony-stimulating factor (GM-CSF) were evaluated. All JMML patients showed monocytosis >1,000/μl. Ten out of the 27 NS patients showed monocytosis >1,000/μl, which included the 3 NS/MPD patients. In JMML patients, circulating CD34+ cells were significantly increased (median, 109.8/μl; range, 44–232) with a low rate of apoptosis (median, 2.1%; range, 0.4–12.1%), and circulating CFU-GMs were hyper-responsive to GM-CSF. NS/MPD patients showed the same flow cytometric pattern as the JMML patients (median, CD34+ cells/μl, 205.7; range, 58–1374; median apoptotic rate, 1.4%; range, 0.2–2.4%) and their circulating CFU-GMs were hyper-responsive to GM-CSF. These functional alterations appeared 10 months before the typical clinical manifestations in 1 NS/MPD patient. In NS, the CD34+ absolute cell count and circulating CFU-GMs showed a normal pattern (median CD34+ cells/μl, 4.9; range, 1.3–17.5), whereas the CD34+ cell apoptotic rate was significantly decreased in

  1. Stromal and Hematopoietic Progenitors from C57/BI/6N Murine Bone Marrow After 30-Day "BION-M1" Spaceflight.

    PubMed

    Markina, Elena; Andreeva, Elena; Andrianova, Irina; Sotnezova, Elena; Buravkova, Ludmila

    2018-05-02

    Elucidation of the spaceflight (SF) effects on the adult stem and progenitor cells is an important goal in space biology and medicine. A unique opportunity for this was provided by project "BION-M1". The purpose of this study was to evaluate the effects of 30-day SF on biosatellite, 7-day recovery (SFR), and subsequent ground control (GC) experiment on the mononuclear cells (MNCs) from C57/BI/6N murine tibia bone marrow. Also, hematopoietic and stromal precursor functions were characterized ex vivo. There was no significant difference in the total MNC number between experimental groups. After SF, immunophenotyping revealed an increase of large-sized CD45 + MNCs corresponded to committed hematopoietic progenitors. The total hematopoietic colony-forming unit (CFU) number decreased after SF and did not restore after 7 day of recovery due to predominant reduction of bi- and multipotent CFUs and primitive burst-forming units in favor of unipotent CFUs. Functional activity of stromal precursors in vitro was only slightly altered. SF cells displayed the enhanced expression of alkaline phosphatase. The data of the GC experiment demonstrated the preservation of the functional activity of progenitor cells from mice bone marrow. The activation of erythropoiesis in expense of burst-forming units of erythrocytes elevation was detected. After 7 days of recovery, the number of colony-forming units of fibroblast (CFUs-f) was similar to the vivarium control, while the proliferative activity of bone marrow stromal precursors decreased. The present study demonstrated that certain hematopoietic progenitors are susceptible to SF factors, while the stromal precursors displayed a certain degree of resistance. These data indicate mild and reversible alterations of bone marrow progenitors after SF.

  2. Multiple myeloma-related deregulation of bone marrow-derived CD34(+) hematopoietic stem and progenitor cells.

    PubMed

    Bruns, Ingmar; Cadeddu, Ron-Patrick; Brueckmann, Ines; Fröbel, Julia; Geyh, Stefanie; Büst, Sebastian; Fischer, Johannes C; Roels, Frederik; Wilk, Christian Matthias; Schildberg, Frank A; Hünerlitürkoglu, Ali-Nuri; Zilkens, Christoph; Jäger, Marcus; Steidl, Ulrich; Zohren, Fabian; Fenk, Roland; Kobbe, Guido; Brors, Benedict; Czibere, Akos; Schroeder, Thomas; Trumpp, Andreas; Haas, Rainer

    2012-09-27

    Multiple myeloma (MM) is a clonal plasma cell disorder frequently accompanied by hematopoietic impairment. We show that hematopoietic stem and progenitor cells (HSPCs), in particular megakaryocyte-erythrocyte progenitors, are diminished in the BM of MM patients. Genomic profiling of HSPC subsets revealed deregulations of signaling cascades, most notably TGFβ signaling, and pathways involved in cytoskeletal organization, migration, adhesion, and cell-cycle regulation in the patients. Functionally, proliferation, colony formation, and long-term self-renewal were impaired as a consequence of activated TGFβ signaling. In accordance, TGFβ levels in the BM extracellular fluid were elevated and mesenchymal stromal cells (MSCs) had a reduced capacity to support long-term hematopoiesis of HSPCs that completely recovered on blockade of TGFβ signaling. Furthermore, we found defective actin assembly and down-regulation of the adhesion receptor CD44 in MM HSPCs functionally reflected by impaired migration and adhesion. Still, transplantation into myeloma-free NOG mice revealed even enhanced engraftment and normal differentiation capacities of MM HSPCs, which underlines that functional impairment of HSPCs depends on MM-related microenvironmental cues and is reversible. Taken together, these data implicate that hematopoietic suppression in MM emerges from the HSPCs as a result of MM-related microenvironmental alterations.

  3. DNA damage tolerance in hematopoietic stem and progenitor cells in mice

    PubMed Central

    Pilzecker, Bas; Buoninfante, Olimpia Alessandra; van den Berk, Paul; Lancini, Cesare; Song, Ji-Ying; Citterio, Elisabetta

    2017-01-01

    DNA damage tolerance (DDT) enables bypassing of DNA lesions during replication, thereby preventing fork stalling, replication stress, and secondary DNA damage related to fork stalling. Three modes of DDT have been documented: translesion synthesis (TLS), template switching (TS), and repriming. TLS and TS depend on site-specific PCNA K164 monoubiquitination and polyubiquitination, respectively. To investigate the role of DDT in maintaining hematopoietic stem cells (HSCs) and progenitors, we used PcnaK164R/K164R mice as a unique DDT-defective mouse model. Analysis of the composition of HSCs and HSC-derived multipotent progenitors (MPPs) revealed a significantly reduced number of HSCs, likely owing to increased differentiation of HSCs toward myeloid/erythroid-associated MPP2s. This skewing came at the expense of the number of lymphoid-primed MPP4s, which appeared to be compensated for by increased MPP4 proliferation. Furthermore, defective DDT decreased the numbers of MPP-derived common lymphoid progenitor (CLP), common myeloid progenitor (CMP), megakaryocyte-erythroid progenitor (MEP), and granulocyte-macrophage progenitor (GMP) cells, accompanied by increased cell cycle arrest in CMPs. The HSC and MPP phenotypes are reminiscent of premature aging and stressed hematopoiesis, and indeed progressed with age and were exacerbated on cisplatin exposure. Bone marrow transplantations revealed a strong cell intrinsic defect of DDT-deficient HSCs in reconstituting lethally irradiated mice and a strong competitive disadvantage when cotransplanted with wild-type HSCs. These findings indicate a critical role of DDT in maintaining HSCs and progenitor cells, and in preventing premature aging. PMID:28761001

  4. Delta-Tocotrienol Protects Mouse and Human Hematopoietic Progenitors from Gamma-Irradiation Through Erk/mTOR Signaling

    DTIC Science & Technology

    2010-01-01

    δ- tocotrienol protects mouse and human hematopoietic progenitors from γ-irradiation through Erk/mTOR signaling by Xiang Hong Li, Dadin Fu, Nabil H...print] Citation: Li XH, Fu D, Latif NH, Mullaney CP, Ney PH, Mog SR, Whitnall MH, Srinivasan V, and Xiao M. δ- tocotrienol protects mouse and human...2010 2. REPORT TYPE 3. DATES COVERED 00-00-2010 to 00-00-2010 4. TITLE AND SUBTITLE !- tocotrienol protects mouse and human hematopoietic

  5. Eotaxin-rich Proangiogenic Hematopoietic Progenitor Cells and CCR3+ Endothelium in the Atopic Asthmatic Response

    PubMed Central

    Asosingh, Kewal; Vasanji, Amit; Tipton, Aaron; Queisser, Kimberly; Wanner, Nicholas; Janocha, Allison; Grandon, Deepa; Anand-Apte, Bela; Rothenberg, Marc. E.; Dweik, Raed; Erzurum, Serpil C.

    2016-01-01

    Angiogenesis is closely linked to and precedes eosinophilic infiltration in asthma. Eosinophils are recruited into the airway by chemoattractant eotaxins, which are expressed by endothelial cells, smooth muscles cells, epithelial cells, and hematopoietic cells. We hypothesized that bone marrow-derived proangiogenic progenitor cells that contain eotaxins contribute to the initiation of angiogenesis and inflammation in asthma. Whole lung allergen challenge of atopic asthma patients revealed vascular activation occurs within hours of challenge, and prior to airway inflammation. The eotaxin receptor CCR3 was expressed at high levels on submucosal endothelial cells in patients and murine model of asthma. Exvivo exposure of murine endothelial cells to eotaxins induced migration and angiogenesis. In mechanistic studies, wildtype mice transplanted with eotaxin-1/2 deficient bone marrow had markedly less angiogenesis and inflammation in an atopic asthma model, while adoptive transfer of proangiogenic progenitor cells from wildtype mice in an atopic asthma model into the eotaxin-1/2 deficient mice led to angiogenesis and airway inflammation. The findings indicate that TH2-promoting hematopoietic progenitor cells are rapidly recruited to the lung upon allergen exposure and release eotaxins that coordinately activate endothelial cells, angiogenesis, and airway inflammation. PMID:26810221

  6. Repurposing Treprostinil for Enhancing Hematopoietic Progenitor Cell Transplantation

    PubMed Central

    Kazemi, Zahra; Bergmayr, Christian; Prchal-Murphy, Michaela; Javaheri, Tahereh; Themanns, Madeleine; Pham, Ha T. T.; Strohmaier, Wolfgang; Sexl, Veronika; Zebedin-Brandl, Eva

    2016-01-01

    Activation of Gs-coupled receptors enhances engraftment of hematopoietic stem and progenitor cells (HSPCs). We tested the hypothesis that treprostinil, a prostacyclin analog approved for the treatment of pulmonary hypertension, can be repurposed to improve hematopoietic stem cell transplantation. Murine and human HSPCs were isolated from bone marrow and umbilical cord blood, respectively. Prostanoid receptor agonists and the combination thereof with forskolin were tested for their capacity to stimulate [3H]cAMP accumulation in HSPCs. Three independent approaches were employed to verify the ability of agonist-activated HSPCs to reconstitute the bone marrow in lethally irradiated recipient mice. The underlying mechanism was explored in cellular migration assays and by blocking C-X-C motif chemokine receptor 4 (CXCR4). Among several prostanoid agonists tested in combination with forskolin, treprostinil was most efficacious in raising intracellular cAMP levels in murine and human HPSCs. Injection of murine and human HSPCs, which had been pretreated with treprostinil and forskolin, enhanced survival of lethally irradiated recipient mice. Survival was further improved if recipient mice were subcutaneously administered treprostinil (0.15 mg kg−1 8 h−1) for 10 days. This regimen also reduced the number of HSPCs required to rescue lethally irradiated mice. Enhanced survival of recipient mice was causally related to treprostinil-enhanced CXCR4-dependent migration of HSPCs. Treprostinil stimulates the engraftment of human and murine hematopoietic stem cells without impairing their capacity for self-renewal. The investigated dose range corresponds to the dose approved for human use. Hence, these findings may be readily translated into a clinical application. PMID:26989084

  7. Demonstration of early functional compromise of bone marrow derived hematopoietic progenitor cells during bovine neonatal pancytopenia through in vitro culture of bone marrow biopsies.

    PubMed

    Laming, Eleanor; Melzi, Eleonora; Scholes, Sandra F E; Connelly, Maira; Bell, Charlotte R; Ballingall, Keith T; Dagleish, Mark P; Rocchi, Mara S; Willoughby, Kim

    2012-10-30

    Bovine neonatal pancytopenia (BNP) is a syndrome characterised by thrombocytopenia associated with marked bone marrow destruction in calves, widely reported since 2007 in several European countries and since 2011 in New Zealand. The disease is epidemiologically associated with the use of an inactivated bovine virus diarrhoea (BVD) vaccine and is currently considered to be caused by absorption of colostral antibody produced by some vaccinated cows ("BNP dams"). Alloantibodies capable of binding to the leukocyte surface have been detected in BNP dams and antibodies recognising bovine MHC class I and β-2-microglobulin have been detected in vaccinated cattle. In this study, calves were challenged with pooled colostrum collected from BNP dams or from non-BNP dams and their bone marrow hematopoietic progenitor cells (HPC) cultured in vitro from sternal biopsies taken at 24 hours and 6 days post-challenge. Clonogenic assay demonstrated that CFU-GEMM (colony forming unit-granulocyte/erythroid/macrophage/megakaryocyte; pluripotential progenitor cell) colony development was compromised from HPCs harvested as early as 24 hour post-challenge. By 6 days post challenge, HPCs harvested from challenged calves failed to develop CFU-E (erythroid) colonies and the development of both CFU-GEMM and CFU-GM (granulocyte/macrophage) was markedly reduced. This study suggests that the bone marrow pathology and clinical signs associated with BNP are related to an insult which compromises the pluripotential progenitor cell within the first 24 hours of life but that this does not initially include all cell types.

  8. Reticular dysgenesis–associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress

    PubMed Central

    Rissone, Alberto; Weinacht, Katja Gabriele; la Marca, Giancarlo; Bishop, Kevin; Giocaliere, Elisa; Jagadeesh, Jayashree; Felgentreff, Kerstin; Dobbs, Kerry; Al-Herz, Waleed; Jones, Marypat; Chandrasekharappa, Settara; Kirby, Martha; Wincovitch, Stephen; Simon, Karen Lyn; Itan, Yuval; DeVine, Alex; Schlaeger, Thorsten; Schambach, Axel; Sood, Raman

    2015-01-01

    Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD. PMID:26150473

  9. Endothelial jagged-2 sustains hematopoietic stem and progenitor reconstitution after myelosuppression.

    PubMed

    Guo, Peipei; Poulos, Michael G; Palikuqi, Brisa; Badwe, Chaitanya R; Lis, Raphael; Kunar, Balvir; Ding, Bi-Sen; Rabbany, Sina Y; Shido, Koji; Butler, Jason M; Rafii, Shahin

    2017-12-01

    Angiocrine factors, such as Notch ligands, supplied by the specialized endothelial cells (ECs) within the bone marrow and splenic vascular niche play an essential role in modulating the physiology of adult hematopoietic stem and progenitor cells (HSPCs). However, the relative contribution of various Notch ligands, specifically jagged-2, to the homeostasis of HSPCs is unknown. Here, we show that under steady state, jagged-2 is differentially expressed in tissue-specific vascular beds, but its expression is induced in hematopoietic vascular niches after myelosuppressive injury. We used mice with EC-specific deletion of the gene encoding jagged-2 (Jag2) to demonstrate that while EC-derived jagged-2 was dispensable for maintaining the capacity of HSPCs to repopulate under steady-state conditions, by activating Notch2 it did contribute to the recovery of HSPCs in response to myelosuppressive conditions. Engraftment and/or expansion of HSPCs was dependent on the expression of endothelial-derived jagged-2 following myeloablation. Additionally, jagged-2 expressed in bone marrow ECs regulated HSPC cell cycle and quiescence during regeneration. Endothelial-deployed jagged-2 triggered Notch2/Hey1, while tempering Notch2/Hes1 signaling in HSPCs. Collectively, these data demonstrate that EC-derived jagged-2 activates Notch2 signaling in HSPCs to promote hematopoietic recovery and has potential as a therapeutic target to accelerate balanced hematopoietic reconstitution after myelosuppression.

  10. Endothelial jagged-2 sustains hematopoietic stem and progenitor reconstitution after myelosuppression

    PubMed Central

    Guo, Peipei; Poulos, Michael G.; Palikuqi, Brisa; Badwe, Chaitanya R.; Lis, Raphael; Kunar, Balvir; Ding, Bi-Sen; Rabbany, Sina Y.; Butler, Jason M.

    2017-01-01

    Angiocrine factors, such as Notch ligands, supplied by the specialized endothelial cells (ECs) within the bone marrow and splenic vascular niche play an essential role in modulating the physiology of adult hematopoietic stem and progenitor cells (HSPCs). However, the relative contribution of various Notch ligands, specifically jagged-2, to the homeostasis of HSPCs is unknown. Here, we show that under steady state, jagged-2 is differentially expressed in tissue-specific vascular beds, but its expression is induced in hematopoietic vascular niches after myelosuppressive injury. We used mice with EC-specific deletion of the gene encoding jagged-2 (Jag2) to demonstrate that while EC-derived jagged-2 was dispensable for maintaining the capacity of HSPCs to repopulate under steady-state conditions, by activating Notch2 it did contribute to the recovery of HSPCs in response to myelosuppressive conditions. Engraftment and/or expansion of HSPCs was dependent on the expression of endothelial-derived jagged-2 following myeloablation. Additionally, jagged-2 expressed in bone marrow ECs regulated HSPC cell cycle and quiescence during regeneration. Endothelial-deployed jagged-2 triggered Notch2/Hey1, while tempering Notch2/Hes1 signaling in HSPCs. Collectively, these data demonstrate that EC-derived jagged-2 activates Notch2 signaling in HSPCs to promote hematopoietic recovery and has potential as a therapeutic target to accelerate balanced hematopoietic reconstitution after myelosuppression. PMID:29058691

  11. JAK2V617F-mutant megakaryocytes contribute to hematopoietic stem/progenitor cell expansion in a model of murine myeloproliferation

    PubMed Central

    Zhan, H; Ma, Y; Lin, CHS; Kaushansky, K

    2016-01-01

    The myeloproliferative neoplasms (MPNs) are characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of mature blood cells. The JAK2V617F mutation is present in hematopoietic cells in a majority of patients with MPNs, but the mechanism(s) responsible for MPN stem cell expansion remain incomplete. One hallmark feature of the marrow in patients with MPNs is megakaryocyte (MK) hyperplasia. We report here that mice bearing a human JAK2V617F gene restricted exclusively to the MK lineage develop many of the features of a MPN. Specifically, these mice exhibit thrombocytosis, splenomegaly, increased numbers of marrow and splenic hematopoietic progenitors and a substantial expansion of HSPCs. In addition, wild-type mice transplanted with cells from JAK2V617F-bearing MK marrow develop a myeloproliferative syndrome with thrombocytosis and erythrocytosis as well as pan-hematopoietic progenitor and stem cell expansion. As marrow histology in this murine model of myeloproliferation reveals a preferentially perivascular localization of JAK2V617F-mutant MKs and an increased marrow sinusoid vascular density, it adds to accumulating data that MKs are an important component of the marrow HSPC niche, and that MK expansion might indirectly contribute to the critical role of the thrombopoietin/c-Mpl signaling pathway in HSPC maintenance and expansion. PMID:27133820

  12. Changes in the frequencies of human hematopoietic stem and progenitor cells with age and site

    PubMed Central

    Farrell, TL; McGuire, TR; Bilek, L; Brusnahan, SK; Jackson, JD; Lane, JT; Garvin, KL; O'Kane, BJ; Berger, AM; Tuljapurkar, SR; Kessinger, MA; Sharp, JG

    2013-01-01

    This study enumerated CD45hi/CD34+ and CD45hi/CD133+ human hematopoietic stem cells (HSC) and granulocyte-monocyte colony forming (GM-CFC) progenitor cells in blood and trochanteric and femoral bone marrow in 233 individuals. Stem cell frequencies were determined by multi-parameter flow cytometry employing an internal control to determine the intrinsic variance of the assays. Progenitor cell frequency was determined using a standard colony assay technique. The frequency of outliers from undetermined methodological causes was highest for blood but less than 5% for all values. The frequency of CD45hi/CD133+ cells correlated highly with the frequency of CD45hi/CD34+ cells in trochanteric and femoral bone marrow. The frequency of these HSC populations in trochanteric and femoral bone marrow rose significantly with age. In contrast, there was no significant trend of either of these cell populations with age in the blood. Trochanteric marrow GM-CFC progenitor cells showed no significant trends with age, but femoral marrow GM-CFC trended downward with age, potentially because of the reported conversion of red marrow at this site to fat with age. Hematopoietic stem and progenitor cells exhibited changes in frequencies with age that differed between blood and bone marrow. We previously reported that side population (SP) multipotential HSC, that include the precursors of CD45hi/CD133+ and CD45hi/CD34+, decline with age. Potentially the increases in stem cell frequencies in the intermediate compartment between SP and GM progenitor cells observed in this study represent a compensatory increase for the loss of more potent members of the HSC hierarchy. PMID:24246745

  13. Eotaxin-Rich Proangiogenic Hematopoietic Progenitor Cells and CCR3+ Endothelium in the Atopic Asthmatic Response.

    PubMed

    Asosingh, Kewal; Vasanji, Amit; Tipton, Aaron; Queisser, Kimberly; Wanner, Nicholas; Janocha, Allison; Grandon, Deepa; Anand-Apte, Bela; Rothenberg, Marc E; Dweik, Raed; Erzurum, Serpil C

    2016-03-01

    Angiogenesis is closely linked to and precedes eosinophilic infiltration in asthma. Eosinophils are recruited into the airway by chemoattractant eotaxins, which are expressed by endothelial cells, smooth muscles cells, epithelial cells, and hematopoietic cells. We hypothesized that bone marrow-derived proangiogenic progenitor cells that contain eotaxins contribute to the initiation of angiogenesis and inflammation in asthma. Whole-lung allergen challenge of atopic asthma patients revealed vascular activation occurs within hours of challenge and before airway inflammation. The eotaxin receptor CCR3 was expressed at high levels on submucosal endothelial cells in patients and a murine model of asthma. Ex vivo exposure of murine endothelial cells to eotaxins induced migration and angiogenesis. In mechanistic studies, wild-type mice transplanted with eotaxin-1/2-deficient bone marrow had markedly less angiogenesis and inflammation in an atopic asthma model, whereas adoptive transfer of proangiogenic progenitor cells from wild-type mice in an atopic asthma model into the eotaxin-1/2-deficient mice led to angiogenesis and airway inflammation. The findings indicate that Th2-promoting hematopoietic progenitor cells are rapidly recruited to the lung upon allergen exposure and release eotaxins that coordinately activate endothelial cells, angiogenesis, and airway inflammation. Copyright © 2016 by The American Association of Immunologists, Inc.

  14. Rapid Lymphocyte Reconstitution of Unconditioned Immunodeficient Mice with Non-Self-Renewing Multipotent Hematopoietic Progenitors

    PubMed Central

    Bhattacharya, Deepta; Bryder, David; Rossi, Derrick J.; Weissman, Irving L.

    2015-01-01

    The replacement of abnormal hematopoietic stem cells (HSCs) with normal transplanted HSCs can correct a wide range of hematologic disorders. Here, we provide evidence that transplantation of more differentiated progenitor cells can be used to more rapidly correct lymphoid deficiencies in unconditioned immunocompromised mice. Transplantation of flk2+ multipotent progenitors led to robust B and T cell reconstitution that was maintained for at least 16 weeks. Antigenic challenge at 16 weeks post-transplantation revealed that reconstituted lymphocytes maintained a functional repertoire. In contrast to the persistent lymphocytic engraftment, myeloid chimerism was lost by 12 weeks post-transplantation consistent with the fact that flk2+ progenitors are non-self-renewing. Thus, while more differentiated progenitors are capable of rescuing lymphoid deficiencies, transplantation of HSCs must be used for the correction of non-lymphoid disorders, and, we propose, very long-term immune reconstitution. Based on recent evidence, we discuss novel strategies to achieve the replacement of abnormal HSCs without the use of cytotoxic conditioning regimens. PMID:16760650

  15. Hematopoietic progenitor cell deficiency in fetuses and children affected by Down's syndrome.

    PubMed

    Holmes, Denise K; Bates, Nicola; Murray, Mary; Ladusans, E J; Morabito, Antonino; Bolton-Maggs, Paula H B; Johnston, Tracey A; Walkenshaw, Steve; Wynn, Robert F; Bellantuono, Ilaria

    2006-12-01

    There is an increased risk of myeloid malignancy in individuals with Down's syndrome (DS), which is associated with a mutation in exon 2 of the transcription factor GATA-1. It is recognized that there is accelerated telomere shortening in blood cells of children with DS similar to that in conditions such as Fanconi anemia and dyskeratosis congenita. The latter conditions are associated with stem cell deficiency and clonal change, including acute myeloid leukemia. In this study we address the questions 1) whether the accelerated telomere shortening is associated with progenitor/stem cell deficiency in individuals with DS, predisposing to clonal change and 2) whether the occurrence of reduced numbers of stem/progenitor cells precede the incidence of mutations in exon 2 of GATA-1. Peripheral blood from fetuses (23-35 weeks gestation) and/or bone marrow from children affected by DS and age-matched hematologically healthy controls were analyzed for telomere length, content of stem/progenitor cells, and mutations in exon 2 of GATA-1. We found that hematopoietic stem/progenitor cell deficiency and telomere shortening occurs in individuals with DS in fetal life. Moreover, the presence of a low number of progenitor cells was not associated with mutations in exon 2 of GATA-1. We propose that stem cell deficiency may be a primary predisposing event to DS leukemia development.

  16. CXCR1 remodels the vascular niche to promote hematopoietic stem and progenitor cell engraftment

    PubMed Central

    Blaser, Bradley W.; Moore, Jessica L.; Hagedorn, Elliott J.; Li, Brian; Riquelme, Raquel; Yang, Song; Zhou, Yi; Tamplin, Owen J.; Binder, Vera

    2017-01-01

    The microenvironment is an important regulator of hematopoietic stem and progenitor cell (HSPC) biology. Recent advances marking fluorescent HSPCs have allowed exquisite visualization of HSPCs in the caudal hematopoietic tissue (CHT) of the developing zebrafish. Here, we show that the chemokine cxcl8 and its receptor, cxcr1, are expressed by zebrafish endothelial cells, and we identify cxcl8/cxcr1 signaling as a positive regulator of HSPC colonization. Single-cell tracking experiments demonstrated that this is a result of increases in HSPC–endothelial cell “cuddling,” HSPC residency time within the CHT, and HSPC mitotic rate. Enhanced cxcl8/cxcr1 signaling was associated with an increase in the volume of the CHT and induction of cxcl12a expression. Finally, using parabiotic zebrafish, we show that cxcr1 acts HSPC nonautonomously to improve the efficiency of donor HSPC engraftment. This work identifies a mechanism by which the hematopoietic niche remodels to promote HSPC engraftment and suggests that cxcl8/cxcr1 signaling is a potential therapeutic target in patients undergoing hematopoietic stem cell transplantation. PMID:28351983

  17. Spheroid Coculture of Hematopoietic Stem/Progenitor Cells and Monolayer Expanded Mesenchymal Stem/Stromal Cells in Polydimethylsiloxane Microwells Modestly Improves In Vitro Hematopoietic Stem/Progenitor Cell Expansion.

    PubMed

    Futrega, Kathryn; Atkinson, Kerry; Lott, William B; Doran, Michael R

    2017-04-01

    While two-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSCs) have been shown to enhance hematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein, we evaluated the capacity of a novel three-dimensional (3D) coculture system to support HSPC expansion in vitro. A high-throughput polydimethylsiloxane (PDMS) microwell platform was used to manufacture thousands of uniform 3D multicellular coculture spheroids. Relative gene expression in 3D spheroid versus 2D adherent BM-derived MSC cultures was characterized and compared with literature reports. We evaluated coculture spheroids, each containing 25-400 MSCs and 10 umbilical cord blood (CB)-derived CD34 + progenitor cells. At low exogenous cytokine concentrations, 2D and 3D MSC coculture modestly improved overall hematopoietic cell and CD34 + cell expansion outcomes. By contrast, a substantial increase in CD34 + CD38 - cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSCs. This outcome indicated that CD34 + CD38 - cell culture yield could be increased using the microwell platform alone, even without MSC coculture support. We found that the increase in CD34 + CD38 - cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NOD/SCID gamma (NSG) mice or a modification in the relative human hematopoietic lineages established in engrafted mice. In summary, there was no statistical difference in CD34 + cell yield from 2D or 3D cocultures, and MSC coculture support provided only modest benefit in either geometry. While the high-throughput 3D microwell platform may provide a useful model system for studying cells in coculture, further optimization will be required to generate HSPC yields suitable for use in clinical applications.

  18. Spheroid Coculture of Hematopoietic Stem/Progenitor Cells and Monolayer Expanded Mesenchymal Stem/Stromal Cells in Polydimethylsiloxane Microwells Modestly Improves In Vitro Hematopoietic Stem/Progenitor Cell Expansion

    PubMed Central

    Futrega, Kathryn; Atkinson, Kerry; Lott, William B.

    2017-01-01

    While two-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSCs) have been shown to enhance hematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein, we evaluated the capacity of a novel three-dimensional (3D) coculture system to support HSPC expansion in vitro. A high-throughput polydimethylsiloxane (PDMS) microwell platform was used to manufacture thousands of uniform 3D multicellular coculture spheroids. Relative gene expression in 3D spheroid versus 2D adherent BM-derived MSC cultures was characterized and compared with literature reports. We evaluated coculture spheroids, each containing 25–400 MSCs and 10 umbilical cord blood (CB)-derived CD34+ progenitor cells. At low exogenous cytokine concentrations, 2D and 3D MSC coculture modestly improved overall hematopoietic cell and CD34+ cell expansion outcomes. By contrast, a substantial increase in CD34+CD38− cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSCs. This outcome indicated that CD34+CD38− cell culture yield could be increased using the microwell platform alone, even without MSC coculture support. We found that the increase in CD34+CD38− cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NOD/SCID gamma (NSG) mice or a modification in the relative human hematopoietic lineages established in engrafted mice. In summary, there was no statistical difference in CD34+ cell yield from 2D or 3D cocultures, and MSC coculture support provided only modest benefit in either geometry. While the high-throughput 3D microwell platform may provide a useful model system for studying cells in coculture, further optimization will be required to generate HSPC yields suitable for use in clinical applications. PMID:28406754

  19. Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood-derived hematopoietic progenitor cells.

    PubMed

    Martin, Colin H; Woll, Petter S; Ni, Zhenya; Zúñiga-Pflücker, Juan Carlos; Kaufman, Dan S

    2008-10-01

    Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34(+) cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34(+) cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34(+) cells also did not produce B cells in vitro. In contrast, CD34(+) cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor-induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs.

  20. Lipoprotein lipase regulates hematopoietic stem progenitor cell maintenance through DHA supply.

    PubMed

    Liu, Chao; Han, Tianxu; Stachura, David L; Wang, Huawei; Vaisman, Boris L; Kim, Jungsu; Klemke, Richard L; Remaley, Alan T; Rana, Tariq M; Traver, David; Miller, Yury I

    2018-04-03

    Lipoprotein lipase (LPL) mediates hydrolysis of triglycerides (TGs) to supply free fatty acids (FFAs) to tissues. Here, we show that LPL activity is also required for hematopoietic stem progenitor cell (HSPC) maintenance. Knockout of Lpl or its obligatory cofactor Apoc2 results in significantly reduced HSPC expansion during definitive hematopoiesis in zebrafish. A human APOC2 mimetic peptide or the human very low-density lipoprotein, which carries APOC2, rescues the phenotype in apoc2 but not in lpl mutant zebrafish. Creating parabiotic apoc2 and lpl mutant zebrafish rescues the hematopoietic defect in both. Docosahexaenoic acid (DHA) is identified as an important factor in HSPC expansion. FFA-DHA, but not TG-DHA, rescues the HSPC defects in apoc2 and lpl mutant zebrafish. Reduced blood cell counts are also observed in Apoc2 mutant mice at the time of weaning. These results indicate that LPL-mediated release of the essential fatty acid DHA regulates HSPC expansion and definitive hematopoiesis.

  1. Combining G-CSF with a blockade of adhesion strongly improves the reconstitutive capacity of mobilized hematopoietic progenitor cells.

    PubMed

    Christ, O; Kronenwett, R; Haas, R; Zöller, M

    2001-03-01

    Mobilization of hematopoietic progenitor cells is achieved mainly by application of growth factors and, more recently, by blockade of adhesion. In this report, we describe the advantages of a combined treatment with granulocyte colony-stimulating factor (G-CSF) and anti-VLA4 (CD49d)/anti-CD44 as compared to treatment with the individual components. Mobilization by intravenous injection of anti-CD44, anti-VLA4, or G-CSF was controlled in spleen and bone marrow with regard to frequencies of multipotential colony-forming unit (C-CFU), marrow repopulating ability, long-term reconstitution, recovery of myelopoiesis, and regain of immunocompetence. Mobilization by anti-CD44 had a strong effect on expansion of early progenitor cells in the bone marrow, while the recovery in the spleen was poor. In anti-CD49d-mobilized noncommitted and committed progenitors, progenitor expansion was less pronounced, but settlement in the spleen was quite efficient. Thus, anti-CD44 and anti-CD49d differently influenced mobilization. Accordingly, mobilization and recovery after transfer were improved by combining anti-CD44 with anti-CD49d treatment. Mobilization by G-CSF was most efficient with respect to recovery of progenitor cells in the spleen. However, when transferring G-CSF-mobilized cells, regain of immunocompetence was strongly delayed. This disadvantage could be overridden when progenitor cells were mobilized via blockade of adhesion and when expansion of these mobilized progenitor cells was supported by low-dose G-CSF only during the last 24 hours before transfer. Mobilization of pluripotent progenitor cells via antibody blockade of CD44 or CD49d or via G-CSF relies on distinct mechanisms. Therefore, the reconstitutive capacity of a transplant can be significantly improved by mobilization regimens combining antibody with low-dose G-CSF treatment.

  2. Stromalized microreactor supports murine hematopoietic progenitor enrichment.

    PubMed

    Khong, Danika; Li, Matthew; Singleton, Amy; Chin, Ling-Yee; Parekkadan, Biju

    2018-01-20

    There is an emerging need to process, expand, and even genetically engineer hematopoietic stem and progenitor cells (HSPCs) prior to administration for blood reconstitution therapy. A closed-system and automated solution for ex vivo HSC processing can improve adoption and standardize processing techniques. Here, we report a recirculating flow bioreactor where HSCs are stabilized and enriched for short-term processing by indirect fibroblast feeder coculture. Mouse 3 T3 fibroblasts were seeded on the extraluminal membrane surface of a hollow fiber micro-bioreactor and were found to support HSPC cell number compared to unsupported BMCs. CFSE analysis indicates that 3 T3-support was essential for the enhanced intrinsic cell cycling of HSPCs. This enhanced support was specific to the HSPC population with little to no effect seen with the Lineage positive and Lineage negative cells. Together, these data suggest that stromal-seeded hollow fiber micro-reactors represent a platform to screening various conditions that support the expansion and bioprocessing of HSPCs ex vivo.

  3. Knockdown of Fanconi anemia genes in human embryonic stem cells reveals early developmental defects in the hematopoietic lineage.

    PubMed

    Tulpule, Asmin; Lensch, M William; Miller, Justine D; Austin, Karyn; D'Andrea, Alan; Schlaeger, Thorsten M; Shimamura, Akiko; Daley, George Q

    2010-04-29

    Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by pediatric bone marrow failure and congenital anomalies. The effect of FA gene deficiency on hematopoietic development in utero remains poorly described as mouse models of FA do not develop hematopoietic failure and such studies cannot be performed on patients. We have created a human-specific in vitro system to study early hematopoietic development in FA using a lentiviral RNA interference (RNAi) strategy in human embryonic stem cells (hESCs). We show that knockdown of FANCA and FANCD2 in hESCs leads to a reduction in hematopoietic fates and progenitor numbers that can be rescued by FA gene complementation. Our data indicate that hematopoiesis is impaired in FA from the earliest stages of development, suggesting that deficiencies in embryonic hematopoiesis may underlie the progression to bone marrow failure in FA. This work illustrates how hESCs can provide unique insights into human development and further our understanding of genetic disease.

  4. Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood–derived hematopoietic progenitor cells

    PubMed Central

    Martin, Colin H.; Woll, Petter S.; Ni, Zhenya; Zúñiga-Pflücker, Juan Carlos

    2008-01-01

    Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34+ cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34+ cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34+ cells also did not produce B cells in vitro. In contrast, CD34+ cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor–induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs. PMID:18621931

  5. Antagonism between MCL-1 and PUMA governs stem/progenitor cell survival during hematopoietic recovery from stress

    PubMed Central

    Delbridge, Alex R. D.; Opferman, Joseph T.; Grabow, Stephanie

    2015-01-01

    Understanding the critical factors that govern recovery of the hematopoietic system from stress, such as during anticancer therapy and bone marrow transplantation, is of clinical significance. We investigated the importance of the prosurvival proteins myeloid cell leukemia-1 (MCL-1) and B-cell lymphoma–extra large (BCL-XL) in stem/progenitor cell survival and fitness during hematopoietic recovery from stress. Loss of a single Mcl-1 allele, which reduced MCL-1 protein levels, severely compromised hematopoietic recovery from myeloablative challenge and following bone marrow transplantation, whereas BCL-XL was dispensable in both contexts. We identified inhibition of proapoptotic p53 upregulated modulator of apoptosis (PUMA) as the key role of MCL-1 in both settings, with Mcl-1+/−;Puma−/− mice completely protected from the deleterious effects of loss of 1 Mcl-1 allele. These results reveal the molecular mechanisms that govern cell survival during hematopoietic recovery from stress. PMID:25847014

  6. M-CSF improves protection against bacterial and fungal infections after hematopoietic stem/progenitor cell transplantation

    PubMed Central

    Sarrazin, Sandrine; Redelberger, David

    2016-01-01

    Myeloablative treatment preceding hematopoietic stem cell (HSC) and progenitor cell (HS/PC) transplantation results in severe myeloid cytopenia and susceptibility to infections in the lag period before hematopoietic recovery. We have previously shown that macrophage colony-stimulating factor (CSF-1; M-CSF) directly instructed myeloid commitment in HSCs. In this study, we tested whether this effect had therapeutic benefit in improving protection against pathogens after HS/PC transplantation. M-CSF treatment resulted in an increased production of mature myeloid donor cells and an increased survival of recipient mice infected with lethal doses of clinically relevant opportunistic pathogens, namely the bacteria Pseudomonas aeruginosa and the fungus Aspergillus fumigatus. M-CSF treatment during engraftment or after infection efficiently protected from these pathogens as early as 3 days after transplantation and was effective as a single dose. It was more efficient than granulocyte CSF (G-CSF), a common treatment of severe neutropenia, which showed no protective effect under the tested conditions. M-CSF treatment showed no adverse effect on long-term lineage contribution or stem cell activity and, unlike G-CSF, did not impede recovery of HS/PCs, thrombocyte numbers, or glucose metabolism. These results encourage potential clinical applications of M-CSF to prevent severe infections after HS/PC transplantation. PMID:27811055

  7. Integration of adeno-associated virus vectors in CD34+ human hematopoietic progenitor cells after transduction.

    PubMed

    Fisher-Adams, G; Wong, K K; Podsakoff, G; Forman, S J; Chatterjee, S

    1996-07-15

    Gene transfer vectors based on adeno-associated virus (AAV) appear promising because of their high transduction frequencies regardless of cell cycle status and ability to integrate into chromosomal DNA. We tested AAV-mediated gene transfer into a panel of human bone marrow or umbilical cord-derived CD34+ hematopoietic progenitor cells, using vectors encoding several transgenes under the control of viral and cellular promoters. Gene transfer was evaluated by (1) chromosomal integration of vector sequences and (2) analysis of transgene expression. Southern hybridization and fluorescence in situ hybridization analysis of transduced CD34 genomic DNA showed the presence of integrated vector sequences in chromosomal DNA in a portion of transduced cells and showed that integrated vector sequences were replicated along with cellular DNA during mitosis. Transgene expression in transduced CD34 cells in suspension cultures and in myeloid colonies differentiating in vitro from transduced CD34 cells approximated that predicted by the multiplicity of transduction. This was true in CD34 cells from different donors, regardless of the transgene or selective pressure. Comparisons of CD34 cell transduction either before or after cytokine stimulation showed similar gene transfer frequencies. Our findings suggest that AAV transduction of CD34+ hematopoietic progenitor cells is efficient, can lead to stable integration in a population of transduced cells, and may therefore provide the basis for safe and efficient ex vivo gene therapy of the hematopoietic system.

  8. Cord blood-derived CD34+ hematopoietic cells with low mitochondrial mass are enriched in hematopoietic repopulating stem cell function.

    PubMed

    Romero-Moya, Damia; Bueno, Clara; Montes, Rosa; Navarro-Montero, Oscar; Iborra, Francisco J; López, Luis Carlos; Martin, Miguel; Menendez, Pablo

    2013-07-01

    The homeostasis of the hematopoietic stem/progenitor cell pool relies on a fine-tuned balance between self-renewal, differentiation and proliferation. Recent studies have proposed that mitochondria regulate these processes. Although recent work has contributed to understanding the role of mitochondria during stem cell differentiation, it remains unclear whether the mitochondrial content/function affects human hematopoietic stem versus progenitor function. We found that mitochondrial mass correlates strongly with mitochondrial membrane potential in CD34(+) hematopoietic stem/progenitor cells. We, therefore, sorted cord blood CD34(+) cells on the basis of their mitochondrial mass and analyzed the in vitro homeostasis and clonogenic potential as well as the in vivo repopulating potential of CD34(+) cells with high (CD34(+) Mito(High)) versus low (CD34(+) Mito(Low)) mitochondrial mass. The CD34(+) Mito(Low) fraction contained 6-fold more CD34(+)CD38(-) primitive cells and was enriched in hematopoietic stem cell function, as demonstrated by its significantly greater hematopoietic reconstitution potential in immuno-deficient mice. In contrast, the CD34(+) Mito(High) fraction was more enriched in hematopoietic progenitor function with higher in vitro clonogenic capacity. In vitro differentiation of CD34(+) Mito(Low) cells was significantly delayed as compared to that of CD34(+) Mito(High) cells. The eventual complete differentiation of CD34(+) Mito(Low) cells, which coincided with a robust expansion of the CD34(-) differentiated progeny, was accompanied by mitochondrial adaptation, as shown by significant increases in ATP production and expression of the mitochondrial genes ND1 and COX2. In conclusion, cord blood CD34(+) cells with low levels of mitochondrial mass are enriched in hematopoietic repopulating stem cell function whereas high levels of mitochondrial mass identify hematopoietic progenitors. A mitochondrial response underlies hematopoietic stem/progenitor cell

  9. The preferentially expressed antigen in melanoma (PRAME) inhibits myeloid differentiation in normal hematopoietic and leukemic progenitor cells

    PubMed Central

    Guthrie, Katherine A.; Cummings, Carrie L.; Sabo, Kathleen; Wood, Brent L.; Gooley, Ted; Yang, Taimei; Epping, Mirjam T.; Shou, Yaping; Pogosova-Agadjanyan, Era; Ladne, Paula; Stirewalt, Derek L.; Abkowitz, Janis L.; Radich, Jerald P.

    2009-01-01

    The preferentially expressed antigen in melanoma (PRAME) is expressed in several hematologic malignancies, but either is not expressed or is expressed at only low levels in normal hematopoietic cells, making it a target for cancer therapy. PRAME is a tumor-associated antigen and has been described as a corepressor of retinoic acid signaling in solid tumor cells, but its function in hematopoietic cells is unknown. PRAME mRNA expression increased with chronic myeloid leukemia (CML) disease progression and its detection in late chronic-phase CML patients before tyrosine kinase inhibitor therapy was associated with poorer therapeutic responses and ABL tyrosine kinase domain point mutations. In leukemia cell lines, PRAME protein expression inhibited granulocytic differentiation only in cell lines that differentiate along this lineage after all-trans retinoic acid (ATRA) exposure. Forced PRAME expression in normal hematopoietic progenitors, however, inhibited myeloid differentiation both in the presence and absence of ATRA, and this phenotype was reversed when PRAME was silenced in primary CML progenitors. These observations suggest that PRAME inhibits myeloid differentiation in certain myeloid leukemias, and that its function in these cells is lineage and phenotype dependent. Lastly, these observations suggest that PRAME is a target for both prognostic and therapeutic applications. PMID:19625708

  10. E3 ligase FLRF (Rnf41) regulates differentiation of hematopoietic progenitors by governing steady-state levels of cytokine and retinoic acid receptors.

    PubMed

    Jing, Xin; Infante, Jorge; Nachtman, Ronald G; Jurecic, Roland

    2008-09-01

    FLRF (Rnf41) gene was identified through screening of subtracted cDNA libraries form murine hematopoietic stem cells and progenitors. Subsequent work has revealed that FLRF acts as E3 ubiquitin ligase, and that it regulates steady-state levels of neuregulin receptor ErbB3 and participates in degradation of IAP protein BRUCE and parkin. The objective of this study was to start exploring the role of FLRF during hematopoiesis. FLRF was overexpressed in a murine multipotent hematopoietic progenitor cell line EML, which can differentiate into almost all blood cell lineages, and in pro-B progenitor cell line BaF3. The impact of FLRF overexpression on EML cell differentiation into myeloerythroid lineages was studied using hematopoietic colony-forming assays. The interaction of FLRF with cytokine receptors and receptor levels in control cells and EML and BaF3 cells overexpressing FLRF were examined with Western and immunoprecipitation. Remarkably, overexpression of FLRF significantly attenuated erythroid and myeloid differentiation of EML cells in response to cytokines erythropoietin (EPO) and interleukin-3 (IL-3), and retinoic acid (RA), and resulted in significant and constitutive decrease of steady-state levels of IL-3, EPO, and RA receptor-alpha (RARalpha) in EML and BaF3 cells. Immunoprecipitation has revealed that FLRF interacts with IL-3, EPO, and RARalpha receptors in EML and BaF3 cells, and that FLRF-mediated downregulation of these receptors is ligand binding-independent. The results of this study have revealed new FLRF-mediated pathway for ligand-independent receptor level regulation, and support the notion that through maintaining basal levels of cytokine receptors, FLRF is involved in the control of hematopoietic progenitor cell differentiation into myeloerythroid lineages.

  11. Perturbed hematopoietic stem and progenitor cell hierarchy in myelodysplastic syndromes patients with monosomy 7 as the sole cytogenetic abnormality.

    PubMed

    Dimitriou, Marios; Woll, Petter S; Mortera-Blanco, Teresa; Karimi, Mohsen; Wedge, David C; Doolittle, Helen; Douagi, Iyadh; Papaemmanuil, Elli; Jacobsen, Sten Eirik W; Hellström-Lindberg, Eva

    2016-11-08

    The stem and progenitor cell compartments in low- and intermediate-risk myelodysplastic syndromes (MDS) have recently been described, and shown to be highly conserved when compared to those in acute myeloid leukemia (AML). Much less is known about the characteristics of the hematopoietic hierarchy of subgroups of MDS with a high risk of transforming to AML. Immunophenotypic analysis of immature stem and progenitor cell compartments from patients with an isolated loss of the entire chromosome 7 (isolated -7), an independent high-risk genetic event in MDS, showed expansion and dominance of the malignant -7 clone in the granulocyte and macrophage progenitors (GMP), and other CD45RA+ progenitor compartments, and a significant reduction of the LIN-CD34+CD38low/-CD90+CD45RA- hematopoietic stem cell (HSC) compartment, highly reminiscent of what is typically seen in AML, and distinct from low-risk MDS. Established functional in vitro and in vivo stem cell assays showed a poor readout for -7 MDS patients irrespective of marrow blast counts. Moreover, while the -7 clone dominated at all stages of GM differentiation, the -7 clone had a competitive disadvantage in erythroid differentiation. In azacitidine-treated -7 MDS patients with a clinical response, the decreased clonal involvement in mononuclear bone marrow cells was not accompanied by a parallel reduced clonal involvement in the dominant CD45RA+ progenitor populations, suggesting a selective azacitidine-resistance of these distinct -7 progenitor compartments. Our data demonstrate, in a subgroup of high risk MDS with monosomy 7, that the perturbed stem and progenitor cell compartments resemble more that of AML than low-risk MDS.

  12. Enumerating Hematopoietic Stem and Progenitor Cells in Zebrafish Embryos.

    PubMed

    Esain, Virginie; Cortes, Mauricio; North, Trista E

    2016-01-01

    Over the past 20 years, zebrafish have proven to be a valuable model to dissect the signaling pathways involved in hematopoiesis, including Hematopoietic Stem and Progenitor Cell (HSPC) formation and homeostasis. Despite tremendous efforts to generate the tools necessary to characterize HSPCs in vitro and in vivo the zebrafish community still lacks standardized methods to quantify HSPCs across laboratories. Here, we describe three methods used routinely in our lab, and in others, to reliably enumerate HSPCs in zebrafish embryos: large-scale live imaging of transgenic reporter lines, Fluorescence-Activated Cell Sorting (FACS), and in vitro cell culture. While live imaging and FACS analysis allows enumeration of total or site-specific HSPCs, the cell culture assay provides the unique opportunity to test the functional potential of isolated HSPCs, similar to those employed in mammals.

  13. Isolation of pancreatic progenitor cells with the surface marker of hematopoietic stem cells.

    PubMed

    Ma, Fengxia; Chen, Fang; Chi, Ying; Yang, Shaoguang; Lu, Shihong; Han, Zhongchao

    2012-01-01

    To isolate pancreatic progenitor cells with the surface markers of hematopoietic stem cells, the expression of stem cell antigen (Sca-1) and c-Kit and the coexpression of them with pancreatic duodenal homeobox-1 (PDX-1), neurogenin 3 (Ngn3), and insulin were examined in murine embryonic pancreas. Then different pancreatic cell subpopulations were isolated by magnet-activated cell sorting. Isolated cells were cultured overnight in hanging drops. When cells formed spheres, they were laid on floating filters at the air/medium interface. With this new culture system, pancreatic progenitor cells were induced to differentiate to endocrine and exocrine cells. It was shown that c-Kit and Sca-1 were expressed differently in embryonic pancreas at 12.5, 15.5, and 17.5 days of gestation. The expression of c-Kit and Sca-1 was the highest at 15.5 days of gestation. c-Kit rather than Sca-1 coexpressed with PDX-1, Ngn3, and insulin. Cells differentiated from c-Kit-positive cells contained more insulin-producing cells and secreted more insulin in response to glucose stimulation than that from c-Kit-negative cells. These results suggested that c-Kit could be used to isolate pancreatic progenitor cells and our new culture system permitted pancreatic progenitor cells to differentiate to mature endocrine cells.

  14. Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

    PubMed

    Shi, Wei; Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K; Straube, Jasmin; Boyle, Glen M; Al-Ejeh, Fares; Nag, Purba; Jeffery, Jessie; Harris, Janelle L; Bain, Amanda L; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J; Finnie, John; Skaar, Jeffrey R; Walkley, Carl R; Pandita, Tej K; Rowicka, Maga; Ginalski, Krzysztof; Lane, Steven W; Khanna, Kum Kum

    2017-05-04

    Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R -loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. © 2017 by The American Society of Hematology.

  15. Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress

    PubMed Central

    Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K.; Straube, Jasmin; Boyle, Glen M.; Al-Ejeh, Fares; Jeffery, Jessie; Harris, Janelle L.; Bain, Amanda L.; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J.; Finnie, John; Skaar, Jeffrey R.; Walkley, Carl R.; Pandita, Tej K.; Rowicka, Maga; Ginalski, Krzysztof

    2017-01-01

    Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2. Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. PMID:28270450

  16. Radiologic Differences between Bone Marrow Stromal and Hematopoietic Progenitor Cell Lines from Fanconi Anemia (Fancd2−/−) Mice

    PubMed Central

    Berhane, Hebist; Epperly, Michael W.; Goff, Julie; Kalash, Ronny; Cao, Shaonan; Franicola, Darcy; Zhang, Xichen; Shields, Donna; Houghton, Frank; Wang, Hong; Wipf, Peter; Parmar, Kalindi; Greenberger, Joel S.

    2014-01-01

    FancD2 plays a central role in the human Fanconi anemia DNA damage response (DDR) pathway. Fancd2−/− mice exhibit many features of human Fanconi anemia including cellular DNA repair defects. Whether the DNA repair defect in Fancd2−/− mice results in radiologic changes in all cell lineages is unknown. We measured stress of hematopoiesis in long-term marrow cultures and radiosensitivity in clonogenic survival curves, as well as comet tail intensity, total antioxidant stores and radiation-induced gene expression in hematopoietic progenitor compared to bone marrow stromal cell lines. We further evaluated radioprotection by a mitochondrial-targeted antioxidant GS-nitroxide, JP4-039. Hematopoiesis longevity in Fancd2−/− mouse long-term marrow cultures was diminished and bone marrow stromal cell lines were radiosensitive compared to Fancd2+/+ stromal cells (Fancd2−/− D0 = 1.4 ± 0.1 Gy, ñ = 5.0 ± 0.6 vs. Fancd2+/+ D0 = 1.6 ± 0.1 Gy, ñ = 6.7 ± 1.6), P = 0.0124 for D0 and P = 0.0023 for ñ, respectively). In contrast, Fancd2−/− IL-3-dependent hematopoietic progenitor cells were radioresistant (D0 = 1.71 ± 0.04 Gy and ñ = 5.07 ± 0.52) compared to Fancd2+/+ (D0 = 1.39 ± 0.09 Gy and ñ = 2.31 ± 0.85, P = 0.001 for D0). CFU-GM from freshly explanted Fancd2−/− marrow was also radioresistant. Consistent with radiosensitivity, irradiated Fancd2−/− stromal cells had higher DNA damage by comet tail intensity assay compared to Fancd2+/+ cells (P < 0.0001), slower DNA damage recovery, lower baseline total antioxidant capacity, enhanced radiation-induced depletion of antioxidants, and increased CDKN1A-p21 gene transcripts and protein. Consistent with radioresistance, Fancd2−/− IL-3-dependent hematopoietic cells had higher baseline and post irradiation total antioxidant capacity. While, there was no detectable alteration of radiation-induced cell cycle arrest with Fancd2−/− stromal cells, hematopoietic progenitor cells showed reduced G2/M

  17. Time lapse video recordings of highly purified human hematopoietic progenitor cells in culture.

    PubMed

    Denkers, I A; Dragowska, W; Jaggi, B; Palcic, B; Lansdorp, P M

    1993-05-01

    Major hurdles in studies of stem cell biology include the low frequency and heterogeneity of human hematopoietic precursor cells in bone marrow and the difficulty of directly studying the effect of various culture conditions and growth factors on such cells. We have adapted the cell analyzer imaging system for monitoring and recording the morphology of limited numbers of cells under various culture conditions. Hematopoietic progenitor cells with a CD34+ CD45RAlo CD71lo phenotype were purified from previously frozen organ donor bone marrow by fluorescence activated cell sorting. Cultures of such cells were analyzed with the imaging system composed of an inverted microscope contained in an incubator, a video camera, an optical memory disk recorder and a computer-controlled motorized microscope XYZ precision stage. Fully computer-controlled video images at defined XYZ positions were captured at selected time intervals and recorded at a predetermined sequence on an optical memory disk. In this study, the cell analyzer system was used to obtain descriptions and measurements of hematopoietic cell behavior, like cell motility, cell interactions, cell shape, cell division, cell cycle time and cell size changes under different culture conditions.

  18. E3 ligase FLRF (Rnf41) regulates differentiation of hematopoietic progenitors by governing steady-state levels of cytokine and retinoic acid receptors

    PubMed Central

    Jing, Xin; Infante, Jorge; Nachtman, Ronald G.; Jurecic, Roland

    2008-01-01

    Objective FLRF (Rnf41) gene was identified through screening of subtracted cDNA libraries form murine hematopoietic stem cells and progenitors. Subsequent work has revealed that FLRF acts as E3 ubiquitin ligase, and that it regulates steady-state levels of neuregulin receptor ErbB3, and participates in degradation of IAP protein BRUCE and parkin. The objective of this study was to start exploring the role of FLRF during hematopoiesis. Methods FLRF was over-expressed in a murine multipotent hematopoietic progenitor cell line EML, which can differentiate into almost all blood cell lineages, and in pro-B progenitor cell line BaF3. The impact of FLRF over-expression on EML cell differentiation into myelo-erythroid lineages was studied using hematopoietic colony-forming assays. The interaction of FLRF with cytokine receptors and receptor levels in control cells and EML and BaF3 cells over-expressing FLRF were examined with Western and immunoprecipitation. Results Remarkably, over-expression of FLRF significantly attenuated erythroid and myeloid differentiation of EML cells in response to cytokines Epo and IL-3, and retinoic acid (RA), and resulted in significant and constitutive decrease of steady-state levels of IL-3, Epo and RA receptor RARα in EML and BaF3 cells. Immunoprecipitation has revealed that FLRF interacts with IL-3, Epo and RARα receptors in EML and BaF3 cells, and that FLRF-mediated down-regulation of these receptors is ligand binding-independent. Conclusions The results of this study have revealed new FLRF-mediated pathway for ligand-independent receptor level regulation, and support the notion that through maintaining basal levels of cytokine receptors, FLRF is involved in the control of hematopoietic progenitor cell differentiation into myelo-erythroid lineages. PMID:18495327

  19. F4/80+ Host Macrophages Are a Barrier to Murine Embryonic Stem Cell-Derived Hematopoietic Progenitor Engraftment In Vivo.

    PubMed

    Thompson, Heather L; van Rooijen, Nico; McLelland, Bryce T; Manilay, Jennifer O

    2016-01-01

    Understanding how embryonic stem cells and their derivatives interact with the adult host immune system is critical to developing their therapeutic potential. Murine embryonic stem cell-derived hematopoietic progenitors (ESHPs) were generated via coculture with the bone marrow stromal cell line, OP9, and then transplanted into NOD.SCID.Common Gamma Chain (NSG) knockout mice, which lack B, T, and natural killer cells. Compared to control mice transplanted with adult lineage-negative bone marrow (Lin - BM) progenitors, ESHP-transplanted mice attained a low but significant level of donor hematopoietic chimerism. Based on our previous studies, we hypothesized that macrophages might contribute to the low engraftment of ESHPs in vivo . Enlarged spleens were observed in ESHP-transplanted mice and found to contain higher numbers of host F4/80 + macrophages compared to BM-transplanted controls. In vivo depletion of host macrophages using clodronate-loaded liposomes improved the ESHP-derived hematopoietic chimerism in the spleen but not in the BM. F4/80 + macrophages demonstrated a striking propensity to phagocytose ESHP targets in vitro . Taken together, these results suggest that macrophages are a barrier to both syngeneic and allogeneic ESHP engraftment in vivo .

  20. Benzene-Induced Aberrant miRNA Expression Profile in Hematopoietic Progenitor Cells in C57BL/6 Mice.

    PubMed

    Wei, Haiyan; Zhang, Juan; Tan, Kehong; Sun, Rongli; Yin, Lihong; Pu, Yuepu

    2015-11-12

    Benzene is a common environmental pollutant that causes hematological alterations. MicroRNAs (miRNAs) may play a role in benzene-induced hematotoxicity. In this study, C57BL/6 mice showed significant hematotoxicity after exposure to 150 mg/kg benzene for 4 weeks. Benzene exposure decreased not only the number of cells in peripheral blood but also hematopoietic progenitor cells in the bone marrow. Meanwhile, RNA from Lin(-) cells sorted from the bone marrow was applied to aberrant miRNA expression profile using Illumina sequencing. We found that 5 miRNAs were overexpressed and 45 miRNAs were downregulated in the benzene exposure group. Sequencing results were confirmed through qRT-PCR. Furthermore, we also identified five miRNAs which significantly altered in Lin(-)c-Kit⁺ cells obtained from benzene-exposed mice, including mmu-miR-34a-5p; mmu-miR-342-3p; mmu-miR-100-5p; mmu-miR-181a-5p; and mmu-miR-196b-5p. In summary, we successfully established a classical animal model to induce significant hematotoxicity by benzene injection. Benzene exposure may cause severe hematotoxicity not only to blood cells in peripheral circulation but also to hematopoietic cells in bone marrow. Benzene exposure also alters miRNA expression in hematopoietic progenitor cells. This study suggests that benzene induces alteration in hematopoiesis and hematopoiesis-associated miRNAs.

  1. Dimethyl Sulfoxide (DMSO) Increases Percentage of CXCR4(+) Hematopoietic Stem/Progenitor Cells, Their Responsiveness to an SDF-1 Gradient, Homing Capacities, and Survival.

    PubMed

    Jarocha, Danuta; Zuba-Surma, Ewa; Majka, Marcin

    2016-01-01

    Cryopreservation of bone marrow (BM), mobilized peripheral blood (mPB), and cord blood (CB) hematopoietic stem/progenitor cells (HSPCs) is a routine procedure before transplantation. The most commonly used cryoprotectant for HSPCs is dimethyl sulfoxide (DMSO). The objective of this study was to evaluate the influence of DMSO on surface receptor expression and chemotactic activities of HSPCs. We found that 10 min of incubation of human mononuclear cells (MNCs) with 10% DMSO significantly increases the percentage of CXCR4(+), CD38(+), and CD34(+) cells, resulting in an increase of CD34(+), CD34(+)CXCR4(+), and CD34(+)CXCR4(+)CD38(-) subpopulations. Furthermore, DMSO significantly increased chemotactic responsiveness of MNCs and CXCR4(+) human hematopoietic Jurkat cell line to a stromal cell-derived factor-1 (SDF-1) gradient. Furthermore, we demonstrated enhanced chemotaxis of human clonogenic progenitor cells to an SDF-1 gradient, which suggests that DMSO directly enhances the chemotactic responsiveness of early human progenitors. DMSO preincubation also caused lower internalization of the CXCR4 receptor. In parallel experiments, we found that approximately 30% more of DMSO-preincubated human CD45(+) and CD45(+)CD34(+) cells homed to the mouse BM 24 h after transplantation in comparison to control cells. Finally, we demonstrated considerably higher (25 days) survival of mice transplanted with DMSO-exposed MNCs than those transplanted with the control cells. We show in this study an unexpected beneficial influence of DMSO on HSPC homing and suggest that a short priming with DMSO before transplantation could be considered a new strategy to enhance cell homing and engraftment.

  2. Novel pathways to erythropoiesis induced by dimerization of intracellular C-Mpl in human hematopoietic progenitors.

    PubMed

    Parekh, Chintan; Sahaghian, Arineh; Kim, William; Scholes, Jessica; Ge, Shundi; Zhu, Yuhua; Asgharzadeh, Shahab; Hollis, Roger; Kohn, Donald; Ji, Lingyun; Malvar, Jemily; Wang, Xiaoyan; Crooks, Gay

    2012-04-01

    The cytokine thrombopoietin (Tpo) plays a critical role in hematopoiesis by binding to the extracellular domain and inducing homodimerization of the intracellular signaling domain of its receptor, c-Mpl. Mpl homodimerization can also be accomplished by binding of a synthetic ligand to a constitutively expressed fusion protein F36VMpl consisting of a ligand binding domain (F36V) and the intracellular signaling domain of Mpl. Unexpectedly, in contrast to Tpo stimulation, robust erythropoiesis is induced after dimerization of F36VMpl in human CD34+ progenitor cells. The goal of this study was to define the hematopoietic progenitor stages at which dimerization of intracellular Mpl induces erythropoiesis and the downstream molecular events that mediate this unanticipated effect. Dimerization (in the absence of erythropoietin and other cytokines) in human common myeloid progenitors and megakaryocytic erythroid progenitors caused a significant increase in CD34+ cells (p < .01) and induced all stages of erythropoiesis including production of enucleated red blood cells. In contrast, erythropoiesis was not seen with Tpo stimulation. CD34+ cell expansion was the result of increased cell cycling and survival (p < .05). Microarray profiling of CD34+ cells demonstrated that a unique transcriptional pattern is activated in progenitors by F36VMpl dimerization. Ligand-inducible dimerization of intracellular Mpl in human myeloerythroid progenitors induces progenitor expansion and erythropoiesis through molecular mechanisms that are not shared by Tpo stimulation of endogenous Mpl. Copyright © 2012 AlphaMed Press.

  3. Single-cell RNA sequencing reveals developmental heterogeneity among early lymphoid progenitors.

    PubMed

    Alberti-Servera, Llucia; von Muenchow, Lilly; Tsapogas, Panagiotis; Capoferri, Giuseppina; Eschbach, Katja; Beisel, Christian; Ceredig, Rhodri; Ivanek, Robert; Rolink, Antonius

    2017-12-15

    Single-cell RNA sequencing is a powerful technology for assessing heterogeneity within defined cell populations. Here, we describe the heterogeneity of a B220 + CD117 int CD19 - NK1.1 - uncommitted hematopoietic progenitor having combined lymphoid and myeloid potential. Phenotypic and functional assays revealed four subpopulations within the progenitor with distinct lineage developmental potentials. Among them, the Ly6D + SiglecH - CD11c - fraction was lymphoid-restricted exhibiting strong B-cell potential, whereas the Ly6D - SiglecH - CD11c - fraction showed mixed lympho-myeloid potential. Single-cell RNA sequencing of these subsets revealed that the latter population comprised a mixture of cells with distinct lymphoid and myeloid transcriptional signatures and identified a subgroup as the potential precursor of Ly6D + SiglecH - CD11c - Subsequent functional assays confirmed that B220 + CD117 int CD19 - NK1.1 - single cells are, with rare exceptions, not bipotent for lymphoid and myeloid lineages. A B-cell priming gradient was observed within the Ly6D + SiglecH - CD11c - subset and we propose a herein newly identified subgroup as the direct precursor of the first B-cell committed stage. Therefore, the apparent multipotency of B220 + CD117 int CD19 - NK1.1 - progenitors results from underlying heterogeneity at the single-cell level and highlights the validity of single-cell transcriptomics for resolving cellular heterogeneity and developmental relationships among hematopoietic progenitors. © 2017 The Authors.

  4. Deregulation of vital mitotic kinase-phosphatase signaling in hematopoietic stem/progenitor compartment leads to cellular catastrophe in experimental aplastic anemia.

    PubMed

    Chatterjee, Ritam; Chattopadhyay, Sukalpa; Law, Sujata

    2016-11-01

    Aplastic anemia, the paradigm of bone marrow failure, is characterized by pancytopenic peripheral blood and hypoplastic bone marrow. Among various etiologies, inappropriate use of DNA alkylating drugs like cyclophosphamide and busulfan often causes the manifestation of the dreadful disease. Cell cycle impairment in marrow hematopoietic stem/progenitor compartment together with cellular apoptosis has been recognized as culpable factors behind aplastic pathophysiologies. However, the intricate molecular mechanisms remain unrevealed till date. In the present study, we have dealt with the mechanistic intervention of the disease by peripheral blood hemogram, bone marrow histopathology, cytopathology, hematopoietic kinetic study, scanning electron microscopy, DNA damage assessment and flowcytometric analysis of cellular proliferation and apoptosis in hematopoietic stem/progenitor cell (HSPC) rich marrow compartment using busulfan and cyclophosphamidemediated mouse model. To unveil the molecular mechanisms behind aplastic pathophysiology, we further investigated the role of some crucial mitotic and apoptotic regulators like Protein kinase-B (PKB), Gsk-3β, Cyclin-D1, PP2A, Cdc25c, Plk-1, Aurora kinase-A, Chk-1 regarding the hematopoietic catastrophe. Our observations revealed that the alteration of PKB-GSK-3β axis, Plk-1, and Aurora kinase-A expressions in HSPC compartment due to DNA damage response was associated with the proliferative impairment and apoptosis during aplastic anemia. The study established the correlation between the accumulation of DNA damage and alteration of the mentioned molecules in aplastic HSPCs that lead to the hematopoietic catastrophe. We anticipate that our findings will be beneficial for developing better therapeutic strategies for the dreadful disease concerned.

  5. BLOS2 negatively regulates Notch signaling during neural and hematopoietic stem and progenitor cell development

    PubMed Central

    Zhou, Wenwen; He, Qiuping; Zhang, Chunxia; He, Xin; Cui, Zongbin; Liu, Feng; Li, Wei

    2016-01-01

    Notch signaling plays a crucial role in controling the proliferation and differentiation of stem and progenitor cells during embryogenesis or organogenesis, but its regulation is incompletely understood. BLOS2, encoded by the Bloc1s2 gene, is a shared subunit of two lysosomal trafficking complexes, biogenesis of lysosome-related organelles complex-1 (BLOC-1) and BLOC-1-related complex (BORC). Bloc1s2−/− mice were embryonic lethal and exhibited defects in cortical development and hematopoiesis. Loss of BLOS2 resulted in elevated Notch signaling, which consequently increased the proliferation of neural progenitor cells and inhibited neuronal differentiation in cortices. Likewise, ablation of bloc1s2 in zebrafish or mice led to increased hematopoietic stem and progenitor cell production in the aorta-gonad-mesonephros region. BLOS2 physically interacted with Notch1 in endo-lysosomal trafficking of Notch1. Our findings suggest that BLOS2 is a novel negative player in regulating Notch signaling through lysosomal trafficking to control multiple stem and progenitor cell homeostasis in vertebrates. DOI: http://dx.doi.org/10.7554/eLife.18108.001 PMID:27719760

  6. Progenitor cell dose determines the pace and completeness of engraftment in a xenograft model for cord blood transplantation

    PubMed Central

    Liu, Congxiao; Chen, Benny J.; DeOliveira, Divinomar; Sempowski, Gregory D.; Chao, Nelson J.

    2010-01-01

    Two critical concerns in clinical cord blood transplantation are the initial time to engraftment and the subsequent restoration of immune function. These studies measured the impact of progenitor cell dose on both the pace and strength of hematopoietic reconstitution by transplanting nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-gamma–null (NSγ) mice with lineage-depleted aldehyde dehydrogenase-bright CD34+ human cord blood progenitors. The progress of each transplant was monitored over an extended time course by repeatedly analyzing the peripheral blood for human hematopoietic cells. In vivo human hematopoietic development was complete. After long-term transplantation assays (≥ 19 weeks), human T-cell development was documented within multiple tissues in 16 of 32 NSγ mice. Human T-cell differentiation was active within NSγ thymuses, as documented by the presence of CD4+ CD8+ T-cell progenitors as well as T-cell receptor excision circles. It is important to note that although myeloid and B-cell engraftment was detected as early as 4 weeks after transplantation, human T-cell development was exclusively late onset. High progenitor cell doses were associated with a robust human hematopoietic chimerism that accelerated both initial time to engraftment and subsequent T-cell development. At lower progenitor cell doses, the chimerism was weak and the human hematopoietic lineage development was frequently incomplete. PMID:20833978

  7. Benzene-Induced Aberrant miRNA Expression Profile in Hematopoietic Progenitor Cells in C57BL/6 Mice

    PubMed Central

    Wei, Haiyan; Zhang, Juan; Tan, Kehong; Sun, Rongli; Yin, Lihong; Pu, Yuepu

    2015-01-01

    Benzene is a common environmental pollutant that causes hematological alterations. MicroRNAs (miRNAs) may play a role in benzene-induced hematotoxicity. In this study, C57BL/6 mice showed significant hematotoxicity after exposure to 150 mg/kg benzene for 4 weeks. Benzene exposure decreased not only the number of cells in peripheral blood but also hematopoietic progenitor cells in the bone marrow. Meanwhile, RNA from Lin− cells sorted from the bone marrow was applied to aberrant miRNA expression profile using Illumina sequencing. We found that 5 miRNAs were overexpressed and 45 miRNAs were downregulated in the benzene exposure group. Sequencing results were confirmed through qRT-PCR. Furthermore, we also identified five miRNAs which significantly altered in Lin−c-Kit+ cells obtained from benzene-exposed mice, including mmu-miR-34a-5p; mmu-miR-342-3p; mmu-miR-100-5p; mmu-miR-181a-5p; and mmu-miR-196b-5p. In summary, we successfully established a classical animal model to induce significant hematotoxicity by benzene injection. Benzene exposure may cause severe hematotoxicity not only to blood cells in peripheral circulation but also to hematopoietic cells in bone marrow. Benzene exposure also alters miRNA expression in hematopoietic progenitor cells. This study suggests that benzene induces alteration in hematopoiesis and hematopoiesis-associated miRNAs. PMID:26569237

  8. Extracellular matrix functionalized microcavities to control hematopoietic stem and progenitor cell fate.

    PubMed

    Kurth, Ina; Franke, Katja; Pompe, Tilo; Bornhäuser, Martin; Werner, Carsten

    2011-06-14

    Polymeric microcavities functionalized with extracellular matrix components were used as an experimental in vitro model to investigate principles of hematopoietic stem and progenitor cell (HSPC) fate control. Using human CD133+ HSPC we could demonstrate distinct differences in HSPC cycling and differentiation dependence on the adhesion ligand specificity (i.e., heparin, collagen I) and cytokine levels. The presented microcavity platform provides a powerful in vitro approach to explore the role of exogenous cues in HSPC fate decisions and can therefore be instrumental to progress in stem cell biology and translational research toward new therapies. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Preterm Cord Blood Contains a Higher Proportion of Immature Hematopoietic Progenitors Compared to Term Samples.

    PubMed

    Podestà, Marina; Bruschettini, Matteo; Cossu, Claudia; Sabatini, Federica; Dagnino, Monica; Romantsik, Olga; Spaggiari, Grazia Maria; Ramenghi, Luca Antonio; Frassoni, Francesco

    2015-01-01

    Cord blood contains high number of hematopoietic cells that after birth disappear. In this paper we have studied the functional properties of the umbilical cord blood progenitor cells collected from term and preterm neonates to establish whether quantitative and/or qualitative differences exist between the two groups. Our results indicate that the percentage of total CD34+ cells was significantly higher in preterm infants compared to full term: 0.61% (range 0.15-4.8) vs 0.3% (0.032-2.23) p = 0.0001 and in neonates <32 weeks of gestational age (GA) compared to those ≥32 wks GA: 0.95% (range 0.18-4.8) and 0.36% (0.15-3.2) respectively p = 0.0025. The majority of CD34+ cells co-expressed CD71 antigen (p<0.05 preterm vs term) and grew in vitro large BFU-E, mostly in the second generation. The subpopulations CD34+CD38- and CD34+CD45- resulted more represented in preterm samples compared to term, conversely, Side Population (SP) did not show any difference between the two group. The absolute number of preterm colonies (CFCs/10microL) resulted higher compared to term (p = 0.004) and these progenitors were able to grow until the third generation maintaining an higher proportion of CD34+ cells (p = 0.0017). The number of colony also inversely correlated with the gestational age (Pearson r = -0.3001 p<0.0168). We found no differences in the isolation and expansion capacity of Endothelial Colony Forming Cells (ECFCs) from cord blood of term and preterm neonates: both groups grew in vitro large number of endothelial cells until the third generation and showed a transitional phenotype between mesenchymal stem cells and endothelial progenitors (CD73, CD31, CD34 and CD144)The presence, in the cord blood of preterm babies, of high number of immature hematopoietic progenitors and endothelial/mesenchymal stem cells with high proliferative potential makes this tissue an important source of cells for developing new cells therapies.

  10. Early Hematopoietic Zinc Finger Protein Prevents Tumor Cell Recognition by Natural Killer Cells1

    PubMed Central

    La Rocca, Rosanna; Fulciniti, Mariateresa; Lakshmikanth, Tadepally; Mesuraca, Maria; Ali, Talib Hassan; Mazzei, Valerio; Amodio, Nicola; Catalano, Lucio; Rotoli, Bruno; Ouerfelli, Ouathek; Grieco, Michele; Gulletta, Elio; Bond, Heather M.; Morrone, Giovanni; Ferrone, Soldano; Carbone, Ennio

    2009-01-01

    Early hematopoietic zinc finger/zinc finger protein 521 (EHZF/ZNF521) is a novel zinc finger protein expressed in hematopoietic stem and progenitor cells and is down-regulated during their differentiation. Its transcript is also abundant in some hematopoietic malignancies. Analysis of the changes in the antigenic profile of cells transfected with EHZF cDNA revealed up-regulation of HLA class I cell surface expression. This phenotypic change was associated with an increased level of HLA class I H chain, in absence of detectable changes in the expression of other Ag-processing machinery components. Enhanced resistance of target cells to NK cell-mediated cytotoxicity was induced by enforced expression of EHZF in the cervical carcinoma cell line HeLa and in the B lymphoblastoid cell line IM9. Preincubation of transfected cells with HLA class I Ag-specific mAb restored target cell susceptibility to NK cell-mediated lysis, indicating a specific role for HLA class I Ag up-regulation in the NK resistance induced by EHZF. A potential clinical significance of these findings is further suggested by the inverse correlation between EHZF and MHC class I expression levels, and autologous NK susceptibility of freshly explanted multiple myeloma cells. PMID:19342626

  11. Maintenance and expansion of hematopoietic stem/progenitor cells in biomimetic osteoblast niche.

    PubMed

    Tan, Jing; Liu, Ting; Hou, Li; Meng, Wentong; Wang, Yuchun; Zhi, Wei; Deng, Li

    2010-10-01

    In this study, we employed bio-derived bone scaffold and composited with the marrow mesenchymal stem cell induced into osteoblast to replicate a "biomimetic niche." The CD34(+) cells or mononuclear cells (MNC) from umbilical cord blood were cultured for 2-5 weeks in the biomimetic niche (3D system) was compared with conventional two dimensional cultures (2D system) without adding cytokine supplement. After 2 weeks in culture, the CD34(+) cells from umbilical cord blood in the 3D system increased 3.3-4.8 folds when compared with the initial CD34(+) cells. CD34(+)/CD38(-) cells accounted for 82-90% of CD34(+) cells. After 5 weeks, CD34(+)/CD38(-) cells in the 3D system increased when compared with initial (1.3 ± 0.3 × 10(3) vs. 1.0 ± 0.5 × 10(4), p < 0.05), but were decreased in the 2D system (1.3 ± 0.3 × 10(3) vs. 2.5 ± 0.7 × 10(2), p < 0.05). The CFU progenitors were produced more in the 3D system than in the 2D system (4.6-9.3 folds vs. 1.0-1.5 folds) after 2 weeks in culture, and the colony distribution in the 3D system manifested higher percentage of BFU-E and CFU-GEMM, but in the 2D system was mainly CFU-GM. The LTC-ICs in the 3D system showed 5.2-7.2 folds increase over input at 2 weeks in culture, and maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5 weeks. In conclusion, this new 3D hematopoietic progenitor cell culture system is the first to utilize natural cancellous bone as scaffold with osteoblasts as supporting cells; it is mimicry of natural bone marrow HSC niche. Our primary work has demonstrated it could maintain and expand HSC/HPC in vitro.

  12. Proliferative status of primitive hematopoietic progenitors from patients with acute myelogenous leukemia (AML).

    PubMed

    Guan, Y; Hogge, D E

    2000-12-01

    One possible explanation for the competitive advantage that malignant cells in patients with acute myelogenous leukemia (AML) appear to have over normal hematopoietic elements is that leukemic progenitors proliferate more rapidly than their normal progenitor cell counterparts. To test this hypothesis, an overnight 3H-thymidine (3H-Tdr) suicide assay was used to analyze the proliferative status of malignant progenitors detected in both colony-forming cell (CFC) and long-term culture initiating cell (LTC-IC) assays from the peripheral blood of nine patients with newly diagnosed AML. Culture of AML cells in serum-free medium with 100 ng/ml Steel factor (SF), 20 ng/ml interleukin 3 (IL-3) and 20 ng/ml granulocyte colony-stimulating factor (G-CSF) for 16-24 h maintained the number of AML-CFC and LTC-IC at near input values (mean % input +/- s.d. for CFC and LTC-IC were 78 +/- 33 and 126 +/- 53, respectively). The addition of 20 muCi/ml high specific activity 3H-Tdr to these cultures reduced the numbers of both progenitor cell types from most of the patient samples substantially: mean % kill +/- s.d. for AML-CFC and LTC-IC were 64 +/- 27 and 82 +/- 16, respectively, indicating that a large proportion of both progenitor populations were actively cycling. FISH analysis of colonies from CFC and LTC-IC assays confirmed that most cytogenetically abnormal CFC and LTC-IC were actively cycling (mean % kill +/- s.d.: 68 +/- 26 and 85 +/- 13, respectively). Interestingly, in six patient samples where a significant number of cytogenetically normal LTC-ICs were detected, the % kill of these cells (74 +/- 20) was similar to that of the abnormal progenitors. These data contrast with the predominantly quiescent cell cycle status of CFC and LTC-IC previously observed in steady-state peripheral blood from normal individuals but also provide evidence that a significant proportion of primitive malignant progenitors from AML patients are quiescent and therefore may be resistant to standard

  13. Loss of the Liver X Receptors Disrupts the Balance of Hematopoietic Populations, With Detrimental Effects on Endothelial Progenitor Cells.

    PubMed

    Rasheed, Adil; Tsai, Ricky; Cummins, Carolyn L

    2018-05-08

    The liver X receptors (LXRs; α/β) are nuclear receptors known to regulate cholesterol homeostasis and the production of select hematopoietic populations. The objective of this study was to determine the importance of LXRs and a high-fat high-cholesterol diet on global hematopoiesis, with special emphasis on endothelial progenitor cells (EPCs), a vasoreparative cell type that is derived from bone marrow hematopoietic stem cells. Wild-type and LXR double-knockout ( Lxr αβ -/- ) mice were fed a Western diet (WD) to increase plasma cholesterol levels. In WD-fed Lxr αβ -/- mice, flow cytometry and complete blood cell counts revealed that hematopoietic stem cells, a myeloid progenitor, and mature circulating myeloid cells were increased; EPC numbers were significantly decreased. Hematopoietic stem cells from WD-fed Lxr αβ -/- mice showed increased cholesterol content, along with increased myeloid colony formation compared with chow-fed mice. In contrast, EPCs from WD-fed Lxr αβ -/- mice also demonstrated increased cellular cholesterol content that was associated with greater expression of the endothelial lineage markers Cd144 and Vegfr2 , suggesting accelerated differentiation of the EPCs. Treatment of human umbilical vein endothelial cells with conditioned medium collected from these EPCs increased THP-1 monocyte adhesion. Increased monocyte adhesion to conditioned medium-treated endothelial cells was recapitulated with conditioned medium from Lxr αβ -/- EPCs treated with cholesterol ex vivo, suggesting cholesterol is the main component of the WD inducing EPC dysfunction. LXRs are crucial for maintaining the balance of hematopoietic cells in a hypercholesterolemic environment and for mitigating the negative effects of cholesterol on EPC differentiation/secretome changes that promote monocyte-endothelial adhesion. © 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

  14. Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via re-specification of lineage-restricted precursors

    PubMed Central

    Doulatov, Sergei; Vo, Linda T.; Chou, Stephanie S.; Kim, Peter G.; Arora, Natasha; Li, Hu; Hadland, Brandon K.; Bernstein, Irwin D.; Collins, James J.; Zon, Leonard I.; Daley, George Q.

    2013-01-01

    Summary Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor (HSPCs) has limited their characterization to in vitro assays. We report a strategy to re-specify lineage-restricted CD34+CD45+ myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engraft in vivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential in vitro and maintained primitive CD34+CD38− cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, were required for engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching in vivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription factor-mediated engraftment of blood progenitors from human pluripotent cells. PMID:24094326

  15. Human pluripotent stem cells differentiated in fully defined medium generate hematopoietic CD34- and CD34+ progenitors with distinct characteristics.

    PubMed

    Chicha, Laurie; Feki, Anis; Boni, Alessandro; Irion, Olivier; Hovatta, Outi; Jaconi, Marisa

    2011-02-25

    Differentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM) would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells. ESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU) assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34(+) cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB) CD34(+) cells. ESC-derived CD34(+) cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34(-) cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult β-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors. Short-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive hematopoietic progenitors, which, in turn, produce more mature

  16. Single dose of filgrastim (rhG-CSF) increases the number of hematopoietic progenitors in the peripheral blood of adult volunteers.

    PubMed

    Schwinger, W; Mache, C; Urban, C; Beaufort, F; Töglhofer, W

    1993-06-01

    Hematopoietic progenitor cell levels were monitored in the peripheral blood of ten healthy adults receiving a single dose of recombinant human granulocyte colony-stimulating factor (rhG-CSF). The objective was to determine the time and number of progenitor cells released into the peripheral blood, induced by a single dose of 15 micrograms/kg rhG-CSF administered intravenously. In all cases the absolute number of circulating progenitor cells including granulocyte-macrophage and erythroid lineages increased up to 12-fold (median 9.4-fold) 4 days after treatment. These findings were based on flow cytometric quantification of CD34+ cells and on progenitor assays. The relative distribution of granulocyte/macrophage and erythroid progenitors remained unchanged. rhG-CSF was well tolerated; mild to moderate bone pain was the most common side-effect and was noted in 6 of 10 subjects. Thus a single dose of rhG-CSF is effective in mobilizing progenitor cells into the peripheral blood in healthy adults. If these progenitors are capable of reconstituting bone marrow, peripheral progenitor cell separation following rhG-CSF administration could be a reasonable alternative to conventional bone marrow harvest in healthy adults.

  17. Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells.

    PubMed

    Hoban, Megan D; Cost, Gregory J; Mendel, Matthew C; Romero, Zulema; Kaufman, Michael L; Joglekar, Alok V; Ho, Michelle; Lumaquin, Dianne; Gray, David; Lill, Georgia R; Cooper, Aaron R; Urbinati, Fabrizia; Senadheera, Shantha; Zhu, Allen; Liu, Pei-Qi; Paschon, David E; Zhang, Lei; Rebar, Edward J; Wilber, Andrew; Wang, Xiaoyan; Gregory, Philip D; Holmes, Michael C; Reik, Andreas; Hollis, Roger P; Kohn, Donald B

    2015-04-23

    Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the β-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the β-globin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34(+) hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγ(null) mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34(+) cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers. © 2015 by The American Society of Hematology.

  18. Mobilization of Hematopoietic Stem and Progenitor Cells Using Inhibitors of CXCR4 and VLA-4

    PubMed Central

    Rettig, Michael P.; Ansstas, George; DiPersio, John F.

    2012-01-01

    Successful hematopoietic stem cell transplant (HSCT) requires the infusion of a sufficient number of hematopoietic stem/progenitor cells (HSPCs) that are capable of homing to the bone marrow cavity and regenerating durable trilineage hematopoiesis in a timely fashion. Stem cells harvested from peripheral blood are the most commonly used graft source in HSCT. While granulocyte colony-stimulating factor (G-CSF) is the most frequently used agent for stem cell mobilization, the use of G-CSF alone results in suboptimal stem cell yields in a significant proportion of patients. Both the chemokine receptor CXCR4 and the integrin α4β1 (VLA-4) play important roles in the homing and retention of HSPCs within the bone marrow microenvironment. Preclinical and/or clinical studies have shown that targeted disruption of the interaction of CXCR4 or VLA-4 with their ligands results in the rapid and reversible mobilization of hematopoietic stem cells into the peripheral circulation and is synergistic when combined with G-CSF. In this review we discuss the development of small molecule CXCR4 and VLA-4 inhibitors and how they may improve the utility and convenience of peripheral blood stem cell transplantation. PMID:21886173

  19. Serum of myeloproliferative neoplasms stimulates hematopoietic stem and progenitor cells.

    PubMed

    Lubberich, Richard K; Walenda, Thomas; Goecke, Tamme W; Strathmann, Klaus; Isfort, Susanne; Brümmendorf, Tim H; Koschmieder, Steffen; Wagner, Wolfgang

    2018-01-01

    Myeloproliferative neoplasms (MPN)-such as polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF)-are typically diseases of the elderly caused by acquired somatic mutations. However, it is largely unknown how the malignant clone interferes with normal hematopoiesis. In this study, we analyzed if serum of MPN patients comprises soluble factors that impact on hematopoietic stem and progenitor cells (HPCs). CD34+ HPCs were cultured in medium supplemented with serum samples of PV, ET, or MF patients, or healthy controls. The impact on proliferation, maintenance of immature hematopoietic surface markers, and colony forming unit (CFU) potential was systematically analyzed. In addition, we compared serum of healthy young (<25 years) and elderly donors (>50 years) to determine how normal aging impacts on the hematopoiesis-supportive function of serum. Serum from MF, PV and ET patients significantly increased proliferation as compared to controls. In addition, serum from MF and ET patients attenuated the loss of a primitive immunophenotype during in vitro culture. The CFU counts were significantly higher if HPCs were cultured with serum of MPN patients as compared to controls. Furthermore, serum of healthy young versus old donors did not evoke significant differences in proliferation or immunophenotype of HPCs, whereas the CFU frequency was significantly increased by serum from elderly patients. Our results indicate that serum derived from patients with MPN comprises activating feedback signals that stimulate the HPCs-and this stimulatory signal may result in a viscous circle that further accelerates development of the disease.

  20. Low doses of oxygen ion irradiation cause long-term damage to bone marrow hematopoietic progenitor and stem cells in mice

    PubMed Central

    Wang, Yingying; Chang, Jianhui; Li, Xin; Pathak, Rupak; Sridharan, Vijayalakshmi; Jones, Tamako; Mao, Xiao Wen; Nelson, Gregory; Boerma, Marjan; Hauer-Jensen, Martin; Zhou, Daohong

    2017-01-01

    During deep space missions, astronauts will be exposed to low doses of charged particle irradiation. The long-term health effects of these exposures are largely unknown. We previously showed that low doses of oxygen ion (16O) irradiation induced acute damage to the hematopoietic system, including hematopoietic progenitor and stem cells in a mouse model. However, the chronic effects of low dose 16O irradiation remain undefined. In the current study, we investigated the long-term effects of low dose 16O irradiation on the mouse hematopoietic system. Male C57BL/6J mice were exposed to 0.05 Gy, 0.1 Gy, 0.25 Gy and 1.0 Gy whole body 16O (600 MeV/n) irradiation. The effects of 16O irradiation on bone marrow (BM) hematopoietic progenitor cells (HPCs) and hematopoietic stem cells (HSCs) were examined three months after the exposure. The results showed that the frequencies and numbers of BM HPCs and HSCs were significantly reduced in 0.1 Gy, 0.25 Gy and 1.0 Gy irradiated mice compared to 0.05 Gy irradiated and non-irradiated mice. Exposure of mice to low dose 16O irradiation also significantly reduced the clongenic function of BM HPCs determined by the colony-forming unit assay. The functional defect of irradiated HSCs was detected by cobblestone area-forming cell assay after exposure of mice to 0.1 Gy, 0.25 Gy and 1.0 Gy of 16O irradiation, while it was not seen at three months after 0.5 Gy and 1.0 Gy of γ-ray irradiation. These adverse effects of 16O irradiation on HSCs coincided with an increased intracellular production of reactive oxygen species (ROS). However, there were comparable levels of cellular apoptosis and DNA damage between irradiated and non-irradiated HPCs and HSCs. These data suggest that exposure to low doses of 16O irradiation induces long-term hematopoietic injury, primarily via increased ROS production in HSCs. PMID:29232383

  1. Human Pluripotent Stem Cells Differentiated in Fully Defined Medium Generate Hematopoietic CD34+ and CD34− Progenitors with Distinct Characteristics

    PubMed Central

    Chicha, Laurie; Feki, Anis; Boni, Alessandro; Irion, Olivier; Hovatta, Outi; Jaconi, Marisa

    2011-01-01

    Background Differentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM) would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells. Methodology/Principal Findings ESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU) assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34+ cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB) CD34+ cells. ESC-derived CD34+ cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34− cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult β-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors. Conclusions/Significance Short-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive

  2. Preterm Cord Blood Contains a Higher Proportion of Immature Hematopoietic Progenitors Compared to Term Samples

    PubMed Central

    Podestà, Marina; Bruschettini, Matteo; Cossu, Claudia; Sabatini, Federica; Dagnino, Monica; Romantsik, Olga; Spaggiari, Grazia Maria; Ramenghi, Luca Antonio; Frassoni, Francesco

    2015-01-01

    Background Cord blood contains high number of hematopoietic cells that after birth disappear. In this paper we have studied the functional properties of the umbilical cord blood progenitor cells collected from term and preterm neonates to establish whether quantitative and/or qualitative differences exist between the two groups. Methods and Results Our results indicate that the percentage of total CD34+ cells was significantly higher in preterm infants compared to full term: 0.61% (range 0.15–4.8) vs 0.3% (0.032–2.23) p = 0.0001 and in neonates <32 weeks of gestational age (GA) compared to those ≥32 wks GA: 0.95% (range 0.18–4.8) and 0.36% (0.15–3.2) respectively p = 0.0025. The majority of CD34+ cells co-expressed CD71 antigen (p<0.05 preterm vs term) and grew in vitro large BFU-E, mostly in the second generation. The subpopulations CD34+CD38- and CD34+CD45- resulted more represented in preterm samples compared to term, conversely, Side Population (SP) did not show any difference between the two group. The absolute number of preterm colonies (CFCs/10microL) resulted higher compared to term (p = 0.004) and these progenitors were able to grow until the third generation maintaining an higher proportion of CD34+ cells (p = 0.0017). The number of colony also inversely correlated with the gestational age (Pearson r = -0.3001 p<0.0168). Conclusions We found no differences in the isolation and expansion capacity of Endothelial Colony Forming Cells (ECFCs) from cord blood of term and preterm neonates: both groups grew in vitro large number of endothelial cells until the third generation and showed a transitional phenotype between mesenchymal stem cells and endothelial progenitors (CD73, CD31, CD34 and CD144)The presence, in the cord blood of preterm babies, of high number of immature hematopoietic progenitors and endothelial/mesenchymal stem cells with high proliferative potential makes this tissue an important source of cells for developing new cells therapies

  3. Circulating Hematopoietic Stem and Progenitor Cells in Aging Atomic Bomb Survivors.

    PubMed

    Kyoizumi, Seishi; Kubo, Yoshiko; Misumi, Munechika; Kajimura, Junko; Yoshida, Kengo; Hayashi, Tomonori; Imai, Kazue; Ohishi, Waka; Nakachi, Kei; Young, Lauren F; Shieh, Jae-Hung; Moore, Malcolm A; van den Brink, Marcel R M; Kusunoki, Yoichiro

    2016-01-01

    It is not yet known whether hematopoietic stem and progenitor cells (HSPCs) are compromised in the aging population of atomic bomb (A-bomb) survivors after their exposure nearly 70 years ago. To address this, we evaluated age- and radiation-related changes in different subtypes of circulating HSPCs among the CD34-positive/lineage marker-negative (CD34(+)Lin(-)) cell population in 231 Hiroshima A-bomb survivors. We enumerated functional HSPC subtypes, including: cobblestone area-forming cells; long-term culture-initiating cells; erythroid burst-forming units; granulocyte and macrophage colony-forming units; and T-cell and natural killer cell progenitors using cell culture. We obtained the count of each HSPC subtype per unit volume of blood and the proportion of each HSPC subtype in CD34(+)Lin(-) cells to represent the lineage commitment trend. Multivariate analyses, using sex, age and radiation dose as variables, showed significantly decreased counts with age in the total CD34(+)Lin(-) cell population and all HSPC subtypes. As for the proportion, only T-cell progenitors decreased significantly with age, suggesting that the commitment to the T-cell lineage in HSPCs continuously declines with age throughout the lifetime. However, neither the CD34(+)Lin(-) cell population, nor HSPC subtypes showed significant radiation-induced dose-dependent changes in counts or proportions. Moreover, the correlations of the proportions among HSPC subtypes in the survivors properly revealed the hierarchy of lineage commitments. Taken together, our findings suggest that many years after exposure to radiation and with advancing age, the number and function of HSPCs in living survivors as a whole may have recovered to normal levels.

  4. Circulating Hematopoietic Stem and Progenitor Cells in Aging Atomic Bomb Survivors

    PubMed Central

    Kyoizumi, Seishi; Kubo, Yoshiko; Misumi, Munechika; Kajimura, Junko; Yoshida, Kengo; Hayashi, Tomonori; Imai, Kazue; Ohishi, Waka; Nakachi, Kei; Young, Lauren F.; Shieh, Jae-Hung; Moore, Malcolm A.; van den Brink, Marcel R. M.; Kusunoki, Yoichiro

    2016-01-01

    It is not yet known whether hematopoietic stem and progenitor cells (HSPCs) are compromised in the aging population of atomic bomb (A-bomb) survivors after their exposure nearly 70 years ago. To address this, we evaluated age- and radiation-related changes in different subtypes of circulating HSPCs among the CD34-positive/lineage marker-negative (CD34+Lin− ) cell population in 231 Hiroshima A-bomb survivors. We enumerated functional HSPC subtypes, including: cobblestone area-forming cells; long-term culture-initiating cells; erythroid burst-forming units; granulocyte and macrophage colony-forming units; and T-cell and natural killer cell progenitors using cell culture. We obtained the count of each HSPC subtype per unit volume of blood and the proportion of each HSPC subtype in CD34+Lin− cells to represent the lineage commitment trend. Multivariate analyses, using sex, age and radiation dose as variables, showed significantly decreased counts with age in the total CD34+Lin− cell population and all HSPC subtypes. As for the proportion, only T-cell progenitors decreased significantly with age, suggesting that the commitment to the T-cell lineage in HSPCs continuously declines with age throughout the lifetime. However, neither the CD34+Lin− cell population, nor HSPC subtypes showed significant radiation-induced dose-dependent changes in counts or proportions. Moreover, the correlations of the proportions among HSPC subtypes in the survivors properly revealed the hierarchy of lineage commitments. Taken together, our findings suggest that many years after exposure to radiation and with advancing age, the number and function of HSPCs in living survivors as a whole may have recovered to normal levels. PMID:26720799

  5. The RUNX1 +24 enhancer and P1 promoter identify a unique subpopulation of hematopoietic progenitor cells derived from human pluripotent stem cells

    PubMed Central

    Ferrell, Patrick I; Xi, Jiafei; Ma, Chao; Adlakha, Mitali; Kaufman, Dan S.

    2016-01-01

    Derivation of hematopoietic stem cells from human pluripotent stem cells remains a key goal for the fields of developmental biology and regenerative medicine. Here, we use a novel genetic reporter system to prospectively identify and isolate early hematopoietic cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (iPSCs). Cloning the human RUNX1c P1 promoter and +24 enhancer to drive expression of tdTomato (tdTom) in hESCs and iPSCs, we demonstrate that tdTom expression faithfully enriches for RUNX1c-expressing hematopoietic progenitor cells. Time-lapse microscopy demonstrated the tdTom+ hematopoietic cells to emerge from adherent cells. Furthermore, inhibition of primitive hematopoiesis by blocking Activin/Nodal signaling promoted the expansion and/or survival of tdTom+ population. Notably, RUNX1c/tdTom+ cells represent only a limited subpopuation of CD34+CD45+ and CD34+CD43+ cells with a unique genetic signature. Using gene array analysis, we find significantly lower expression of Let-7 and mir181a microRNAs in the RUNX1c/tdTom+ cell population. These phenotypic and genetic analyses comparing the RUNX1c/tdTom+ population to CD34+CD45+ umbilical cord blood and fetal liver demonstrate several key differences that likely impact the development of HSCs capable of long-term multilineage engraftment from hESCs and iPSCs. PMID:25546363

  6. Subsequent donation requests among 2472 unrelated hematopoietic progenitor cell donors are associated with bone marrow harvest

    PubMed Central

    Lown, Robert N.; Tulpule, Sameer; Russell, Nigel H.; Craddock, Charles F.; Roest, Rochelle; Madrigal, J. Alejandro; Shaw, Bronwen E.

    2013-01-01

    Approximately 1 in 20 unrelated donors are asked to make a second donation of hematopoietic progenitor cells, the majority for the same patient. Anthony Nolan undertook a study of subsequent hematopoietic progenitor cell donations made by its donors from 2005 to 2011, with the aims of predicting those donors more likely to be called for a second donation, assessing rates of serious adverse reactions and examining harvest yields. This was not a study of factors predictive of second allografts. During the study period 2591 donations were made, of which 120 (4.6%) were subsequent donations. The median time between donations was 179 days (range, 21–4016). Indications for a second allogeneic transplant included primary graft failure (11.7%), secondary graft failure (53.2%), relapse (30.6%) and others (1.8%). On multivariate analysis, bone marrow harvest at first donation was associated with subsequent donation requests (odds ratio 2.00, P=0.001). The rate of serious adverse reactions in donors making a subsequent donation appeared greater than the rate in those making a first donation (relative risk=3.29, P=0.005). Harvest yields per kilogram recipient body weight were equivalent between donations, although females appeared to have a lower yield at the subsequent donation. Knowledge of these factors will help unrelated donor registries to counsel their donors. PMID:23812935

  7. Hematopoietic cell differentiation from embryonic and induced pluripotent stem cells

    PubMed Central

    2013-01-01

    Pluripotent stem cells, both embryonic stem cells and induced pluripotent stem cells, are undifferentiated cells that can self-renew and potentially differentiate into all hematopoietic lineages, such as hematopoietic stem cells (HSCs), hematopoietic progenitor cells and mature hematopoietic cells in the presence of a suitable culture system. Establishment of pluripotent stem cells provides a comprehensive model to study early hematopoietic development and has emerged as a powerful research tool to explore regenerative medicine. Nowadays, HSC transplantation and hematopoietic cell transfusion have successfully cured some patients, especially in malignant hematological diseases. Owing to a shortage of donors and a limited number of the cells, hematopoietic cell induction from pluripotent stem cells has been regarded as an alternative source of HSCs and mature hematopoietic cells for intended therapeutic purposes. Pluripotent stem cells are therefore extensively utilized to facilitate better understanding in hematopoietic development by recapitulating embryonic development in vivo, in which efficient strategies can be easily designed and deployed for the generation of hematopoietic lineages in vitro. We hereby review the current progress of hematopoietic cell induction from embryonic stem/induced pluripotent stem cells. PMID:23796405

  8. Fetal bovine bone marrow is a rich source of CD34+ hematopoietic progenitors with myelo-monocytic colony-forming activity.

    PubMed

    Pessa-Morikawa, Tiina; Niku, Mikael; Iivanainen, Antti

    2012-03-01

    The CD34 glycoprotein is an important marker of hematopoietic stem cells. We used a polyclonal rabbit anti-bovine CD34 antibody to stain fetal and adult bovine bone marrow cells. Flow cytometry revealed a low side scatter (SSC(low)) population of cells that were CD34(+) but negative for leukocyte lineage markers CD11b, CD14 or CD2. Hematopoietic colony assays with CD34(+) and CD34(-) bone marrow cells suggested that the colony-forming potential in SSC(low) bone marrow cells was confined to the CD34(+) fraction. In contrast, this population was not enriched for cells expressing high aldehyde dehydrogenase activity, a metabolic marker that has been used to characterize hematopoietic stem cells. Thus, the CD34 antigen can be used to identify and isolate bovine bone marrow cells exhibiting clonogenic potential in vitro. Moreover, the proportion of CD34(+) cells is very high in fetal bovine bone marrow, indicating it as a rich source of hematopoietic progenitors. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. l-Arginine is a Radioprotector for Hematopoietic Progenitor Cells

    PubMed Central

    Pearce, Linda L.; Zheng, Xichen; Martinez-Bosch, Sandra; Kerr, Patrick P.; Khlangwiset, Pornsri; Epperly, Michael W.; Fink, Mitchell P.; Greenberger, Joel S.; Peterson, Jim

    2012-01-01

    l-Arginine is shown to protect hematopoietic progenitor (32D cl 3) cells from death due to exposure to γ radiation (137Cs). Some of the other intermediates in the urea cycle, namely ornithine and citrulline, plus urea itself, were not found to have any significant impact on cell survival after irradiation. Intriguingly, supplementation of irradiated cells with l-arginine results in decreased production of peroxynitrite, suggesting that suppression of superoxide generation by nitric oxide synthase in one or more microenvironments is an important factor in the observed radioprotection. The absence of any radioprotective effect of l-arginine in cells at 3% oxygen also confirms the involvement of one or more oxygen-derived species. Knockdown experiments with nitric oxide synthase (NOS) siRNAs in cells and NOS knockout animals confirm that the observed radioprotection is associated with nNOS (NOS-1). l-Arginine also ameliorates the transient inhibition of the electron-transport chain complex I that occurs within 30 min of completing the dose (10 Gy) and that appears to be a functional marker for postirradiation mitochondrial oxidant production. PMID:22175298

  10. Early osteoinductive human bone marrow mesenchymal stromal/stem cells support an enhanced hematopoietic cell expansion with altered chemotaxis- and adhesion-related gene expression profiles

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sugino, Noriko; Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507; Miura, Yasuo, E-mail: ym58f5@kuhp.kyoto-u.ac.jp

    Bone marrow (BM) microenvironment has a crucial role in supporting hematopoiesis. Here, by using a microarray analysis, we demonstrate that human BM mesenchymal stromal/stem cells (MSCs) in an early osteoinductive stage (e-MSCs) are characterized by unique hematopoiesis-associated gene expression with an enhanced hematopoiesis-supportive ability. In comparison to BM-MSCs without osteoinductive treatment, gene expression in e-MSCs was significantly altered in terms of their cell adhesion- and chemotaxis-related profiles, as identified with Gene Ontology and Gene Set Enrichment Analysis. Noteworthy, expression of the hematopoiesis-associated molecules CXCL12 and vascular cell adhesion molecule 1 was remarkably decreased in e-MSCs. e-MSCs supported an enhanced expansionmore » of CD34{sup +} hematopoietic stem and progenitor cells, and generation of myeloid lineage cells in vitro. In addition, short-term osteoinductive treatment favored in vivo hematopoietic recovery in lethally irradiated mice that underwent BM transplantation. e-MSCs exhibited the absence of decreased stemness-associated gene expression, increased osteogenesis-associated gene expression, and apparent mineralization, thus maintaining the ability to differentiate into adipogenic cells. Our findings demonstrate the unique biological characteristics of e-MSCs as hematopoiesis-regulatory stromal cells at differentiation stage between MSCs and osteoprogenitor cells and have significant implications in developing new strategy for using pharmacological osteoinductive treatment to support hematopoiesis in hematopoietic stem and progenitor cell transplantation. - Highlights: • Human BM-MSCs in an early osteoinductive stage (e-MSCs) support hematopoiesis. • Adhesion- and chemotaxis-associated gene signatures are altered in e-MSCs. • Expression of CXCL12 and VCAM1 is remarkably decreased in e-MSCs. • e-MSCs are at differentiation stage between MSCs and osteoprogenitor cells. • Osteoinductive

  11. Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors

    PubMed Central

    Cheng, Hui; Ang, Heather Yin-Kuan; A. EL Farran, Chadi; Li, Pin; Fang, Hai Tong; Liu, Tong Ming; Kong, Say Li; Chin, Michael Lingzi; Ling, Wei Yin; Lim, Edwin Kok Hao; Li, Hu; Huber, Tara; Loh, Kyle M.; Loh, Yuin-Han; Lim, Bing

    2016-01-01

    Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into ‘induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase+ megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit+ hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors. PMID:27869129

  12. Lentiviral-mediated genetic correction of hematopoietic and mesenchymal progenitor cells from Fanconi anemia patients.

    PubMed

    Jacome, Ariana; Navarro, Susana; Río, Paula; Yañez, Rosa M; González-Murillo, Africa; Lozano, M Luz; Lamana, Maria Luisa; Sevilla, Julian; Olive, Teresa; Diaz-Heredia, Cristina; Badell, Isabel; Estella, Jesus; Madero, Luis; Guenechea, Guillermo; Casado, José; Segovia, Jose C; Bueren, Juan A

    2009-06-01

    Previous clinical trials based on the genetic correction of purified CD34(+) cells with gamma-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34(+) cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34(+) cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34(-) mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs).

  13. Lentiviral-mediated Genetic Correction of Hematopoietic and Mesenchymal Progenitor Cells From Fanconi Anemia Patients

    PubMed Central

    Jacome, Ariana; Navarro, Susana; Río, Paula; Yañez, Rosa M; González-Murillo, Africa; Luz Lozano, M; Lamana, Maria Luisa; Sevilla, Julian; Olive, Teresa; Diaz-Heredia, Cristina; Badell, Isabel; Estella, Jesus; Madero, Luis; Guenechea, Guillermo; Casado, José; Segovia, Jose C; Bueren, Juan A

    2009-01-01

    Previous clinical trials based on the genetic correction of purified CD34+ cells with γ-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34+ cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34+ cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34− mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs). PMID:19277017

  14. Transcriptional diversity during lineage commitment of human blood progenitors.

    PubMed

    Chen, Lu; Kostadima, Myrto; Martens, Joost H A; Canu, Giovanni; Garcia, Sara P; Turro, Ernest; Downes, Kate; Macaulay, Iain C; Bielczyk-Maczynska, Ewa; Coe, Sophia; Farrow, Samantha; Poudel, Pawan; Burden, Frances; Jansen, Sjoert B G; Astle, William J; Attwood, Antony; Bariana, Tadbir; de Bono, Bernard; Breschi, Alessandra; Chambers, John C; Consortium, Bridge; Choudry, Fizzah A; Clarke, Laura; Coupland, Paul; van der Ent, Martijn; Erber, Wendy N; Jansen, Joop H; Favier, Rémi; Fenech, Matthew E; Foad, Nicola; Freson, Kathleen; van Geet, Chris; Gomez, Keith; Guigo, Roderic; Hampshire, Daniel; Kelly, Anne M; Kerstens, Hindrik H D; Kooner, Jaspal S; Laffan, Michael; Lentaigne, Claire; Labalette, Charlotte; Martin, Tiphaine; Meacham, Stuart; Mumford, Andrew; Nürnberg, Sylvia; Palumbo, Emilio; van der Reijden, Bert A; Richardson, David; Sammut, Stephen J; Slodkowicz, Greg; Tamuri, Asif U; Vasquez, Louella; Voss, Katrin; Watt, Stephen; Westbury, Sarah; Flicek, Paul; Loos, Remco; Goldman, Nick; Bertone, Paul; Read, Randy J; Richardson, Sylvia; Cvejic, Ana; Soranzo, Nicole; Ouwehand, Willem H; Stunnenberg, Hendrik G; Frontini, Mattia; Rendon, Augusto

    2014-09-26

    Blood cells derive from hematopoietic stem cells through stepwise fating events. To characterize gene expression programs driving lineage choice, we sequenced RNA from eight primary human hematopoietic progenitor populations representing the major myeloid commitment stages and the main lymphoid stage. We identified extensive cell type-specific expression changes: 6711 genes and 10,724 transcripts, enriched in non-protein-coding elements at early stages of differentiation. In addition, we found 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes. We demonstrated experimentally cell-specific isoform usage, identifying nuclear factor I/B (NFIB) as a regulator of megakaryocyte maturation-the platelet precursor. Our data highlight the complexity of fating events in closely related progenitor populations, the understanding of which is essential for the advancement of transplantation and regenerative medicine. Copyright © 2014, American Association for the Advancement of Science.

  15. Endothelial and circulating progenitor cells in hematological diseases and allogeneic hematopoietic stem cell transplantation.

    PubMed

    Ruggeri, Annalisa; Paviglianiti, Annalisa; Volt, Fernanda; Kenzey, Chantal; Rafii, Hanadi; Rocha, Vanderson; Gluckman, Eliane

    2017-10-12

    Circulating endothelial cells (CECs), originated form endothelial progenitors (EPCs) are mature cells which are not associated with vessel walls, and that are detached from the endothelium. Normally, they are present in insignificant amounts in the peripheral blood of healthy individuals. On the other hand, elevated CECs and EPCs levels have been reported in the peripheral blood of patients with different types of cancers and some other diseases. Consequently, CECs and EPCs represent a potential biomarker in several clinical conditions involving endothelial turnover and remodeling, such as hematological diseases. These cells may be involved in disease progression and the neoplastic angiogenesis process. Moreover, CESs and EPCs are probably involved in endothelial damage that is a marker of several complications following allogeneic hematopoietic stem cell transplantation. This review aims to provide an overview on the characterization of CECs and EPCs, describe isolation methods and to identify the potential role of these cells in hematological diseases and hematopoietic stem cell transplantation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Cytohesin 1 regulates homing and engraftment of human hematopoietic stem and progenitor cells.

    PubMed

    Rak, Justyna; Foster, Katie; Potrzebowska, Katarzyna; Talkhoncheh, Mehrnaz Safaee; Miharada, Natsumi; Komorowska, Karolina; Torngren, Therese; Kvist, Anders; Borg, Åke; Svensson, Lena; Bonnet, Dominique; Larsson, Jonas

    2017-02-23

    Adhesion is a key component of hematopoietic stem cell regulation mediating homing and retention to the niche in the bone marrow. Here, using an RNA interference screen, we identify cytohesin 1 (CYTH1) as a critical mediator of adhesive properties in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs). Knockdown of CYTH1 disrupted adhesion of HSPCs to primary human mesenchymal stroma cells. Attachment to fibronectin and ICAM1, 2 integrin ligands, was severely impaired, and CYTH1-deficient cells showed a reduced integrin β1 activation response, suggesting that CYTH1 mediates integrin-dependent functions. Transplantation of CYTH1-knockdown cells to immunodeficient mice resulted in significantly lower long-term engraftment levels, associated with a reduced capacity of the transplanted cells to home to the bone marrow. Intravital microscopy showed that CYTH1 deficiency profoundly affects HSPC mobility and localization within the marrow space and thereby impairs proper lodgment into the niche. Thus, CYTH1 is a novel major regulator of adhesion and engraftment in human HSPCs through mechanisms that, at least in part, involve the activation of integrins. © 2017 by The American Society of Hematology.

  17. Transfection of Murine and Human Hematopoietic Progenitors with Rearranged Immunoglobulin Genes

    DTIC Science & Technology

    1991-01-01

    fluorouracil (SFU) to eliminate most cycling progenitors. Previous studies have shown that 5FU -treatment enriches for one early progenitor with high...Table I shows a time course of SCA-I positive cell expression various times post- 5FU treatment. Table 1 clearly shows that 5FU treatment can increase...the percentage of SCA-l-positive cells to 6-7% by day 7 post- 5FU treatment. The level of SCA-I expression falls to approximately 1% of total nucleated

  18. Comparison of cryopreservation bags for hematopoietic progenitor cells using a WBC-enriched product.

    PubMed

    Dijkstra-Tiekstra, Margriet J; Hazelaar, Sandra; Gkoumassi, Effimia; Weggemans, Margienus; de Wildt-Eggen, Janny

    2015-04-01

    Hematopoietic progenitor cells (HPC) are stored in cryopreservation bags that are resistant to liquid nitrogen. Since Cryocyte bags of Baxter (B-bags) are no longer available, an alternative bag was sought. Also, the influence of freezing volume was studied. Miltenyi Biotec (MB)- and MacoPharma (MP)-bags passed the integrity tests without failure. Comparing MB- and MP-bags with B-bags, no difference in WBC recovery or viability was found when using a WBC-enriched product as a "dummy" HPC product. Further, a freezing volume of 30 mL resulted in better WBC recovery and viability than 60 mL. Additonal studies using real HPC might be necessary. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Expression of Fas and Fas-ligand in donor hematopoietic stem and progenitor cells is dissociated from the sensitivity to apoptosis.

    PubMed

    Pearl-Yafe, Michal; Yolcu, Esma S; Stein, Jerry; Kaplan, Ofer; Shirwan, Haval; Yaniv, Isaac; Askenasy, Nadir

    2007-10-01

    The interaction between the Fas receptor and its cognate ligand (FasL) has been implicated in the mutual suppression of donor and host hematopoietic cells after transplantation. Following the observation of deficient early engraftment of Fas and FasL-defective donor cells and recipients, we determined the role of the Fas-FasL interaction. Donor cells were recovered after syngeneic (CD45.1-->CD45.2) transplants from various organs and assessed for expression of Fas/FasL in reference to lineage markers, carboxyfluorescein succinimidyl ester dilution, Sca-1 and c-kit expression. Naïve and bone marrow-homed cells were challenged for apoptosis ex vivo. The Fas receptor and ligand were markedly upregulated to 40% to 60% (p < 0.001 vs 5-10% in naïve cells) within 2 days after syngeneic transplantation, while residual host cells displayed modest and delayed upregulation of these molecules ( approximately 10%). All lin(-)Sca(+)c-kit(+) cells were Fas(+)FasL(+), including 95% of Sca-1(+) and 30% of c-kit(+) cells. Fas and FasL expression varied in donor cells that homed to bone marrow, spleen, liver and lung, and was induced by interaction with the stroma, irradiation, cell cycling, and differentiation. Bone marrow-homed donor cells challenged with supralethal doses of FasL were insensitive to apoptosis (3.2% +/- 1% vs 38% +/- 5% in naïve bone marrow cells), and engraftment was not affected by pretransplantation exposure of donor cells to an apoptotic challenge with FasL. There was no evidence of Fas-mediated suppression of donor and host cell activity after transplantation. Resistance to Fas-mediated apoptosis evolves as a functional characteristic of hematopoietic reconstituting stem and progenitor cells, providing them competitive engraftment advantage over committed progenitors.

  20. Contribution of endothelial progenitors and proangiogenic hematopoietic cells to vascularization of tumor and ischemic tissue

    PubMed Central

    Kopp, Hans-Georg; Ramos, Carlos A.; Rafii, Shahin

    2010-01-01

    Purpose of review During the last several years, a substantial amount of evidence from animal as well as human studies has advanced our knowledge of how bone marrow derived cells contribute to neoangiogenesis. In the light of recent findings, we may have to redefine our thinking of endothelial cells as well as of perivascular mural cells. Recent findings Inflammatory hematopoietic cells, such as macrophages, have been shown to promote neoangiogenesis during tumor growth and wound healing. Dendritic cells, B lymphocytes, monocytes, and other immune cells have also been found to be recruited to neoangiogenic niches and to support neovessel formation. These findings have led to the concept that subsets of hematopoietic cells comprise proangiogenic cells that drive adult revascularization processes. While evidence of the importance of endothelial progenitor cells in adult vasculogenesis increased further, the role of these comobilized hematopoietic cells has been intensely studied in the last few years. Summary Angiogenic factors promote mobilization of vascular endothelial growth factor receptor 1-positive hematopoietic cells through matrix metalloproteinase-9 mediated release of soluble kit-ligand and recruit these proangiogenic cells to areas of hypoxia, where perivascular mural cells present stromal-derived factor 1 (CXCL-12) as an important retention signal. The same factors are possibly involved in mobilization of vascular endothelial growth factor receptor 2-positive endothelial precursors that may participate in neovessel formation. The complete characterization of mechanisms, mediators and signaling pathways involved in these processes will provide novel targets for both anti and proangiogenic therapeutic strategies. PMID:16567962

  1. Hematologic changes after total body irradiation and autologous transplantation of hematopoietic peripheral blood progenitor cells in dogs with lymphoma.

    PubMed

    Escobar, C; Grindem, C; Neel, J A; Suter, S E

    2012-03-01

    Dogs with and without lymphoma have undergone hematopoietic cell transplantation in a research setting for decades. North Carolina State University is currently treating dogs with B- and T-cell lymphoma in a clinical setting with autologous peripheral blood progenitor cell transplants, using peripheral blood CD34+ progenitor cells harvested using an apheresis machine. Complete blood counts were performed daily for 15 to 19 days posttransplantation to monitor peripheral blood cell nadirs and subsequent CD34+ cell engraftment. This study documents the hematologic toxicities of total body irradiation in 10 dogs and the subsequent recovery of the affected cell lines after peripheral blood progenitor cell transplant, indicating successful CD34+ engraftment. All peripheral blood cell lines, excluding red blood cells, experienced grade 4 toxicities. All dogs had ≥ 500 neutrophils/μl by day 12, while thrombocytopenia persisted for many weeks. All dogs were clinically normal at discharge.

  2. Expansion of Human and Murine Hematopoietic Stem and Progenitor Cells Ex Vivo without Genetic Modification Using MYC and Bcl-2 Fusion Proteins

    PubMed Central

    Bird, Gregory A.; Polsky, Avital; Estes, Patricia; Hanlon, Teri; Hamilton, Haley; Morton, John J.; Gutman, Jonathan; Jimeno, Antonio

    2014-01-01

    The long-term repopulating hematopoietic stem cell (HSC) population can self-renew in vivo, support hematopoiesis for the lifetime of the individual, and is of critical importance in the context of bone marrow stem cell transplantation. The mechanisms that regulate the expansion of HSCs in vivo and in vitro remain unclear to date. Since the current set of surface markers only allow for the identification of a population of cells that is highly enriched for HSC activity, we will refer to the population of cells we expand as Hematopoietic Stem and Progenitor cells (HSPCs). We describe here a novel approach to expand a cytokine-dependent Hematopoietic Stem and Progenitor Cell (HSPC) population ex vivo by culturing primary adult human or murine HSPCs with fusion proteins including the protein transduction domain of the HIV-1 transactivation protein (Tat) and either MYC or Bcl-2. HSPCs obtained from either mouse bone marrow, human cord blood, human G-CSF mobilized peripheral blood, or human bone marrow were expanded an average of 87 fold, 16.6 fold, 13.6 fold, or 10 fold, respectively. The expanded cell populations were able to give rise to different types of colonies in methylcellulose assays in vitro, as well as mature hematopoietic populations in vivo upon transplantation into irradiated mice. Importantly, for both the human and murine case, the ex vivo expanded cells also gave rise to a self-renewing cell population in vivo, following initial transplantation, that was able to support hematopoiesis upon serial transplantation. Our results show that a self-renewing cell population, capable of reconstituting the hematopoietic compartment, expanded ex vivo in the presence of Tat-MYC and Tat-Bcl-2 suggesting that this may be an attractive approach to expand human HSPCs ex vivo for clinical use. PMID:25170611

  3. An abnormal bone marrow microenvironment contributes to hematopoietic dysfunction in Fanconi anemia.

    PubMed

    Zhou, Yuan; He, Yongzheng; Xing, Wen; Zhang, Peng; Shi, Hui; Chen, Shi; Shi, Jun; Bai, Jie; Rhodes, Steven D; Zhang, Fengqui; Yuan, Jin; Yang, Xianlin; Zhu, Xiaofan; Li, Yan; Hanenberg, Helmut; Xu, Mingjiang; Robertson, Kent A; Yuan, Weiping; Nalepa, Grzegorz; Cheng, Tao; Clapp, D Wade; Yang, Feng-Chun

    2017-06-01

    Fanconi anemia is a complex heterogeneous genetic disorder with a high incidence of bone marrow failure, clonal evolution to acute myeloid leukemia and mesenchymal-derived congenital anomalies. Increasing evidence in Fanconi anemia and other genetic disorders points towards an interdependence of skeletal and hematopoietic development, yet the impact of the marrow microenvironment in the pathogenesis of the bone marrow failure in Fanconi anemia remains unclear. Here we demonstrated that mice with double knockout of both Fancc and Fancg genes had decreased bone formation at least partially due to impaired osteoblast differentiation from mesenchymal stem/progenitor cells. Mesenchymal stem/progenitor cells from the double knockout mice showed impaired hematopoietic supportive activity. Mesenchymal stem/progenitor cells of patients with Fanconi anemia exhibited similar cellular deficits, including increased senescence, reduced proliferation, impaired osteoblast differentiation and defective hematopoietic stem/progenitor cell supportive activity. Collectively, these studies provide unique insights into the physiological significance of mesenchymal stem/progenitor cells in supporting the marrow microenvironment, which is potentially of broad relevance in hematopoietic stem cell transplantation. Copyright© Ferrata Storti Foundation.

  4. An abnormal bone marrow microenvironment contributes to hematopoietic dysfunction in Fanconi anemia

    PubMed Central

    Zhou, Yuan; He, Yongzheng; Xing, Wen; Zhang, Peng; Shi, Hui; Chen, Shi; Shi, Jun; Bai, Jie; Rhodes, Steven D.; Zhang, Fengqui; Yuan, Jin; Yang, Xianlin; Zhu, Xiaofan; Li, Yan; Hanenberg, Helmut; Xu, Mingjiang; Robertson, Kent A.; Yuan, Weiping; Nalepa, Grzegorz; Cheng, Tao; Clapp, D. Wade; Yang, Feng-Chun

    2017-01-01

    Fanconi anemia is a complex heterogeneous genetic disorder with a high incidence of bone marrow failure, clonal evolution to acute myeloid leukemia and mesenchymal-derived congenital anomalies. Increasing evidence in Fanconi anemia and other genetic disorders points towards an interdependence of skeletal and hematopoietic development, yet the impact of the marrow microenvironment in the pathogenesis of the bone marrow failure in Fanconi anemia remains unclear. Here we demonstrated that mice with double knockout of both Fancc and Fancg genes had decreased bone formation at least partially due to impaired osteoblast differentiation from mesenchymal stem/progenitor cells. Mesenchymal stem/progenitor cells from the double knockout mice showed impaired hematopoietic supportive activity. Mesenchymal stem/progenitor cells of patients with Fanconi anemia exhibited similar cellular deficits, including increased senescence, reduced proliferation, impaired osteoblast differentiation and defective hematopoietic stem/progenitor cell supportive activity. Collectively, these studies provide unique insights into the physiological significance of mesenchymal stem/progenitor cells in supporting the marrow microenvironment, which is potentially of broad relevance in hematopoietic stem cell transplantation. PMID:28341737

  5. Rad18 confers hematopoietic progenitor cell DNA damage tolerance independently of the Fanconi Anemia pathway in vivo

    PubMed Central

    Yang, Yang; Poe, Jonathan C.; Yang, Lisong; Fedoriw, Andrew; Desai, Siddhi; Magnuson, Terry; Li, Zhiguo; Fedoriw, Yuri; Araki, Kimi; Gao, Yanzhe; Tateishi, Satoshi; Sarantopoulos, Stefanie; Vaziri, Cyrus

    2016-01-01

    In cultured cancer cells the E3 ubiquitin ligase Rad18 activates Trans-Lesion Synthesis (TLS) and the Fanconi Anemia (FA) pathway. However, physiological roles of Rad18 in DNA damage tolerance and carcinogenesis are unknown and were investigated here. Primary hematopoietic stem and progenitor cells (HSPC) co-expressed RAD18 and FANCD2 proteins, potentially consistent with a role for Rad18 in FA pathway function during hematopoiesis. However, hematopoietic defects typically associated with fanc-deficiency (decreased HSPC numbers, reduced engraftment potential of HSPC, and Mitomycin C (MMC) -sensitive hematopoiesis), were absent in Rad18−/− mice. Moreover, primary Rad18−/− mouse embryonic fibroblasts (MEF) retained robust Fancd2 mono-ubiquitination following MMC treatment. Therefore, Rad18 is dispensable for FA pathway activation in untransformed cells and the Rad18 and FA pathways are separable in hematopoietic cells. In contrast with responses to crosslinking agents, Rad18−/− HSPC were sensitive to in vivo treatment with the myelosuppressive agent 7,12 Dimethylbenz[a]anthracene (DMBA). Rad18-deficient fibroblasts aberrantly accumulated DNA damage markers after DMBA treatment. Moreover, in vivo DMBA treatment led to increased incidence of B cell malignancy in Rad18−/− mice. These results identify novel hematopoietic functions for Rad18 and provide the first demonstration that Rad18 confers DNA damage tolerance and tumor-suppression in a physiological setting. PMID:26883629

  6. Insulin-Like Growth Factor 1 Mitigates Hematopoietic Toxicity After Lethal Total Body Irradiation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhou, Dunhua; Deoliveira, Divino; Kang, Yubin

    2013-03-15

    Purpose: To investigate whether and how insulin-like growth factor 1 (IGF-1) mitigates hematopoietic toxicity after total body irradiation. Methods and Materials: BALB/c mice were irradiated with a lethal dose of radiation (7.5 Gy) and treated with IGF-1 at a dose of 100 μg/dose intravenously once a day for 5 consecutive days starting within 1 hour after exposure. Survival and hematopoietic recovery were monitored. The mechanisms by which IGF-1 promotes hematopoietic recovery were also studied by use of an in vitro culture system. Results: IGF-1 protected 8 of 20 mice (40%) from lethal irradiation, whereas only 2 of 20 mice (10%) inmore » the saline control group survived for more than 100 days after irradiation. A single dose of IGF-1 (500 μg) was as effective as daily dosing for 5 days. Positive effects were noted even when the initiation of treatment was delayed as long as 6 hours after irradiation. In comparison with the saline control group, treatment with IGF-1 significantly accelerated the recovery of both platelets and red blood cells in peripheral blood, total cell numbers, hematopoietic stem cells, and progenitor cells in the bone marrow when measured at day 14 after irradiation. IGF-1 protected both hematopoietic stem cells and progenitor cells from radiation-induced apoptosis and cell death. In addition, IGF-1 was able to facilitate the proliferation and differentiation of nonirradiated and irradiated hematopoietic progenitor cells. Conclusions: IGF-1 mitigates radiation-induced hematopoietic toxicity through protecting hematopoietic stem cells and progenitor cells from apoptosis and enhancing proliferation and differentiation of the surviving hematopoietic progenitor cells.« less

  7. Identifying the progenitors of present-day early-type galaxies in observational surveys: correcting `progenitor bias' using the Horizon-AGN simulation

    NASA Astrophysics Data System (ADS)

    Martin, G.; Kaviraj, S.; Devriendt, J. E. G.; Dubois, Y.; Pichon, C.; Laigle, C.

    2018-03-01

    As endpoints of the hierarchical mass-assembly process, the stellar populations of local early-type galaxies encode the assembly history of galaxies over cosmic time. We use Horizon-AGN, a cosmological hydrodynamical simulation, to study the merger histories of local early-type galaxies and track how the morphological mix of their progenitors evolves over time. We provide a framework for alleviating `progenitor bias' - the bias that occurs if one uses only early-type galaxies to study the progenitor population. Early types attain their final morphology at relatively early epochs - by z ˜ 1, around 60 per cent of today's early types have had their last significant merger. At all redshifts, the majority of mergers have one late-type progenitor, with late-late mergers dominating at z > 1.5 and early-early mergers becoming significant only at z < 0.5. Progenitor bias is severe at all but the lowest redshifts - e.g. at z ˜ 0.6, less than 50 per cent of the stellar mass in today's early types is actually in progenitors with early-type morphology, while, at z ˜ 2, studying only early types misses almost all (80 per cent) of the stellar mass that eventually ends up in local early-type systems. At high redshift, almost all massive late-type galaxies, regardless of their local environment or star formation rate, are progenitors of local early-type galaxies, as are lower mass (M⋆ < 1010.5 M_{⊙}) late-types as long as they reside in high-density environments. In this new era of large observational surveys (e.g. LSST, JWST), this study provides a framework for studying how today's early-type galaxies have been built up over cosmic time.

  8. An update on ABO incompatible hematopoietic progenitor cell transplantation.

    PubMed

    Staley, Elizabeth M; Schwartz, Joseph; Pham, Huy P

    2016-06-01

    Hematopoietic progenitor cell (HPC) transplantation has long been established as the optimal treatment for many hematologic malignancies. In the setting of allogenic HLA matched HPC transplantation, greater than 50% of unrelated donors and 30% of related donors demonstrate some degree of ABO incompatibility (ABOi), which is classified in one of three ways: major, minor, or bidirectional. Major ABOi refers to the presence of recipient isoagglutinins against the donor's A and/or B antigen. Minor ABOi occurs when the HPC product contains the isoagglutinins targeting the recipient's A and/or B antigen. Bidirectional refers to the presence of both major and minor ABOi. Major adverse events associated with ABOi HPC transplantation includes acute and delayed hemolysis, pure red cell aplasia, and delayed engraftment. ABOi HPC transplantation poses a unique challenge to the clinical transplantation unit, the HPC processing lab, and the transfusion medicine service. Therefore, it is essential that these services actively communicate with one another to ensure patient safety. This review will attempt to globally address the challenges related to ABOi HPC transplantation, with an increased focus on aspects related to the laboratory and transfusion medicine services. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture.

    PubMed

    Schmal, Olga; Seifert, Jan; Schäffer, Tilman E; Walter, Christina B; Aicher, Wilhelm K; Klein, Gerd

    2016-01-01

    Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34(+) hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard.

  10. Transient loading of CD34+ hematopoietic progenitor cells with polystyrene nanoparticles.

    PubMed

    Deville, Sarah; Hadiwikarta, Wahyu Wijaya; Smisdom, Nick; Wathiong, Bart; Ameloot, Marcel; Nelissen, Inge; Hooyberghs, Jef

    2017-01-01

    CD34 + hematopoietic progenitor cells (HPCs) offer great opportunities to develop new treatments for numerous malignant and non-malignant diseases. Nanoparticle (NP)-based strategies can further enhance this potential, and therefore a thorough understanding of the loading behavior of HPCs towards NPs is essential for a successful application. The present study focusses on the interaction kinetics of 40 nm sized carboxylated polystyrene (PS) NPs with HPCs. Interestingly, a transient association of the NPs with HPCs is observed, reaching a maximum within 1 hour and declining afterwards. This behavior is not seen in dendritic cells (CD34-DCs) differentiated from HPCs, which display a monotonic increase in NP load. We demonstrate that this transient interaction requires an energy-dependent cellular process, suggesting active loading and release of NPs by HPCs. This novel observation offers a unique approach to transiently equip HPCs. A simple theoretical approach modeling the kinetics of NP loading and release is presented, contributing to a framework of describing this phenomenon.

  11. Thrombopoietin contributes to the formation and the maintenance of hematopoietic progenitor-containing cell clusters in the aorta-gonad-mesonephros region.

    PubMed

    Harada, Kaho; Nobuhisa, Ikuo; Anani, Maha; Saito, Kiyoka; Taga, Tetsuya

    2017-07-01

    In the midgestation mouse embryo, hematopoietic cell clusters containing hematopoietic stem/progenitor cells arise in the aorta-gonad-mesonephros (AGM) region. We have previously reported that forced expression of the Sox17 transcription factor in CD45 low c-Kit high AGM cells, which are the hematopoietic cellular component of the cell clusters, and subsequent coculture with OP9 stromal cells in the presence of three cytokines, stem cell factor (SCF), interleukin-3 (IL-3), and thrombopoietin (TPO), led to the formation and the maintenance of cell clusters with cells at an undifferentiated state in vitro. In this study, we investigated the role of each cytokine in the formation of hematopoietic cell clusters. We cultured Sox17-transduced AGM cells with each of the 7 possible combinations of the three cytokines. The size and the number of Sox17-transduced cell clusters in the presence of TPO, either alone or in combination, were comparable to that observed with the complete set of the three cytokines. Expression of TPO receptor, c-Mpl was almost ubiquitously expressed and maintained in Sox17-transduced hematopoietic cell clusters. In addition, the expression level of c-Mpl was highest in the CD45 low c-Kit high cells among the Sox17-transduced cell clusters. Moreover, c-Mpl protein was highly expressed in the intra-aortic hematopoietic cell clusters in comparison with endothelial cells of dorsal aorta. Finally, stimulation of the endothelial cells prepared from the AGM region by TPO induced the production of hematopoietic cells. These results suggest that TPO contributes to the formation and the maintenance of hematopoietic cell clusters in the AGM region. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Low-dose radiation (LDR) induces hematopoietic hormesis: LDR-induced mobilization of hematopoietic progenitor cells into peripheral blood circulation.

    PubMed

    Li, Wei; Wang, Guanjun; Cui, Jiuwei; Xue, Lu; Cai, Lu

    2004-11-01

    The aim of this study was to investigate the stimulating effect of low-dose radiation (LDR) on bone marrow hematopoietic progenitor cell (HPC) proliferation and peripheral blood mobilization. Mice were exposed to 25- to 100-mGy x-rays. Bone marrow and peripheral blood HPCs (BFU-E, CFU-GM, and c-kit+ cells) were measured, and GM-CSF, G-CSF, and IL-3 protein and mRNA expression were detected using ELISA, slot blot hybridization, and Northern blot methods. To functionally evaluate LDR-stimulated and -mobilized HPCs, repopulation of peripheral blood cells in lethally irradiated recipients after transplantation of LDR-treated donor HPCs was examined by WBC counts, animal survival, and colony-forming units in the recipient spleens (CFUs-S). 75-mGy x-rays induced a maximal stimulation for bone marrow HPC proliferation (CFU-GM and BFU-E formation) 48 hours postirradiation, along with a significant increase in HPC mobilization into peripheral blood 48 to 72 hours postradiation, as shown by increases in CFU-GM formation and proportion of c-kit+ cells in the peripheral mononuclear cells. 75-mGy x-rays also maximally induced increases in G-CSF and GM-CSF mRNA expression in splenocytes and levels of serum GM-CSF. To define the critical role of these hematopoietic-stimulating factors in HPC peripheral mobilization, direct administration of G-CSF at a dose of 300 microg/kg/day or 150 microg/kg/day was applied and found to significantly stimulate GM-CFU formation and increase c-kit+ cells in the peripheral mononuclear cells. More importantly, 75-mGy x-rays plus 150 microg/kg/day G-CSF (LDR/150-G-CSF) produced a similar effect to that of 300 microg/kg/day G-CSF alone. Furthermore, the capability of LDR-mobilized donor HPCs to repopulate blood cells was confirmed in lethally irradiated recipient mice by counting peripheral WBC and CFUs-S. These results suggest that LDR induces hematopoietic hormesis, as demonstrated by HPC proliferation and peripheral mobilization, providing a

  13. Beta-blockade prevents hematopoietic progenitor cell suppression after hemorrhagic shock.

    PubMed

    Elhassan, Ihab O; Hannoush, Edward J; Sifri, Ziad C; Jones, Eyone; Alzate, Walter D; Rameshwar, Pranela; Livingston, David H; Mohr, Alicia M

    2011-08-01

    Severe injury is accompanied by sympathetic stimulation that induces bone marrow (BM) dysfunction by both suppression of hematopoietic progenitor cell (HPC) growth and loss of cells via HPC mobilization to the peripheral circulation and sites of injury. Previous work demonstrated that beta-blockade (BB) given prior to tissue injury both reduces HPC mobilization and restores HPC colony growth within the BM. This study examined the effect and timing of BB on BM function in a hemorrhagic shock (HS) model. Male Sprague-Dawley rats underwent HS via blood withdrawal, maintaining the mean arterial blood pressure at 30-40 mm Hg for 45 min, after which the extracted blood was reinfused. Propranolol (10 mg/kg) was given either prior to or immediately after HS. Blood pressure, heart rate, BM cellularity, and death were recorded. Bone marrow HPC growth was assessed by counting colony-forming unit-granulocyte-, erythrocyte-, monocyte-, megakaryocyte (CFU-GEMM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-erythroid (CFU-E) cells. Administration of BB prior to injury restored HPC growth to that of naïve animals (CFU-GEMM 59 ± 11 vs. 61 ± 4, BFU-E 68 ± 9 vs. 73 ± 3, and CFU-E 81 ± 35 vs. 78 ± 14 colonies/plate). Beta-blockade given after HS increased the growth of CFU-GEMM, BFU-E, and CFU-E significantly and improved BM cellularity compared with HS alone. The mortality rate was not increased in the groups receiving BB. Administration of propranolol either prior to injury or immediately after resuscitation significantly reduced post-shock BM suppression. After HS, BB may improve BM cellularity by decreasing HPC mobilization. Therefore, the early use of BB post-injury may play an important role in attenuating the BM dysfunction accompanying HS.

  14. Stromal Progenitor Cells in Mitigation of Non-Hematopoietic Radiation Injuries

    PubMed Central

    Kulkarni, Shilpa; Wang, Timothy C.; Guha, Chandan

    2016-01-01

    Purpose of review Therapeutic exposure to high doses of radiation can severely impair organ function due to ablation of stem cells. Normal tissue injury is a dose-limiting toxicity for radiation therapy (RT). Although advances in the delivery of high precision conformal RT has increased normal tissue sparing, mitigating and therapeutic strategies that could alleviate early and chronic radiation effects are urgently needed in order to deliver curative doses of RT, especially in abdominal, pelvic and thoracic malignancies. Radiation-induced gastrointestinal injury is also a major cause of lethality from accidental or intentional exposure to whole body irradiation in the case of nuclear accidents or terrorism. This review examines the therapeutic options for mitigation of non-hematopoietic radiation injuries. Recent findings We have developed stem cell based therapies for the mitigation of acute radiation syndrome (ARS) and radiation-induced gastrointestinal syndrome (RIGS). This is a promising option because of the robustness of standardized isolation and transplantation of stromal cells protocols, and their ability to support and replace radiation-damaged stem cells and stem cell niche. Stromal progenitor cells (SPC) represent a unique multipotent and heterogeneous cell population with regenerative, immunosuppressive, anti-inflammatory, and wound healing properties. SPC are also known to secrete various key cytokines and growth factors such as platelet derived growth factors (PDGF), keratinocyte growth factor (KGF), R-spondins (Rspo), and may consequently exert their regenerative effects via paracrine function. Additionally, secretory vesicles such as exosomes or microparticles can potentially be a cell-free alternative replacing the cell transplant in some cases. Summary This review highlights the beneficial effects of SPC on tissue regeneration with their ability to (a) target the irradiated tissues, (b) recruit host stromal cells, (c) regenerate endothelium and

  15. Relationship between spontaneous γH2AX foci formation and progenitor functions in circulating hematopoietic stem and progenitor cells among atomic-bomb survivors.

    PubMed

    Kajimura, Junko; Kyoizumi, Seishi; Kubo, Yoshiko; Misumi, Munechika; Yoshida, Kengo; Hayashi, Tomonori; Imai, Kazue; Ohishi, Waka; Nakachi, Kei; Weng, Nan-Ping; Young, Lauren F; Shieh, Jae-Hung; Moore, Malcolm A; van den Brink, Marcel R M; Kusunoki, Yoichiro

    2016-05-01

    Accumulated DNA damage in hematopoietic stem cells is a primary mechanism of aging-associated dysfunction in human hematopoiesis. About 70 years ago, atomic-bomb (A-bomb) radiation induced DNA damage and functional decreases in the hematopoietic system of A-bomb survivors in a radiation dose-dependent manner. The peripheral blood cell populations then recovered to a normal range, but accompanying cells derived from hematopoietic stem cells still remain that bear molecular changes possibly caused by past radiation exposure and aging. In the present study, we evaluated radiation-related changes in the frequency of phosphorylated (Ser-139) H2AX (γH2AX) foci formation in circulating CD34-positive/lineage marker-negative (CD34+Lin-) hematopoietic stem and progenitor cells (HSPCs) among 226Hiroshima A-bomb survivors. An association between the frequency of γH2AX foci formation in HSPCs and the radiation dose was observed, but the γH2AX foci frequency was not significantly elevated by past radiation. We found a negative correlation between the frequency of γH2AX foci formation and the length of granulocyte telomeres. A negative interaction effect between the radiation dose and the frequency of γH2AX foci was suggested in a proportion of a subset of HSPCs as assessed by the cobblestone area-forming cell assay (CAFC), indicating that the self-renewability of HSPCs may decrease in survivors who were exposed to a higher radiation dose and who had more DNA damage in their HSPCs. Thus, although many years after radiation exposure and with advancing age, the effect of DNA damage on the self-renewability of HSPCs may be modified by A-bomb radiation exposure. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Relationship between spontaneous γH2AX foci formation and progenitor functions in circulating hematopoietic stem and progenitor cells among atomic-bomb survivors

    PubMed Central

    Kajimura, Junko; Kyoizumi, Seishi; Kubo, Yoshiko; Misumi, Munechika; Yoshida, Kengo; Hayashi, Tomonori; Imai, Kazue; Ohishi, Waka; Nakachi, Kei; Weng, Nan-ping; Young, Lauren F.; Shieh, Jae-Hung; Moore, Malcolm A.; van den Brink, Marcel R.M.; Kusunoki, Yoichiro

    2016-01-01

    Accumulated DNA damage in hematopoietic stem cells is a primary mechanism of aging-associated dysfunction in human hematopoiesis. About 70 years ago, atomic-bomb (A-bomb) radiation induced DNA damage and functional decreases in the hematopoietic system of A-bomb survivors in a radiation dose-dependent manner. The peripheral blood cell populations then recovered to a normal range, but accompanying cells derived from hematopoietic stem cells still remain that bear molecular changes possibly caused by past radiation exposure and aging. In the present study, we evaluated radiation-related changes in the frequency of phosphorylated (Ser-139) H2AX (γH2AX) foci formation in circulating CD34-positive/lineage marker-negative (CD34 + Lin−) hematopoietic stem and progenitor cells (HSPCs) among 226Hiroshima A-bomb survivors. An association between the frequency of γH2AX foci formation in HSPCs and the radiation dose was observed, but the γH2AX foci frequency was not significantly elevated by past radiation. We found a negative correlation between the frequency of γH2AX foci formation and the length of granulocyte telomeres. A negative interaction effect between the radiation dose and the frequency of γH2AX foci was suggested in a proportion of a subset of HSPCs as assessed by the cobblestone area-forming cell assay (CAFC), indicating that the self-renewability of HSPCs may decrease in survivors who were exposed to a higher radiation dose and who had more DNA damage in their HSPCs. Thus, although many years after radiation exposure and with advancing age, the effect of DNA damage on the self-renewability of HSPCs may be modified by A-bomb radiation exposure. PMID:27169377

  17. Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture

    PubMed Central

    Schmal, Olga; Seifert, Jan; Schäffer, Tilman E.; Walter, Christina B.; Aicher, Wilhelm K.; Klein, Gerd

    2016-01-01

    Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard. PMID:26839560

  18. Distinct Brca1 Mutations Differentially Reduce Hematopoietic Stem Cell Function.

    PubMed

    Mgbemena, Victoria E; Signer, Robert A J; Wijayatunge, Ranjula; Laxson, Travis; Morrison, Sean J; Ross, Theodora S

    2017-01-24

    BRCA1 is a well-known DNA repair pathway component and a tissue-specific tumor suppressor. However, its role in hematopoiesis is uncertain. Here, we report that a cohort of patients heterozygous for BRCA1 mutations experienced more hematopoietic toxicity from chemotherapy than those with BRCA2 mutations. To test whether this reflects a requirement for BRCA1 in hematopoiesis, we generated mice with Brca1 mutations in hematopoietic cells. Mice homozygous for a null Brca1 mutation in the embryonic hematopoietic system (Vav1-iCre;Brca1 F22-24/F22-24 ) developed hematopoietic defects in early adulthood that included reduced hematopoietic stem cells (HSCs). Although mice homozygous for a huBRCA1 knockin allele (Brca1 BRCA1/BRCA1 ) were normal, mice with a mutant huBRCA1/5382insC allele and a null allele (Mx1-Cre;Brca1 F22-24/5382insC ) had severe hematopoietic defects marked by a complete loss of hematopoietic stem and progenitor cells. Our data show that Brca1 is necessary for HSC maintenance and normal hematopoiesis and that distinct mutations lead to different degrees of hematopoietic dysfunction. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. Related-to-receptor tyrosine kinase receptor regulates hematopoietic stem and progenitor sensitivity to myelosuppressive injury in mice.

    PubMed

    Povinelli, Benjamin J; Srivastava, Pragya; Nemeth, Michael J

    2015-03-01

    Maintaining a careful balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) is necessary for lifelong blood formation. Previously, we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a noncanonical Wnt ligand receptor termed 'related-to-receptor tyrosine kinase' (Ryk). Expression of Ryk on HSPCs in vivo is associated with a lower rate of proliferation, and, following treatment with fluorouracil (5-FU), the percentage of Ryk(+/high) HSPCs increased and the percentage of Ryk(-/low) HSPCs decreased. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. We found that Ryk expression on HSPCs is associated with lower rates of apoptosis following 5-FU and radiation. Transient inhibition of Ryk signaling in vivo resulted in increased hematopoietic-stem-cell proliferation and decreased hematopoietic-stem-cell function in bone marrow transplant assays. Furthermore, inhibition of Ryk signaling sensitized HSPCs to 5-FU treatment in association with increased levels of reactive oxygen species. Together, these results demonstrated an association between Ryk expression and survival of HSPCs following suppressive injury. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  20. Notch2 blockade enhances hematopoietic stem cell mobilization and homing.

    PubMed

    Wang, Weihuan; Yu, Shuiliang; Myers, Jay; Wang, Yiwei; Xin, William W; Albakri, Marwah; Xin, Alison W; Li, Ming; Huang, Alex Y; Xin, Wei; Siebel, Christian W; Lazarus, Hillard M; Zhou, Lan

    2017-10-01

    Despite use of newer approaches, some patients being considered for autologous hematopoietic cell transplantation (HCT) may only mobilize limited numbers of hematopoietic progenitor cells (HPCs) into blood, precluding use of the procedure, or being placed at increased risk of complications due to slow hematopoietic reconstitution. Developing more efficacious HPC mobilization regimens and strategies may enhance the mobilization process and improve patient outcome. Although Notch signaling is not essential for homeostasis of adult hematopoietic stem cells (HSCs), Notch-ligand adhesive interaction maintains HSC quiescence and niche retention. Using Notch receptor blocking antibodies, we report that Notch2 blockade, but not Notch1 blockade, sensitizes hematopoietic stem cells and progenitors (HSPCs) to mobilization stimuli and leads to enhanced egress from marrow to the periphery. Notch2 blockade leads to transient myeloid progenitor expansion without affecting HSC homeostasis and self-renewal. We show that transient Notch2 blockade or Notch2-loss in mice lacking Notch2 receptor lead to decreased CXCR4 expression by HSC but increased cell cycling with CXCR4 transcription being directly regulated by the Notch transcriptional protein RBPJ. In addition, we found that Notch2-blocked or Notch2-deficient marrow HSPCs show an increased homing to the marrow, while mobilized Notch2-blocked, but not Notch2-deficient stem cells and progenitors, displayed a competitive repopulating advantage and enhanced hematopoietic reconstitution. These findings suggest that blocking Notch2 combined with the current clinical regimen may further enhance HPC mobilization and improve engraftment during HCT. Copyright© 2017 Ferrata Storti Foundation.

  1. Open the gates: vascular neurocrine signaling mobilizes hematopoietic stem and progenitor cells.

    PubMed

    Itkin, Tomer; Gómez-Salinero, Jesús María; Rafii, Shahin

    2017-12-01

    Mobilization of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) into the peripheral blood is a complex process that is enhanced dramatically under stress-induced conditions. A better understanding of how the mobilization process is regulated will likely facilitate the development of improved clinical protocols for stem cell harvesting and transplantation. In this issue of the JCI, Singh et al. (1) showed that the truncated cleaved form of neurotransmitter neuropeptide Y (NPY) actively promotes a breach of BM vascular sinusoidal portals, thereby augmenting HSPC trafficking to the circulation. The authors report a previously unrecognized axis, in which expression of the enzyme dipeptidylpeptidase-4 (DPP4)/CD26 by endothelial cells activates NPY-mediated signaling by increasing the bioavailability of the truncated form of NPY. These findings underscore the importance of and urgency to develop pharmacological therapies that target the vasculature and regulate diverse aspects of hematopoiesis, such as HSPC trafficking, in steady-state and stress-induced conditions.

  2. Are hematopoietic stem cells involved in hepatocarcinogenesis?

    PubMed

    Facciorusso, Antonio; Antonino, Matteo; Del Prete, Valentina; Neve, Viviana; Scavo, Maria Principia; Barone, Michele

    2014-08-01

    THE LIVER HAS THREE CELL LINEAGES ABLE TO PROLIFERATE AFTER A HEPATIC INJURY: the mature hepatocyte, the ductular "bipolar" progenitor cell termed "oval cell" and the putative periductular stem cell. Hepatocytes can only produce other hepatocytes whereas ductular progenitor cells are considerate bipolar since they can give rise to biliary cells or hepatocytes. Periductular stem cells are rare in the liver, have a very long proliferation potential and may be multipotent, being this aspect still under investigation. They originate in the bone marrow since their progeny express genetic markers of donor hematopoietic cells after bone marrow transplantation. Since the liver is the hematopoietic organ of the fetus, it is possible that hematopoietic stem cells may reside in the liver of the adult. This assumption is proved by the finding that oval cells express hematopoietic markers like CD34, CD45, CD 109, Thy-1, c-kit, and others, which are also expressed by bone marrow-derived hematopoietic stem cells (BMSCs). Few and discordant studies have evaluated the role of BMSC in hepatocarcinogenesis so far and further studies in vitro and in vivo are warranted in order to definitively clarify such an issue.

  3. Are hematopoietic stem cells involved in hepatocarcinogenesis?

    PubMed Central

    Antonino, Matteo; Del Prete, Valentina; Neve, Viviana; Scavo, Maria Principia; Barone, Michele

    2014-01-01

    The liver has three cell lineages able to proliferate after a hepatic injury: the mature hepatocyte, the ductular “bipolar” progenitor cell termed “oval cell” and the putative periductular stem cell. Hepatocytes can only produce other hepatocytes whereas ductular progenitor cells are considerate bipolar since they can give rise to biliary cells or hepatocytes. Periductular stem cells are rare in the liver, have a very long proliferation potential and may be multipotent, being this aspect still under investigation. They originate in the bone marrow since their progeny express genetic markers of donor hematopoietic cells after bone marrow transplantation. Since the liver is the hematopoietic organ of the fetus, it is possible that hematopoietic stem cells may reside in the liver of the adult. This assumption is proved by the finding that oval cells express hematopoietic markers like CD34, CD45, CD 109, Thy-1, c-kit, and others, which are also expressed by bone marrow-derived hematopoietic stem cells (BMSCs). Few and discordant studies have evaluated the role of BMSC in hepatocarcinogenesis so far and further studies in vitro and in vivo are warranted in order to definitively clarify such an issue. PMID:25202697

  4. Hhex Regulates Hematopoietic Stem Cell Self-Renewal and Stress Hematopoiesis via Repression of Cdkn2a.

    PubMed

    Jackson, Jacob T; Shields, Benjamin J; Shi, Wei; Di Rago, Ladina; Metcalf, Donald; Nicola, Nicos A; McCormack, Matthew P

    2017-08-01

    The hematopoietically expressed homeobox transcription factor (Hhex) is important for the maturation of definitive hematopoietic progenitors and B-cells during development. We have recently shown that in adult hematopoiesis, Hhex is dispensable for maintenance of hematopoietic stem cells (HSCs) and myeloid lineages but essential for the commitment of common lymphoid progenitors (CLPs) to lymphoid lineages. Here, we show that during serial bone marrow transplantation, Hhex-deleted HSCs are progressively lost, revealing an intrinsic defect in HSC self-renewal. Moreover, Hhex-deleted mice show markedly impaired hematopoietic recovery following myeloablation, due to a failure of progenitor expansion. In vitro, Hhex-null blast colonies were incapable of replating, implying a specific requirement for Hhex in immature progenitors. Transcriptome analysis of Hhex-null Lin - Sca + Kit + cells showed that Hhex deletion leads to derepression of polycomb repressive complex 2 (PRC2) and PRC1 target genes, including the Cdkn2a locus encoding the tumor suppressors p16 Ink 4 a and p19 Arf . Indeed, loss of Cdkn2a restored the capacity of Hhex-null blast colonies to generate myeloid progenitors in vitro, as well as hematopoietic reconstitution following myeloablation in vivo. Thus, HSCs require Hhex to promote PRC2-mediated Cdkn2a repression to enable continued self-renewal and response to hematopoietic stress. Stem Cells 2017;35:1948-1957. © 2017 AlphaMed Press.

  5. The ability of multipotent mesenchymal stromal cells from the bone marrow of patients with leukemia to maintain normal hematopoietic progenitor cells.

    PubMed

    Sorokina, Tamara; Shipounova, Irina; Bigildeev, Alexey; Petinati, Nataliya; Drize, Nina; Turkina, Anna; Chelysheva, Ekaterina; Shukhov, Oleg; Kuzmina, Larisa; Parovichnikova, Elena; Savchenko, Valery

    2016-09-01

    The development of leukemia impairs normal hematopoiesis and marrow stromal microenvironment. The aim of the investigation was to study the ability of multipotent mesenchymal stromal cells (MSCs) derived from the bone marrow of patients with leukemia to maintain normal hematopoietic progenitor cells. MSCs were obtained from the bone marrow of 14 patients with acute lymphoblastic (ALL), 25 with myeloid (AML), and 15 with chronic myeloid (CML) leukemia. As a control, MSCs from 22 healthy donors were used. The incidence of cobblestone area forming cells (CAFC 7-8 d) in the bone marrow of healthy donor cultivated on the supportive layer of patients MSCs was measured. The ability of MSCs from AML and ALL patients at the moment of diagnosis to maintain normal CAFC was significantly decreased when compared to donors. After chemotherapy, the restoration of ALL patients' MSCs functions was slower than that of AML. CML MSCs maintained CAFC better than donors' at the moment of diagnosis and this ability increased with treatment. The ability of patients' MSCs to maintain normal hematopoietic progenitor cells was shown to change in comparison with MSCs from healthy donors and depended on nosology. During treatment, the functional capacity of patients' MSCs had been partially restored. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Do autologous peripheral blood cell transplants provide more than hematopoietic recovery?

    PubMed

    Kessinger, A

    1995-07-01

    Bone marrow damage caused by myeloablative radiation therapy and/or chemotherapy can be repaired by intravenously infusing viable stem/progenitor cells collected from either blood or bone marrow. The hematopoietic graft product contains both stem/progenitor cells and populations of hematopoietic and nonhematopoietic (accessory) cells. The frequency of accessory cell types varies with the source of the graft product; marrow or blood. Reinfusion of these accessory cells causes effects other than the hematopoietic restoration provided by the stem/progenitor cells such as graft versus host disease and graft versus leukemia effect after allogeneic transplants. Effects of infused accessory cells in the autologous setting are less well studied and could provide ancillary advantages and/or disadvantages to the patient. Do these additional effects actually occur, and, if they do, are they more likely to appear following peripheral blood cell transplants (PBCT) or after autologous bone marrow transplants (AMBT)? Preliminary data are beginning to accumulate which suggest that reinfusion of occult tumor cells is less likely with PBCT, that immune reconstitution is different depending on the source of the autograft and that, for certain diseases, patient event-free survival following PBCT rather than ABMT may be better. However, infusion of occult tumor cells may result in re-establishment of the malignancy. If the accessory cells (including potential occult tumor cells) are eliminated from the product before transplant, will the patient have a better clinical outcome, or would benefits provided by infused accessory cells outweigh the risks of infused occult tumor cells? These controversial issues are in the very early stages of investigation.

  7. FGF7 supports hematopoietic stem and progenitor cells and niche-dependent myeloblastoma cells via autocrine action on bone marrow stromal cells in vitro

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ishino, Ruri; Minami, Kaori; Tanaka, Satowa

    2013-10-11

    Highlights: •FGF7 is downregulated in MED1-deficient mesenchymal cells. •FGF7 produced by mesenchymal stromal cells is a novel hematopoietic niche molecule. •FGF7 supports hematopoietic progenitor cells and niche-dependent leukemia cells. •FGF7 activates FGFR2IIIb of bone marrow stromal cells in an autocrine manner. •FGF7 indirectly acts on hematopoietic cells lacking FGFR2IIIb via stromal cells. -- Abstract: FGF1 and FGF2 support hematopoietic stem and progenitor cells (HSPCs) under stress conditions. In this study, we show that fibroblast growth factor (FGF7) may be a novel niche factor for HSPC support and leukemic growth. FGF7 expression was attenuated in mouse embryonic fibroblasts (MEFs) deficient formore » the MED1 subunit of the Mediator transcriptional coregulator complex. When normal mouse bone marrow (BM) cells were cocultured with Med1{sup +/+} MEFs or BM stromal cells in the presence of anti-FGF7 antibody, the growth of BM cells and the number of long-time culture-initiating cells (LTC-ICs) decreased significantly. Anti-FGF7 antibody also attenuated the proliferation and cobblestone formation of MB1 stromal cell-dependent myeloblastoma cells. The addition of recombinant FGF7 to the coculture of BM cells and Med1{sup −/−} MEFs increased BM cells and LTC-ICs. FGF7 and its cognate receptor, FGFR2IIIb, were undetectable in BM cells, but MEFs and BM stromal cells expressed both. FGF7 activated downstream targets of FGFR2IIIb in Med1{sup +/+} and Med1{sup −/−} MEFs and BM stromal cells. Taken together, we propose that FGF7 supports HSPCs and leukemia-initiating cells indirectly via FGFR2IIIb expressed on stromal cells.« less

  8. Multiple prethymic defects underlie age-related loss of T progenitor competence

    PubMed Central

    Zediak, Valerie P.; Maillard, Ivan

    2007-01-01

    Aging in mice and humans is characterized by declining T-lymphocyte production in the thymus, yet it is unclear whether aging impacts the T-lineage potential of hematopoietic progenitors. Although alterations in the lymphoid progenitor content of aged mouse bone marrow (BM) have been described, irradiation-reconstitution experiments have failed to reveal defects in T-lineage potential of BM hematopoietic progenitors or purified hematopoietic stem cells (HSCs) from aged mice. Here, we assessed T-progenitor potential in unmanipulated recipient mice without conditioning irradiation. T-progenitor potential was reduced in aged BM compared with young BM, and this reduction was apparent at the earliest stages of intrathymic differentiation. Further, enriched populations of aged HSCs or multipotent progenitors (MPPs) gave rise to fewer T-lineage cells than their young counterparts. Whereas the T-precursor frequency within the MPP pool was unchanged, there was a 4-fold decline in T-precursor frequency within the HSC pool. In addition, among the T-competent HSC clones, there were fewer highly proliferative clones in the aged HSC pool than in the young HSC pool. These results identify T-compromised aged HSCs and define the nature and cellular sites of prethymic, age-related defects in T-lineage differentiation potential. PMID:17456721

  9. CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo.

    PubMed

    Sebastian, Nadia T; Zaikos, Thomas D; Terry, Valeri; Taschuk, Frances; McNamara, Lucy A; Onafuwa-Nuga, Adewunmi; Yucha, Ryan; Signer, Robert A J; Riddell, James; Bixby, Dale; Markowitz, Norman; Morrison, Sean J; Collins, Kathleen L

    2017-07-01

    Latent HIV infection of long-lived cells is a barrier to viral clearance. Hematopoietic stem and progenitor cells are a heterogeneous population of cells, some of which are long-lived. CXCR4-tropic HIVs infect a broad range of HSPC subtypes, including hematopoietic stem cells, which are multi-potent and long-lived. However, CCR5-tropic HIV infection is limited to more differentiated progenitor cells with life spans that are less well understood. Consistent with emerging data that restricted progenitor cells can be long-lived, we detected persistent HIV in restricted HSPC populations from optimally treated people. Further, genotypic and phenotypic analysis of amplified env alleles from donor samples indicated that both CXCR4- and CCR5-tropic viruses persisted in HSPCs. RNA profiling confirmed expression of HIV receptor RNA in a pattern that was consistent with in vitro and in vivo results. In addition, we characterized a CD4high HSPC sub-population that was preferentially targeted by a variety of CXCR4- and CCR5-tropic HIVs in vitro. Finally, we present strong evidence that HIV proviral genomes of both tropisms can be transmitted to CD4-negative daughter cells of multiple lineages in vivo. In some cases, the transmitted proviral genomes contained signature deletions that inactivated the virus, eliminating the possibility that coincidental infection explains the results. These data support a model in which both stem and non-stem cell progenitors serve as persistent reservoirs for CXCR4- and CCR5-tropic HIV proviral genomes that can be passed to daughter cells.

  10. Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells

    PubMed Central

    Chan, Charles K. F.; Lindau, Paul; Jiang, Wen; Chen, James Y.; Zhang, Lillian F.; Chen, Ching-Cheng; Seita, Jun; Sahoo, Debashis; Kim, Jae-Beom; Lee, Andrew; Park, Sujin; Nag, Divya; Gong, Yongquan; Kulkarni, Subhash; Luppen, Cynthia A.; Theologis, Alexander A.; Wan, Derrick C.; DeBoer, Anthony; Seo, Eun Young; Vincent-Tompkins, Justin D.; Loh, Kyle; Walmsley, Graham G.; Kraft, Daniel L.; Wu, Joseph C.; Longaker, Michael T.; Weissman, Irving L.

    2013-01-01

    Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells. PMID:23858471

  11. Mapping gene expression patterns during myeloid differentiation using the EML hematopoietic progenitor cell line.

    PubMed

    Du, Yang; Campbell, Janee L; Nalbant, Demet; Youn, Hyewon; Bass, Ann C Hughes; Cobos, Everardo; Tsai, Schickwann; Keller, Jonathan R; Williams, Simon C

    2002-07-01

    The detailed examination of the molecular events that control the early stages of myeloid differentiation has been hampered by the relative scarcity of hematopoietic stem cells and the lack of suitable cell line models. In this study, we examined the expression of several myeloid and nonmyeloid genes in the murine EML hematopoietic stem cell line. Expression patterns for 19 different genes were examined by Northern blotting and RT-PCR in RNA samples from EML, a variety of other immortalized cell lines, and purified murine hematopoietic stem cells. Representational difference analysis (RDA) was performed to identify differentially expressed genes in EML. Expression patterns of genes encoding transcription factors (four members of the C/EBP family, GATA-1, GATA-2, PU.1, CBFbeta, SCL, and c-myb) in EML were examined and were consistent with the proposed functions of these proteins in hematopoietic differentiation. Expression levels of three markers of terminal myeloid differentiation (neutrophil elastase, proteinase 3, and Mac-1) were highest in EML cells at the later stages of differentiation. In a search for genes that were differentially expressed in EML cells during myeloid differentiation, six cDNAs were isolated. These included three known genes (lysozyme, histidine decarboxylase, and tryptophan hydroxylase) and three novel genes. Expression patterns of known genes in differentiating EML cells accurately reflected their expected expression patterns based on previous studies. The identification of three novel genes, two of which encode proteins that may act as regulators of hematopoietic differentiation, suggests that EML is a useful model system for the molecular analysis of hematopoietic differentiation.

  12. Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos.

    PubMed

    Berrun, A C; Stachura, D L

    2017-11-30

    Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

  13. Accelerated hematopoietic recovery with angiotensin-(1-7) after total body radiation.

    PubMed

    Rodgers, Kathleen E; Espinoza, Theresa; Roda, Norma; Meeks, Christopher J; Hill, Colin; Louie, Stan G; Dizerega, Gere S

    2012-06-01

    Angiotensin (1-7) [A(1-7)] is a component of the renin angiotensin system (RAS) that stimulates hematopoietic recovery after myelosuppression. In a Phase I/IIa clinical trial, thrombocytopenia after chemotherapy was reduced by A(1-7). In this study, the ability of A(1-7) to improve recovery after total body irradiation (TBI) is shown with specific attention to radiation-induced hematopoietic injury. Mice were exposed to TBI (doses of 2-7 Gray [Gy]) of cesium 137 gamma rays, followed by treatment with A(1-7), typical doses were 100-1000 μg/kg given once or once daily for a specified number of days depending on the study. Animals are injected subcutaneously via the nape of the neck with 0.1 ml drug in saline. The recovery of blood and bone marrow cells was determined. Effects of TBI and A(1-7) on survival and bleeding time was also evaluated. Daily administration of A(1-7) after radiation exposure improved survival (from 60% to 92-97%) and reduced bleeding time at day 30 after TBI. Further, A(1-7) increased early mixed progenitors (3- to 5-fold), megakaryocyte (2- to 3-fold), myeloid (3- to 6-fold) and erythroid (2- to 5-fold) progenitors in the bone marrow and reduced radiation-induced thrombocytopenia (RIT) (up to 2-fold). Reduction in the number of treatments to 3 per week also improved bone marrow recovery and reduced RIT. As emergency responder and healthcare systems in case of nuclear accident or/and terrorist attack may be overwhelmed, the consequence of delayed initiation of treatment was ascertained. Treatment with A(1-7) can be delayed up to 5 days and still be effective in the reduction of RIT or acceleration of bone marrow recovery. The data presented in this paper indicate that A(1-7) reduces the consequences of critical radiation exposure and can be initiated well after initial exposure with maximal effects on early responding hematopoietic progenitors when treatment is initiated 2 days after exposure and 5 days after exposure for the later responding

  14. Pleiotrophin mediates hematopoietic regeneration via activation of RAS

    PubMed Central

    Himburg, Heather A.; Yan, Xiao; Doan, Phuong L.; Quarmyne, Mamle; Micewicz, Eva; McBride, William; Chao, Nelson J.; Slamon, Dennis J.; Chute, John P.

    2014-01-01

    Hematopoietic stem cells (HSCs) are highly susceptible to ionizing radiation–mediated death via induction of ROS, DNA double-strand breaks, and apoptotic pathways. The development of therapeutics capable of mitigating ionizing radiation–induced hematopoietic toxicity could benefit both victims of acute radiation sickness and patients undergoing hematopoietic cell transplantation. Unfortunately, therapies capable of accelerating hematopoietic reconstitution following lethal radiation exposure have remained elusive. Here, we found that systemic administration of pleiotrophin (PTN), a protein that is secreted by BM-derived endothelial cells, substantially increased the survival of mice following radiation exposure and after myeloablative BM transplantation. In both models, PTN increased survival by accelerating the recovery of BM hematopoietic stem and progenitor cells in vivo. PTN treatment promoted HSC regeneration via activation of the RAS pathway in mice that expressed protein tyrosine phosphatase receptor-zeta (PTPRZ), whereas PTN treatment did not induce RAS signaling in PTPRZ-deficient mice, suggesting that PTN-mediated activation of RAS was dependent upon signaling through PTPRZ. PTN strongly inhibited HSC cycling following irradiation, whereas RAS inhibition abrogated PTN-mediated induction of HSC quiescence, blocked PTN-mediated recovery of hematopoietic stem and progenitor cells, and abolished PTN-mediated survival of irradiated mice. These studies demonstrate the therapeutic potential of PTN to improve survival after myeloablation and suggest that PTN-mediated hematopoietic regeneration occurs in a RAS-dependent manner. PMID:25250571

  15. Pleiotrophin mediates hematopoietic regeneration via activation of RAS.

    PubMed

    Himburg, Heather A; Yan, Xiao; Doan, Phuong L; Quarmyne, Mamle; Micewicz, Eva; McBride, William; Chao, Nelson J; Slamon, Dennis J; Chute, John P

    2014-11-01

    Hematopoietic stem cells (HSCs) are highly susceptible to ionizing radiation-mediated death via induction of ROS, DNA double-strand breaks, and apoptotic pathways. The development of therapeutics capable of mitigating ionizing radiation-induced hematopoietic toxicity could benefit both victims of acute radiation sickness and patients undergoing hematopoietic cell transplantation. Unfortunately, therapies capable of accelerating hematopoietic reconstitution following lethal radiation exposure have remained elusive. Here, we found that systemic administration of pleiotrophin (PTN), a protein that is secreted by BM-derived endothelial cells, substantially increased the survival of mice following radiation exposure and after myeloablative BM transplantation. In both models, PTN increased survival by accelerating the recovery of BM hematopoietic stem and progenitor cells in vivo. PTN treatment promoted HSC regeneration via activation of the RAS pathway in mice that expressed protein tyrosine phosphatase receptor-zeta (PTPRZ), whereas PTN treatment did not induce RAS signaling in PTPRZ-deficient mice, suggesting that PTN-mediated activation of RAS was dependent upon signaling through PTPRZ. PTN strongly inhibited HSC cycling following irradiation, whereas RAS inhibition abrogated PTN-mediated induction of HSC quiescence, blocked PTN-mediated recovery of hematopoietic stem and progenitor cells, and abolished PTN-mediated survival of irradiated mice. These studies demonstrate the therapeutic potential of PTN to improve survival after myeloablation and suggest that PTN-mediated hematopoietic regeneration occurs in a RAS-dependent manner.

  16. Regulated Apoptosis of Genetically-Modified Hematopoietic Stem and Progenitor Cells via an Inducible Caspase-9 Suicide Gene in Rhesus Macaques

    PubMed Central

    Barese, Cecilia N.; Felizardo, Tania C.; Sellers, Stephanie E.; Keyvanfar, Keyvan; Di Stasi, Antonio; Metzger, Mark E.; Krouse, Allen E.; Donahue, Robert E.; Spencer, David M.; Dunbar, Cynthia E.

    2014-01-01

    The high risk of insertional oncogenesis reported in clinical trials utilizing integrating retroviral vectors to genetically-modify hematopoietic stem and progenitor cells (HSPC) requires the development of safety strategies to minimize risks associated with novel cell and gene therapies. The ability to ablate genetically modified cells in vivo is desirable, should an abnormal clone emerge. Inclusion of “suicide genes” in vectors to facilitate targeted ablation of vector-containing abnormal clones in vivo is one potential safety approach. We tested whether the inclusion of the “inducible Caspase-9” (iCasp9) suicide gene in a gamma-retroviral vector facilitated efficient elimination of vector-containing HSPCs and their hematopoietic progeny in vivo long-term, in an autologous non-human primate transplantation model. Following stable engraftment of iCasp9 expressing hematopoietic cells in rhesus macaques, administration of AP1903, a chemical inducer of dimerization able to activate iCasp9, specifically eliminated vector-containing cells in all hematopoietic lineages long-term, suggesting activity at the HSPC level. Between 75–94% of vector-containing cells were eliminated by well-tolerated AP1903 dosing, but lack of complete ablation was linked to lower iCasp9 expression in residual cells. Further investigation of resistance mechanisms demonstrated upregulation of Bcl-2 in hematopoietic cell lines transduced with the vector and resistant to AP1903 ablation. These results demonstrate both the potential and the limitations of safety approaches utilizing iCasp9 to HSPC-targeted gene therapy settings, in a model with great relevance to clinical development. PMID:25330775

  17. Whole body proton irradiation causes acute damage to bone marrow hematopoietic progenitor and stem cells in mice.

    PubMed

    Chang, Jianhui; Wang, Yingying; Pathak, Rupak; Sridharan, Vijayalakshmi; Jones, Tamako; Mao, Xiao Wen; Nelson, Gregory; Boerma, Marjan; Hauer-Jensen, Martin; Zhou, Daohong; Shao, Lijian

    2017-12-01

    Exposure to proton irradiation during missions in deep space can lead to bone marrow injury. The acute effects of proton irradiation on hematopoietic stem and progenitor cells remain undefined and thus were investigated. We exposed male C57BL/6 mice to 0.5 and 1.0 Gy proton total body irradiation (proton-TBI, 150 MeV) and examined changes in peripheral blood cells and bone marrow (BM) progenitors and LSK cells 2 weeks after exposure. 1.0 Gy proton-TBI significantly reduced the numbers of peripheral blood cells compared to 0.5 Gy proton-TBI and unirradiated animals, while the numbers of peripheral blood cell counts were comparable between 0.5 Gy proton-TBI and unirradiated mice. The frequencies and numbers of LSK cells and CMPs in BM of 0.5 and 1.0 Gy irradiated mice were decreased in comparison to those of normal controls. LSK cells and CMPs and their progeny exhibited a radiation-induced impairment in clonogenic function. Exposure to 1.0 Gy increased cellular apoptosis but not the production of reactive oxygen species (ROS) in CMPs two weeks after irradiation. LSK cells from irradiated mice exhibited an increase in ROS production and apoptosis. Exposure to proton-TBI can induce acute damage to BM progenitors and LSK cells.

  18. AML1/ETO accelerates cell migration and impairs cell-to-cell adhesion and homing of hematopoietic stem/progenitor cells

    PubMed Central

    Saia, Marco; Termanini, Alberto; Rizzi, Nicoletta; Mazza, Massimiliano; Barbieri, Elisa; Valli, Debora; Ciana, Paolo; Gruszka, Alicja M.; Alcalay, Myriam

    2016-01-01

    The AML1/ETO fusion protein found in acute myeloid leukemias functions as a transcriptional regulator by recruiting co-repressor complexes to its DNA binding site. In order to extend the understanding of its role in preleukemia, we expressed AML1/ETO in a murine immortalized pluripotent hematopoietic stem/progenitor cell line, EML C1, and found that genes involved in functions such as cell-to-cell adhesion and cell motility were among the most significantly regulated as determined by RNA sequencing. In functional assays, AML1/ETO-expressing cells showed a decrease in adhesion to stromal cells, an increase of cell migration rate in vitro, and displayed an impairment in homing and engraftment in vivo upon transplantation into recipient mice. Our results suggest that AML1/ETO expression determines a more mobile and less adherent phenotype in preleukemic cells, therefore altering the interaction with the hematopoietic niche, potentially leading to the migration across the bone marrow barrier and to disease progression. PMID:27713544

  19. Modulation of hematopoietic progenitor cell fate in vitro by varying collagen oligomer matrix stiffness in the presence or absence of osteoblasts.

    PubMed

    Chitteti, Brahmananda Reddy; Kacena, Melissa A; Voytik-Harbin, Sherry L; Srour, Edward F

    2015-10-01

    To recreate the in vivo hematopoietic cell microenvironment or niche and to study the impact of extracellular matrix (ECM) biophysical properties on hematopoietic progenitor cell (HPC) proliferation and function, mouse bone-marrow derived HPC (Lin-Sca1+cKit+/(LSK) were cultured within three-dimensional (3D) type I collagen oligomer matrices. To generate a more physiologic milieu, 3D cultures were established in both the presence and absence of calvariae-derived osteoblasts (OB). Collagen oligomers were polymerized at varying concentration to give rise to matrices of different fibril densities and therefore matrix stiffness (shear storage modulus, 50-800 Pa). Decreased proliferation and increased clonogenicity of LSK cells was associated with increase of matrix stiffness regardless of whether OB were present or absent from the 3D culture system. Also, regardless of whether OB were or were not added to the 3D co-culture system, LSK within 800 Pa collagen oligomer matrices maintained the highest percentage of Lin-Sca1+ cells as well as higher percentage of cells in quiescent state (G0/G1) compared to 50 Pa or 200Pa matrices. Collectively, these data illustrate that biophysical features of collagen oligomer matrices, specifically fibril density-induced modulation of matrix stiffness, provide important guidance cues in terms of LSK expansion and differentiation and therefore maintenance of progenitor cell function. Copyright © 2015. Published by Elsevier B.V.

  20. Clinical-scale validation of a new efficient procedure for cryopreservation of ex vivo expanded cord blood hematopoietic stem and progenitor cells.

    PubMed

    Duchez, Pascale; Rodriguez, Laura; Chevaleyre, Jean; De La Grange, Philippe Brunet; Ivanovic, Zoran

    2016-12-01

    Survival of ex vivo expanded hematopoietic stem cells (HSC) and progenitor cells is low with the standard cryopreservation procedure. We recently showed that the efficiency of cryopreservation of these cells may be greatly enhanced by adding a serum-free xeno-free culture medium (HP01 Macopharma), which improves the antioxidant and biochemical properties of the cryopreservation solution. Here we present the clinical-scale validation of this cryopreservation procedure. The hematopoietic cells expanded in clinical-scale cultures were cryopreserved applying the new HP01-based procedure. The viability, apoptosis rate and number of functional committed progenitors (methyl-cellulose colony forming cell test), short-term repopulating HSCs (primary recipient NSG mice) and long-term HSCs (secondary recipient NSG mice) were tested before and after thawing. The efficiency of clinical-scale procedure reproduced the efficiency of cryopreservation obtained earlier in miniature sample experiments. Furthermore, the full preservation of short- and long-term HSCs was obtained in clinical scale conditions. Because the results obtained in clinical-scale volume are comparable to our earlier results in miniature-scale cultures, the clinical-scale procedure should be considered validated. It allows cryopreservation of the whole ex vivo expanded culture content, conserving full short- and long-term HSC activity. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  1. Early patterning and specification of cardiac progenitors in gastrulating mesoderm

    PubMed Central

    Devine, W Patrick; Wythe, Joshua D; George, Matthew; Koshiba-Takeuchi, Kazuko; Bruneau, Benoit G

    2014-01-01

    Mammalian heart development requires precise allocation of cardiac progenitors. The existence of a multipotent progenitor for all anatomic and cellular components of the heart has been predicted but its identity and contribution to the two cardiac progenitor ‘fields’ has remained undefined. Here we show, using clonal genetic fate mapping, that Mesp1+ cells in gastrulating mesoderm are rapidly specified into committed cardiac precursors fated for distinct anatomic regions of the heart. We identify Smarcd3 as a marker of early specified cardiac precursors and identify within these precursors a compartment boundary at the future junction of the left and right ventricles that arises prior to morphogenesis. Our studies define the timing and hierarchy of cardiac progenitor specification and demonstrate that the cellular and anatomical fate of mesoderm-derived cardiac cells is specified very early. These findings will be important to understand the basis of congenital heart defects and to derive cardiac regeneration strategies. DOI: http://dx.doi.org/10.7554/eLife.03848.001 PMID:25296024

  2. ROS-mediated iron overload injures the hematopoiesis of bone marrow by damaging hematopoietic stem/progenitor cells in mice

    PubMed Central

    Chai, Xiao; Li, Deguan; Cao, Xiaoli; Zhang, Yuchen; Mu, Juan; Lu, Wenyi; Xiao, Xia; Li, Chengcheng; Meng, Juanxia; Chen, Jie; Li, Qing; Wang, Jishi; Meng, Aimin; Zhao, Mingfeng

    2015-01-01

    Iron overload, caused by hereditary hemochromatosis or repeated blood transfusions in some diseases, such as beta thalassemia, bone marrow failure and myelodysplastic syndrome, can significantly induce injured bone marrow (BM) function as well as parenchyma organ dysfunctions. However, the effect of iron overload and its mechanism remain elusive. In this study, we investigated the effects of iron overload on the hematopoietic stem and progenitor cells (HSPCs) from a mouse model. Our results showed that iron overload markedly decreased the ratio and clonogenic function of murine HSPCs by the elevation of reactive oxygen species (ROS). This finding is supported by the results of NAC or DFX treatment, which reduced ROS level by inhibiting NOX4 and p38MAPK and improved the long-term and multi-lineage engrafment of iron overload HSCs after transplantation. Therefore, all of these data demonstrate that iron overload injures the hematopoiesis of BM by enhancing ROS through NOX4 and p38MAPK. This will be helpful for the treatment of iron overload in patients with hematopoietic dysfunction. PMID:25970748

  3. In vitro and in vivo assessment of direct effects of simulated solar and galactic cosmic radiation on human hematopoietic stem/progenitor cells.

    PubMed

    Rodman, C; Almeida-Porada, G; George, S K; Moon, J; Soker, S; Pardee, T; Beaty, M; Guida, P; Sajuthi, S P; Langefeld, C D; Walker, S J; Wilson, P F; Porada, C D

    2017-06-01

    Future deep space missions to Mars and near-Earth asteroids will expose astronauts to chronic solar energetic particles (SEP) and galactic cosmic ray (GCR) radiation, and likely one or more solar particle events (SPEs). Given the inherent radiosensitivity of hematopoietic cells and short latency period of leukemias, space radiation-induced hematopoietic damage poses a particular threat to astronauts on extended missions. We show that exposing human hematopoietic stem/progenitor cells (HSC) to extended mission-relevant doses of accelerated high-energy protons and iron ions leads to the following: (1) introduces mutations that are frequently located within genes involved in hematopoiesis and are distinct from those induced by γ-radiation; (2) markedly reduces in vitro colony formation; (3) markedly alters engraftment and lineage commitment in vivo; and (4) leads to the development, in vivo, of what appears to be T-ALL. Sequential exposure to protons and iron ions (as typically occurs in deep space) proved far more deleterious to HSC genome integrity and function than either particle species alone. Our results represent a critical step for more accurately estimating risks to the human hematopoietic system from space radiation, identifying and better defining molecular mechanisms by which space radiation impairs hematopoiesis and induces leukemogenesis, as well as for developing appropriately targeted countermeasures.

  4. In vitro and in vivo assessment of direct effects of simulated solar and galactic cosmic radiation on human hematopoietic stem/progenitor cells

    DOE PAGES

    Rodman, C.; Almeida-Porada, G.; George, S. K.; ...

    2016-11-24

    Future deep space missions to Mars and near-Earth asteroids will expose astronauts to chronic solar energetic particles (SEP) and galactic cosmic ray (GCR) radiation,and likely one or more solar particle events (SPEs).Given the inherent radiosensitivity of hematopoietic cells and short latency period of leukemias, space radiation-induced hematopoietic damage poses a particular threat to astronauts on extended missions.We show that exposing human hematopoietic stem/progenitor cells(HSC) toextended mission-relevant doses of accelerated high-energyprotons andiron ions leads to the following: (1) introduces mutations that are frequently located within genes involved in hematopoiesis and are distinct from those induced by γ-radiation; (2) markedly reduces inmore » vitro colony formation; (3)markedly alters engraftment and lineage commitment in vivo; and (4) leads to the development, in vivo, ofwhat appears to be T-ALL. Sequential exposure to protons and iron ions (as typically occurs in deep space) proved far more deleterious to HSC genome integrity and function than either particle species alone.Our results represent a critical step for more accurately estimating risks to the human hematopoietic system from space radiation, identifying and better defining molecular mechanisms by which space radiation impairs hematopoiesis and induces leukemogenesis, as well as for developing appropriately targeted countermeasures.« less

  5. In vitro and in vivo assessment of direct effects of simulated solar and galactic cosmic radiation on human hematopoietic stem/progenitor cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rodman, C.; Almeida-Porada, G.; George, S. K.

    Future deep space missions to Mars and near-Earth asteroids will expose astronauts to chronic solar energetic particles (SEP) and galactic cosmic ray (GCR) radiation,and likely one or more solar particle events (SPEs).Given the inherent radiosensitivity of hematopoietic cells and short latency period of leukemias, space radiation-induced hematopoietic damage poses a particular threat to astronauts on extended missions.We show that exposing human hematopoietic stem/progenitor cells(HSC) toextended mission-relevant doses of accelerated high-energyprotons andiron ions leads to the following: (1) introduces mutations that are frequently located within genes involved in hematopoiesis and are distinct from those induced by γ-radiation; (2) markedly reduces inmore » vitro colony formation; (3)markedly alters engraftment and lineage commitment in vivo; and (4) leads to the development, in vivo, ofwhat appears to be T-ALL. Sequential exposure to protons and iron ions (as typically occurs in deep space) proved far more deleterious to HSC genome integrity and function than either particle species alone.Our results represent a critical step for more accurately estimating risks to the human hematopoietic system from space radiation, identifying and better defining molecular mechanisms by which space radiation impairs hematopoiesis and induces leukemogenesis, as well as for developing appropriately targeted countermeasures.« less

  6. Hematopoietic progenitor cells grow on 3T3 fibroblast monolayers that overexpress growth arrest-specific gene-6 (GAS6)

    PubMed Central

    Dormady, Shane P.; Zhang, Xin-Min; Basch, Ross S.

    2000-01-01

    Pluripotential hematopoietic stem cells grow in close association with bone marrow stromal cells, which play a critical role in sustaining hematopoiesis in long-term bone marrow cultures. The mechanisms through which stromal cells act to support pluripotential hematopoietic stem cells are largely unknown. This study demonstrates that growth arrest-specific gene-6 (GAS6) plays an important role in this process. GAS6 is a ligand for the Axl (Ufo/Ark), Sky (Dtk/Tyro3/Rse/Brt/Tif), and Mer (Eyk) family of tyrosine kinase receptors and binds to these receptors via tandem G domains at its C terminus. After translation, GAS6 moves to the lumen of the endoplasmic reticulum, where it is extensively γ-carboxylated. The carboxylation process is vitamin K dependent, and current evidence suggests that GAS6 must be γ-carboxylated to bind and activate any of the cognate tyrosine kinase receptors. Here, we show that expression of GAS6 is highly correlated with the capacity of bone marrow stromal cells to support hematopoiesis in culture. Nonsupportive stromal cell lines express little to no GAS6, whereas supportive cell lines express high levels of GAS6. Transfection of the cDNA encoding GAS6 into 3T3 fibroblasts is sufficient to render this previously nonsupportive cell line capable of supporting long-term hematopoietic cultures. 3T3 cells, genetically engineered to stably express GAS6 (GAS6-3T3), produce a stromal layer that supports the generation of colony-forming units in culture (CFU-c) for up to 6 wk. Hematopoietic support by genetically engineered 3T3 is not vitamin K dependent, and soluble recombinant GAS6 does not substitute for coculturing the hematopoietic progenitors with genetically modified 3T3 cells. PMID:11050245

  7. Endothelial Cells Promote Expansion of Long‐Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates

    PubMed Central

    Gori, Jennifer L.; Butler, Jason M.; Kunar, Balvir; Poulos, Michael G.; Ginsberg, Michael; Nolan, Daniel J.; Norgaard, Zachary K.; Adair, Jennifer E.; Rafii, Shahin

    2016-01-01

    Abstract Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self‐renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self‐renewal. To test this hypothesis, BM autologous CD34+ cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34+C38− HSPCs cocultured with ECs expanded up to 17‐fold, with a significant increase in hematopoietic colony‐forming activity compared with cells cultured with cytokines alone (colony‐forming unit‐granulocyte‐erythroid‐macrophage‐monocyte; p < .005). BM CD34+ cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34+ cells without impeding the long‐term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs. Stem Cells Translational Medicine 2017;6:864–876 PMID:28297579

  8. Sowing the Seeds of a Fruitful Harvest: Hematopoietic Stem Cell Mobilization

    PubMed Central

    Hoggatt, Jonathan; Speth, Jennifer M.; Pelus, Louis M.

    2014-01-01

    Hematopoietic stem cell transplantation is the only curative option for a number of malignant and non-malignant diseases. As the use of hematopoietic transplant has expanded, so too has the source of stem and progenitor cells. The predominate source of stem and progenitors today, particularly in settings of autologous transplantation, is mobilized peripheral blood. This review will highlight the historical advances which lead to the widespread use of peripheral blood stem cells for transplantation, with a look towards future enhancements to mobilization strategies. PMID:24123398

  9. Phenotypic characterization of aberrant stem and progenitor cell populations in myelodysplastic syndromes.

    PubMed

    Ostendorf, Benjamin N; Flenner, Eva; Flörcken, Anne; Westermann, Jörg

    2018-01-01

    Recent reports have revealed myelodysplastic syndromes (MDS) to arise from cancer stem cells phenotypically similar to physiological hematopoietic stem cells. Myelodysplastic hematopoiesis maintains a hierarchical organization, but the proportion of several hematopoietic compartments is skewed and multiple surface markers are aberrantly expressed. These aberrant antigen expression patterns hold diagnostic and therapeutic promise. However, eradication of MDS requires targeting of early myelodysplasia propagating stem cells. This warrants an exact assessment of the differentiation stage at which aberrant expression occurs in transformed hematopoiesis. Here, we report results on the prospective and extensive dissection of the hematopoietic hierarchy in 20 patients with either low-risk MDS or MDS with excess blasts and compare it to hematopoiesis in patients with non-malignancy-associated cytopenia or B cell lymphoma without bone marrow infiltration. We found patients with MDS with excess blasts to exhibit characteristic expansions of specific immature progenitor compartments. We also identified the aberrant expression of several markers including ALDH, CLL-1, CD44, and CD47 to be specific features of hematopoiesis in MDS with excess blasts. We show that amongst these, aberrant CLL-1 expression manifested at the early uncommitted hematopoietic stem cell level, suggesting a potential role as a therapeutic target.

  10. A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

    PubMed

    Nestorowa, Sonia; Hamey, Fiona K; Pijuan Sala, Blanca; Diamanti, Evangelia; Shepherd, Mairi; Laurenti, Elisa; Wilson, Nicola K; Kent, David G; Göttgens, Berthold

    2016-08-25

    Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice. © 2016 by The American Society of Hematology.

  11. Brief report: a human induced pluripotent stem cell model of cernunnos deficiency reveals an important role for XLF in the survival of the primitive hematopoietic progenitors.

    PubMed

    Tilgner, Katarzyna; Neganova, Irina; Singhapol, Chatchawan; Saretzki, Gabriele; Al-Aama, Jumana Yousuf; Evans, Jerome; Gorbunova, Vera; Gennery, Andrew; Przyborski, Stefan; Stojkovic, Miodrag; Armstrong, Lyle; Jeggo, Penny; Lako, Majlinda

    2013-09-01

    Cernunnos (also known as XLF) deficiency syndrome is a rare recessive autosomal disorder caused by mutations in the XLF gene, a key factor involved in the end joining step of DNA during nonhomologous end joining (NHEJ) process. Human patients with XLF mutations display microcephaly, developmental and growth delays, and severe immunodeficiency. While the clinical phenotype of DNA damage disorders, including XLF Syndrome, has been described extensively, the underlying mechanisms of disease onset, are as yet, undefined. We have been able to generate an induced pluripotent stem cell (iPSC) model of XLF deficiency, which accurately replicates the double-strand break repair deficiency observed in XLF patients. XLF patient-specific iPSCs (XLF-iPSC) show typical expression of pluripotency markers, but have altered in vitro differentiation capacity and an inability to generate teratomas comprised of all three germ layers in vivo. Our results demonstrate that XLF-iPSCs possess a weak NHEJ-mediated DNA repair capacity that is incapable of coping with the DNA lesions introduced by physiological stress, normal metabolism, and ionizing radiation. XLF-iPSC lines are capable of hematopoietic differentiation; however, the more primitive subsets of hematopoietic progenitors display increased apoptosis in culture and an inability to repair DNA damage. Together, our findings highlight the importance of NHEJ-mediated-DNA repair in the maintenance of a pristine pool of hematopoietic progenitors during human embryonic development. © AlphaMed Press.

  12. Gene Editing of Human Hematopoietic Stem and Progenitor Cells: Promise and Potential Hurdles.

    PubMed

    Yu, Kyung-Rok; Natanson, Hannah; Dunbar, Cynthia E

    2016-10-01

    Hematopoietic stem and progenitor cells (HSPCs) have great therapeutic potential because of their ability to both self-renew and differentiate. It has been proposed that, given their unique properties, a small number of genetically modified HSPCs could accomplish lifelong, corrective reconstitution of the entire hematopoietic system in patients with various hematologic disorders. Scientists have demonstrated that gene addition therapies-targeted to HSPCs and using integrating retroviral vectors-possess clear clinical benefits in multiple diseases, among them immunodeficiencies, storage disorders, and hemoglobinopathies. Scientists attempting to develop clinically relevant gene therapy protocols have, however, encountered a number of unexpected hurdles because of their incomplete knowledge of target cells, genomic control, and gene transfer technologies. Targeted gene-editing technologies using engineered nucleases such as ZFN, TALEN, and/or CRISPR/Cas9 RGEN show great clinical promise, allowing for the site-specific correction of disease-causing mutations-a process with important applications in autosomal dominant or dominant-negative genetic disorders. The relative simplicity of the CRISPR/Cas9 system, in particular, has sparked an exponential increase in the scientific community's interest in and use of these gene-editing technologies. In this minireview, we discuss the specific applications of gene-editing technologies in human HSPCs, as informed by prior experience with gene addition strategies. HSPCs are desirable but challenging targets; the specific mechanisms these cells evolved to protect themselves from DNA damage render them potentially more susceptible to oncogenesis, especially given their ability to self-renew and their long-term proliferative potential. We further review scientists' experience with gene-editing technologies to date, focusing on strategies to move these techniques toward implementation in safe and effective clinical trials.

  13. Enhanced Ex Vivo Expansion of Human Hematopoietic Progenitors on Native and Spin Coated Acellular Matrices Prepared from Bone Marrow Stromal Cells

    PubMed Central

    Wasnik, Samiksha; Kantipudi, Suma; Kirkland, Mark A.; Pande, Gopal

    2016-01-01

    The extracellular microenvironment in bone marrow (BM) is known to regulate the growth and differentiation of hematopoietic stem and progenitor cells (HSPC). We have developed cell-free matrices from a BM stromal cell line (HS-5), which can be used as substrates either in native form or as tissue engineered coatings, for the enhanced ex vivo expansion of umbilical cord blood (UCB) derived HSPC. The physicochemical properties (surface roughness, thickness, and uniformity) of native and spin coated acellular matrices (ACM) were studied using scanning and atomic force microscopy (SEM and AFM). Lineage-specific expansion of HSPC, grown on these substrates, was evaluated by immunophenotypic (flow cytometry) and functional (colony forming) assays. Our results show that the most efficient expansion of lineage-specific HSPC occurred on spin coated ACM. Our method provides an improved protocol for ex vivo HSPC expansion and it offers a system to study the in vivo roles of specific molecules in the hematopoietic niche that influence HSPC expansion. PMID:26981135

  14. Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates.

    PubMed

    Gori, Jennifer L; Butler, Jason M; Kunar, Balvir; Poulos, Michael G; Ginsberg, Michael; Nolan, Daniel J; Norgaard, Zachary K; Adair, Jennifer E; Rafii, Shahin; Kiem, Hans-Peter

    2017-03-01

    Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self-renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self-renewal. To test this hypothesis, BM autologous CD34 + cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34 + C38 - HSPCs cocultured with ECs expanded up to 17-fold, with a significant increase in hematopoietic colony-forming activity compared with cells cultured with cytokines alone (colony-forming unit-granulocyte-erythroid-macrophage-monocyte; p < .005). BM CD34 + cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34 + cells without impeding the long-term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs. Stem Cells Translational Medicine 2017;6:864-876. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  15. Runx1 and Cbfβ regulate the development of Flt3+ dendritic cell progenitors and restrict myeloproliferative disorder

    PubMed Central

    Satpathy, Ansuman T.; Briseño, Carlos G.; Cai, Xiongwei; Michael, Drew G.; Chou, Chun; Hsiung, Sunnie; Bhattacharya, Deepta; Speck, Nancy A.

    2014-01-01

    Runx1 and Cbfβ are critical for the establishment of definitive hematopoiesis and are implicated in leukemic transformation. Despite the absolute requirements for these factors in the development of hematopoietic stem cells and lymphocytes, their roles in the development of bone marrow progenitor subsets have not been defined. Here, we demonstrate that Cbfβ is essential for the development of Flt3+ macrophage-dendritic cell (DC) progenitors in the bone marrow and all DC subsets in the periphery. Besides the loss of DC progenitors, pan-hematopoietic Cbfb-deficient mice also lack CD105+ erythroid progenitors, leading to severe anemia at 3 to 4 months of age. Instead, Cbfb deficiency results in aberrant progenitor differentiation toward granulocyte-macrophage progenitors (GMPs), resulting in a myeloproliferative phenotype with accumulation of GMPs in the periphery and cellular infiltration of the liver. Expression of the transcription factor Irf8 is severely reduced in Cbfb-deficient progenitors, and overexpression of Irf8 restors DC differentiation. These results demonstrate that Runx proteins and Cbfβ restrict granulocyte lineage commitment to facilitate multilineage hematopoietic differentiation and thus identify their novel tumor suppressor function in myeloid leukemia. PMID:24677539

  16. Melanoma induced immunosuppression is mediated by hematopoietic dysregulation.

    PubMed

    Kamran, Neha; Li, Youping; Sierra, Maria; Alghamri, Mahmoud S; Kadiyala, Padma; Appelman, Henry D; Edwards, Marta; Lowenstein, Pedro R; Castro, Maria G

    2018-01-01

    Tumors are associated with expansion of immunosuppressive cells such as tumor associated macrophages (TAMs), regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs). These cells promote tumor growth, angiogenesis, metastasis and immune escape. Cancer patients frequently present symptoms such as anemia, leukocytosis and/or cytopenia; associated with poor prognosis. To uncover tumor-mediated hematopoietic abnormalities and identify novel targets that can be harnessed to improve tumor-specific immune responses, we investigated the hematopoietic stem and progenitor cell compartment in melanoma bearing mice. We show that melanoma growth results in expansion of myeloid lineages such as MDSCs, macrophages and DCs along with a reduction in mature RBCs and platelets. Mature B lymphocytes in the blood and BM of melanoma mice were also reduced. Mice bearing melanoma showed extramedullary hematopoiesis in the spleen. Increased expansion of myeloid lineages occurred directly at the level of stem and progenitor cells. The reduction in mature B lymphocytes resulted from a block at the Pro-B cell stage in the bone marrow. Addition of recombinant IL-3 to bone marrow cells resulted in the expansion of committed myeloid progenitors including common myeloid precursors, granulocyte-monocyte precursors and megakaryocyte-erythrocyte precursors. In vivo , IL-3 receptor stimulation in melanoma bearing mice using an IL-3 antibody also resulted in a robust expansion of committed myeloid progenitors and hematopoietic stem cells. Collectively our findings demonstrate that tumor growth plays a pivotal role in reprogramming the host immune system by impacting hematopoiesis directly at the level of stem cell compartment.

  17. Validation of a Pediatric Early Warning Score in Hospitalized Pediatric Oncology and Hematopoietic Stem Cell Transplant Patients.

    PubMed

    Agulnik, Asya; Forbes, Peter W; Stenquist, Nicole; Rodriguez-Galindo, Carlos; Kleinman, Monica

    2016-04-01

    To evaluate the correlation of a Pediatric Early Warning Score with unplanned transfer to the PICU in hospitalized oncology and hematopoietic stem cell transplant patients. We performed a retrospective matched case-control study, comparing the highest documented Pediatric Early Warning Score within 24 hours prior to unplanned PICU transfers in hospitalized pediatric oncology and hematopoietic stem cell transplant patients between September 2011 and December 2013. Controls were patients who remained on the inpatient unit and were matched 2:1 using age, condition (oncology vs hematopoietic stem cell transplant), and length of hospital stay. Pediatric Early Warning Scores were documented by nursing staff at least every 4 hours as part of routine care. Need for transfer was determined by a PICU physician called to evaluate the patient. A large tertiary/quaternary free-standing academic children's hospital. One hundred ten hospitalized pediatric oncology patients (42 oncology, 68 hematopoietic stem cell transplant) requiring unplanned PICU transfer and 220 matched controls. None. Using the highest score in the 24 hours prior to transfer for cases and a matched time period for controls, the Pediatric Early Warning Score was highly correlated with the need for PICU transfer overall (area under the receiver operating characteristic = 0.96), and in the oncology and hematopoietic stem cell transplant groups individually (area under the receiver operating characteristic = 0.95 and 0.96, respectively). The difference in Pediatric Early Warning Score results between the cases and controls was noted as early as 24 hours prior to PICU admission. Seventeen patients died (15.4%). Patients with higher Pediatric Early Warning Scores prior to transfer had increased PICU mortality (p = 0.028) and length of stay (p = 0.004). We demonstrate that our institution's Pediatric Early Warning Score is highly correlated with the need for unplanned PICU transfer in hospitalized oncology and

  18. Malat1 as an evolutionarily conserved lncRNA, plays a positive role in regulating proliferation and maintaining undifferentiated status of early-stage hematopoietic cells.

    PubMed

    Ma, Xian-Yong; Wang, Jian-Hui; Wang, Jing-Lan; Ma, Charles X; Wang, Xiao-Chun; Liu, Feng-Song

    2015-09-03

    The metastasis-associated lung adenocarcinoma transcription 1 (Malat1) is a highly conserved long non-coding RNA (lncRNA) gene. Previous studies showed that Malat1 is abundantly expressed in many tissues and involves in promoting tumor growth and metastasis by modulating gene expression and target protein activities. However, little is known about the biological function and regulation mechanism of Malat1 in normal cell proliferation. In this study we conformed that Malat1 is highly conserved across vast evolutionary distances amongst 20 species of mammals in terms of sequence, and found that mouse Malat1 expresses in tissues of liver, kidney, lung, heart, testis, spleen and brain, but not in skeletal muscle. After treating erythroid myeloid lymphoid (EML) cells with All-trans Retinoic Acid (ATRA), we investigated the expression and regulation of Malat1 during hematopoietic differentiation, the results showed that ATRA significantly down regulates Malat1 expression during the differentiation of EML cells. Mouse LRH (Lin-Rhodamine(low) Hoechst(low)) cells that represent the early-stage progenitor cells show a high level of Malat1 expression, while LRB (Lin - Hoechst(Low) Rhodamine(Bright)) cells that represent the late-stage progenitor cells had no detectable expression of Malat1. Knockdown experiment showed that depletion of Malat1 inhibits the EML cell proliferation. Along with the down regulation of Malat1, the tumor suppressor gene p53 was up regulated during the differentiation. Interestingly, we found two p53 binding motifs with help of bioinformatic tools, and the following chromatin immunoprecipitation (ChIP) test conformed that p53 acts as a transcription repressor that binds to Malat1's promoter. Furthermore, we testified that p53 over expression in EML cells causes down regulation of Malat1. In summary, this study indicates Malat1 plays a critical role in maintaining the proliferation potential of early-stage hematopoietic cells. In addition to its

  19. Generation of Transgenic Mouse Fluorescent Reporter Lines for Studying Hematopoietic Development

    PubMed Central

    Vacaru, Andrei M.; Vitale, Joseph; Nieves, Johnathan; Baron, Margaret H.

    2015-01-01

    During the development of the hematopoietic system, at least 8 distinct lineages are generated in the mouse embryo. Transgenic mice expressing fluorescent proteins at various points in the hematopoietic hierarchy, from hematopoietic stem cell to multipotent progenitors to each of the final differentiated cell types, have provided valuable tools for tagging, tracking, and isolating these cells. In this chapter, we discuss general considerations in designing a transgene, survey available fluorescent probes, and methods for confirming and analyzing transgene expression in the hematopoietic systems of the embryo, fetus, and postnatal/adult animal. PMID:25064110

  20. Identification of Three Molecular and Functional Subtypes in Canine Hemangiosarcoma through Gene Expression Profiling and Progenitor Cell Characterization

    PubMed Central

    Gorden, Brandi H.; Kim, Jong-Hyuk; Sarver, Aaron L.; Frantz, Aric M.; Breen, Matthew; Lindblad-Toh, Kerstin; O'Brien, Timothy D.; Sharkey, Leslie C.; Modiano, Jaime F.; Dickerson, Erin B.

    2015-01-01

    Canine hemangiosarcomas have been ascribed to an endothelial origin based on histologic appearance; however, recent findings suggest that these tumors may arise instead from hematopoietic progenitor cells. To clarify this ontogenetic dilemma, we used genome-wide expression profiling of primary hemangiosarcomas and identified three distinct tumor subtypes associated with angiogenesis (group 1), inflammation (group 2), and adipogenesis (group 3). Based on these findings, we hypothesized that a common progenitor may differentiate into the three tumor subtypes observed in our gene profiling experiment. To investigate this possibility, we cultured hemangiosarcoma cell lines under normal and sphere-forming culture conditions to enrich for tumor cell progenitors. Cells from sphere-forming cultures displayed a robust self-renewal capacity and exhibited genotypic, phenotypic, and functional properties consistent with each of the three molecular subtypes seen in primary tumors, including expression of endothelial progenitor cell (CD133 and CD34) and endothelial cell (CD105, CD146, and αvβ3 integrin) markers, expression of early hematopoietic (CD133, CD117, and CD34) and myeloid (CD115 and CD14) differentiation markers in parallel with increased phagocytic capacity, and acquisition of adipogenic potential. Collectively, these results suggest that canine hemangiosarcomas arise from multipotent progenitors that differentiate into distinct subtypes. Improved understanding of the mechanisms that determine the molecular and phenotypic differentiation of tumor cells in vivo could change paradigms regarding the origin and progression of endothelial sarcomas. PMID:24525151

  1. Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

    PubMed Central

    Seet, Christopher S.; He, Chongbin; Bethune, Michael T.; Li, Suwen; Chick, Brent; Gschweng, Eric H.; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B.; Baltimore, David; Crooks, Gay M.; Montel-Hagen, Amélie

    2017-01-01

    Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies. PMID:28369043

  2. Efficient Ex Vivo Engineering and Expansion of Highly Purified Human Hematopoietic Stem and Progenitor Cell Populations for Gene Therapy.

    PubMed

    Zonari, Erika; Desantis, Giacomo; Petrillo, Carolina; Boccalatte, Francesco E; Lidonnici, Maria Rosa; Kajaste-Rudnitski, Anna; Aiuti, Alessandro; Ferrari, Giuliana; Naldini, Luigi; Gentner, Bernhard

    2017-04-11

    Ex vivo gene therapy based on CD34 + hematopoietic stem cells (HSCs) has shown promising results in clinical trials, but genetic engineering to high levels and in large scale remains challenging. We devised a sorting strategy that captures more than 90% of HSC activity in less than 10% of mobilized peripheral blood (mPB) CD34 + cells, and modeled a transplantation protocol based on highly purified, genetically engineered HSCs co-infused with uncultured progenitor cells. Prostaglandin E 2 stimulation allowed near-complete transduction of HSCs with lentiviral vectors during a culture time of less than 38 hr, mitigating the negative impact of standard culture on progenitor cell function. Exploiting the pyrimidoindole derivative UM171, we show that transduced mPB CD34 + CD38 - cells with repopulating potential could be expanded ex vivo. Implementing these findings in clinical gene therapy protocols will improve the efficacy, safety, and sustainability of gene therapy and generate new opportunities in the field of gene editing. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  3. Are neural crest stem cells the missing link between hematopoietic and neurogenic niches?

    PubMed

    Coste, Cécile; Neirinckx, Virginie; Gothot, André; Wislet, Sabine; Rogister, Bernard

    2015-01-01

    Hematopoietic niches are defined as cellular and molecular microenvironments that regulate hematopoietic stem cell (HSC) function together with stem cell autonomous mechanisms. Many different cell types have been characterized as contributors to the formation of HSC niches, such as osteoblasts, endothelial cells, Schwann cells, and mesenchymal progenitors. These mesenchymal progenitors have themselves been classified as CXC chemokine ligand (CXCL) 12-abundant reticular (CAR) cells, stem cell factor expressing cells, or nestin-positive mesenchymal stem cells (MSCs), which have been recently identified as neural crest-derived cells (NCSCs). Together, these cells are spatially associated with HSCs and believed to provide appropriate microenvironments for HSC self-renewal, differentiation, mobilization and hibernation both by cell-cell contact and soluble factors. Interestingly, it appears that regulatory pathways governing the hematopoietic niche homeostasis are operating in the neurogenic niche as well. Therefore, this review paper aims to compare both the regulation of hematopoietic and neurogenic niches, in order to highlight the role of NCSCs and nervous system components in the development and the regulation of the hematopoietic system.

  4. Angiogenin Defines Heterogeneity at the Core of the Hematopoietic Niche.

    PubMed

    Di Scala, Marianna; Hidalgo, Andrés

    2016-09-01

    Successful hematopoietic regeneration demands preservation of stemness while enabling expansion and differentiation into blood lineages. Now, Silberstein et al. (2016) and Goncalves et al. (2016) identify a ribonuclease secreted by proximal niche cells that simultaneously drives quiescence of HSCs and proliferation of myeloid progenitors and dramatically enhances hematopoietic recovery after HSC transplantation. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. A review of transfusion practice before, during, and after hematopoietic progenitor cell transplantation

    PubMed Central

    Johnson, Viviana V.; Sandler, S. Gerald; Sayegh, Antoine; Klumpp, Thomas R.

    2008-01-01

    The increased use of hematopoietic progenitor cell (HPC) transplantation has implications and consequences for transfusion services: not only in hospitals where HPC transplantations are performed, but also in hospitals that do not perform HPC transplantations but manage patients before or after transplantation. Candidates for HPC transplantation have specific and specialized transfusion requirements before, during, and after transplantation that are necessary to avert the adverse consequences of alloimmunization to human leukocyte antigens, immunohematologic consequences of ABO-mismatched transplantations, or immunosuppression. Decisions concerning blood transfusions during any of these times may compromise the outcome of an otherwise successful transplantation. Years after an HPC transplantation, and even during clinical remission, recipients may continue to be immunosuppressed and may have critically important, special transfusion requirements. Without a thorough understanding of these special requirements, provision of compatible blood components may be delayed and often urgent transfusion needs prohibit appropriate consultation with the patient's transplantation specialist. To optimize the relevance of issues and communication between clinical hematologists, transplantation physicians, and transfusion medicine physicians, the data and opinions presented in this review are organized by sequence of patient presentation, namely, before, during, and after transplantation. PMID:18583566

  6. Ryk receptor regulates hematopoietic stem and progenitor sensitivity to myelosuppressive injury in mice

    PubMed Central

    Povinelli, Benjamin J.; Srivastava, Pragya; Nemeth, Michael J.

    2017-01-01

    Maintaining a careful balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) is necessary for lifelong blood formation. Previously, we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a non-canonical Wnt ligand receptor termed Ryk. Expression of Ryk on HSPCs in vivo is associated with a lower rate of proliferation and following treatment with fluorouracil (5-FU), the percentage of Ryk+/high HSPCs increased while the percent of Ryk−/low HSPCs decreased. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. We found that Ryk expression on HSPCs is associated with lower rates of apoptosis following 5-FU and radiation. Transient inhibition of Ryk signaling in vivo resulted in increased HSC proliferation and decreased HSC function in bone marrow transplant assays. Furthermore, inhibition of Ryk signaling sensitized HSPCs to 5-FU treatment in association with increased levels of reactive oxygen species. Together, these results demonstrated an association between Ryk expression and survival of HSPCs following suppressive injury. PMID:25461251

  7. Aid is a key regulator of myeloid/erythroid differentiation and DNA methylation in hematopoietic stem/progenitor cells

    PubMed Central

    Kunimoto, Hiroyoshi; McKenney, Anna Sophia; Meydan, Cem; Shank, Kaitlyn; Nazir, Abbas; Rapaport, Franck; Durham, Benjamin; Garrett-Bakelman, Francine E.; Pronier, Elodie; Shih, Alan H.; Melnick, Ari; Chaudhuri, Jayanta

    2017-01-01

    Recent studies have reported that activation-induced cytidine deaminase (AID) and ten-eleven-translocation (TET) family members regulate active DNA demethylation. Genetic alterations of TET2 occur in myeloid malignancies, and hematopoietic-specific loss of Tet2 induces aberrant hematopoietic stem cell (HSC) self-renewal/differentiation, implicating TET2 as a master regulator of normal and malignant hematopoiesis. Despite the functional link between AID and TET in epigenetic gene regulation, the role of AID loss in hematopoiesis and myeloid transformation remains to be investigated. Here, we show that Aid loss in mice leads to expansion of myeloid cells and reduced erythroid progenitors resulting in anemia, with dysregulated expression of Cebpa and Gata1, myeloid/erythroid lineage-specific transcription factors. Consistent with data in the murine context, silencing of AID in human bone marrow cells skews differentiation toward myelomonocytic lineage. However, in contrast to Tet2 loss, Aid loss does not contribute to enhanced HSC self-renewal or cooperate with Flt3-ITD to induce myeloid transformation. Genome-wide transcription and differential methylation analysis uncover the critical role of Aid as a key epigenetic regulator. These results indicate that AID and TET2 share common effects on myeloid and erythroid lineage differentiation, however, their role is nonredundant in regulating HSC self-renewal and in myeloid transformation. PMID:28077417

  8. WT1 controls antagonistic FGF and BMP-pSMAD pathways in early renal progenitors.

    PubMed

    Motamedi, Fariba Jian; Badro, Danielle A; Clarkson, Michael; Lecca, M Rita; Bradford, Stephen T; Buske, Fabian A; Saar, Kathrin; Hübner, Norbert; Brändli, André W; Schedl, Andreas

    2014-07-17

    Kidney organogenesis requires the tight control of proliferation, differentiation and apoptosis of renal progenitor cells. How the balance between these cellular decisions is achieved remains elusive. The Wilms' tumour suppressor Wt1 is required for progenitor survival, but the molecular cause for renal agenesis in mutants is poorly understood. Here we demonstrate that lack of Wt1 abolishes fibroblast growth factor (FGF) and induces BMP/pSMAD signalling within the metanephric mesenchyme. Addition of recombinant FGFs or inhibition of pSMAD signalling rescues progenitor cell apoptosis induced by the loss of Wt1. We further show that recombinant BMP4, but not BMP7, induces an apoptotic response within the early kidney that can be suppressed by simultaneous addition of FGFs. These data reveal a hitherto unknown sensitivity of early renal progenitors to pSMAD signalling, establishes FGF and pSMAD signalling as antagonistic forces in early kidney development and places WT1 as a key regulator of pro-survival FGF signalling pathway genes.

  9. AML1/ETO induces self-renewal in hematopoietic progenitor cells via the Groucho-related amino-terminal AES protein.

    PubMed

    Steffen, Björn; Knop, Markus; Bergholz, Ulla; Vakhrusheva, Olesya; Rode, Miriam; Köhler, Gabriele; Henrichs, Marcel-Philipp; Bulk, Etmar; Hehn, Sina; Stehling, Martin; Dugas, Martin; Bäumer, Nicole; Tschanter, Petra; Brandts, Christian; Koschmieder, Steffen; Berdel, Wolfgang E; Serve, Hubert; Stocking, Carol; Müller-Tidow, Carsten

    2011-04-21

    The most frequent translocation t(8;21) in acute myeloid leukemia (AML) generates the chimeric AML1/ETO protein, which blocks differentiation and induces self-renewal in hematopoietic progenitor cells. The underlying mechanisms mediating AML1/ETO-induced self-renewal are largely unknown. Using expression microarray analysis, we identified the Groucho-related amino-terminal enhancer of split (AES) as a consistently up-regulated AML1/ETO target. Elevated levels of AES mRNA and protein were confirmed in AML1/ETO-expressing leukemia cells, as well as in other AML specimens. High expression of AES mRNA or protein was associated with improved survival of AML patients, even in the absence of t(8;21). On a functional level, knockdown of AES by RNAi in AML1/ETO-expressing cell lines inhibited colony formation. Similarly, self-renewal induced by AML1/ETO in primary murine progenitors was inhibited when AES was decreased or absent. High levels of AES expression enhanced formation of immature colonies, serial replating capacity of primary cells, and colony formation in colony-forming unit-spleen assays. These findings establish AES as a novel AML1/ETO-induced target gene that plays an important role in the self-renewal phenotype of t(8;21)-positive AML.

  10. Altered Expression of Genes in Signaling Pathways Regulating Proliferation of Hematopoietic Stem and Progenitor Cells in Mice with Subchronic Benzene Exposure

    PubMed Central

    Sun, Rongli; Zhang, Juan; Xiong, Mengzhen; Wei, Haiyan; Tan, Kehong; Yin, Lihong; Pu, Yuepu

    2015-01-01

    Leukemias and hematopoietic disorders induced by benzene may arise from the toxicity of benzene to hematopoietic stem or progenitor cells (HS/PCs). Since there is a latency period between initial benzene exposure and the development of leukemia, subsequent impact of benzene on HS/PCs are crucial for a deeper understanding of the carcinogenicity and hematotoxicity in post-exposure stage. This study aims to explore the effects of benzene on HS/PCs and gene-expression in Wnt, Notch and Hh signaling pathways in post-exposure stage. The C3H/He mice were injected subcutaneously with benzene (0, 150, 300 mg/kg/day) for three months and were monitored for another 10 months post-exposure. The body weights were monitored, the relative organ weights, blood parameters and bone marrow smears were examined. Frequency of lineage- sca-1+ c-kit+ (LSK) cells, capability of colony forming and expression of genes in Wnt, Notch and Hedghog (Hh) signaling pathways were also analyzed. The colony formation of the progenitor cells for BFU-E, CFU-GEMM and CFU-GM was significantly decreased with increasing benzene exposure relative to controls, while no significant difference was observed in colonies for CFU-G and CFU-M. The mRNA level of cyclin D1 was increased and Notch1 and p53 were decreased in LSK cells in mice exposed to benzene but with no statistical significance. These results suggest that subsequent toxic effects of benzene on LSK cells and gene expression in Wnt, Notch and Hh signaling pathways persist in post-exposure stage and may play roles in benzene-induced hematotoxicity. PMID:26262635

  11. Altered Expression of Genes in Signaling Pathways Regulating Proliferation of Hematopoietic Stem and Progenitor Cells in Mice with Subchronic Benzene Exposure.

    PubMed

    Sun, Rongli; Zhang, Juan; Xiong, Mengzhen; Wei, Haiyan; Tan, Kehong; Yin, Lihong; Pu, Yuepu

    2015-08-07

    Leukemias and hematopoietic disorders induced by benzene may arise from the toxicity of benzene to hematopoietic stem or progenitor cells (HS/PCs). Since there is a latency period between initial benzene exposure and the development of leukemia, subsequent impact of benzene on HS/PCs are crucial for a deeper understanding of the carcinogenicity and hematotoxicity in post-exposure stage. This study aims to explore the effects of benzene on HS/PCs and gene-expression in Wnt, Notch and Hh signaling pathways in post-exposure stage. The C3H/He mice were injected subcutaneously with benzene (0, 150, 300 mg/kg/day) for three months and were monitored for another 10 months post-exposure. The body weights were monitored, the relative organ weights, blood parameters and bone marrow smears were examined. Frequency of lineage(-) sca-1(+) c-kit(+) (LSK) cells, capability of colony forming and expression of genes in Wnt, Notch and Hedghog (Hh) signaling pathways were also analyzed. The colony formation of the progenitor cells for BFU-E, CFU-GEMM and CFU-GM was significantly decreased with increasing benzene exposure relative to controls, while no significant difference was observed in colonies for CFU-G and CFU-M. The mRNA level of cyclin D1 was increased and Notch 1 and p53 were decreased in LSK cells in mice exposed to benzene but with no statistical significance. These results suggest that subsequent toxic effects of benzene on LSK cells and gene expression in Wnt, Notch and Hh signaling pathways persist in post-exposure stage and may play roles in benzene-induced hematotoxicity.

  12. Interleukin-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the osteoclastogenic process

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hong, Huixian; Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294; Shi, Zhenqi

    2013-11-01

    Highlights: •IL-3 treatment of bone marrow cells generates a population of hematopoietic cells. •IL-3-dependent hematopoietic cells are capable of differentiating into osteoclasts. •Osteoclasts derived from IL-3-dependent hematopoietic cells are functional. •IL-3 promotes the development of osteoclast progenitors. •IL-3 inhibits the osteoclastogenic process. -- Abstract: Interleukin (IL)-3, a multilineage hematopoietic growth factor, is implicated in the regulation of osteoclastogenesis. However, the role of IL-3 in osteoclastogenesis remains controversial; whereas early studies showed that IL-3 stimulates osteoclastogenesis, recent investigations demonstrated that IL-3 inhibits osteoclast formation. The objective of this work is to further address the role of IL-3 in osteoclastogenesis. We foundmore » that IL-3 treatment of bone marrow cells generated a population of cells capable of differentiating into osteoclasts in tissue culture dishes in response to the stimulation of the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of nuclear factor kappa B ligand (RANKL). The IL-3-dependent hematopoietic cells were able to further proliferate and differentiate in response to M-CSF stimulation and the resulting cells were also capable of forming osteoclasts with M-CSF and RANKL treatment. Interestingly, IL-3 inhibits M-CSF-/RANKL-induced differentiation of the IL-3-dependent hematopoietic cells into osteoclasts. The flow cytometry analysis indicates that while IL-3 treatment of bone marrow cells slightly affected the percentage of osteoclast precursors in the surviving populations, it considerably increased the percentage of osteoclast precursors in the populations after subsequent M-CSF treatment. Moreover, osteoclasts derived from IL-3-dependent hematopoietic cells were fully functional. Thus, we conclude that IL-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting

  13. Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene.

    PubMed

    Perez-Cunningham, Jessica; Boyer, Scott W; Landon, Mark; Forsberg, E Camilla

    2016-08-01

    Selective labeling of specific cell types by expression of green fluorescent protein (GFP) within the hematopoietic system would have great utility in identifying, localizing, and tracking different cell populations in flow cytometry, microscopy, lineage tracing, and transplantation assays. In this report, we describe the generation and characterization of a new transgenic mouse line with specific GFP labeling of all nucleated hematopoietic cells and platelets. This new "Vav-GFP" mouse line labels the vast majority of hematopoietic cells with GFP during both embryonic development and adulthood, with particularly high expression in hematopoietic stem and progenitor cells (HSPCs). With the exception of transient labeling of fetal endothelial cells, GFP expression is highly selective for hematopoietic cells and persists in donor-derived progeny after transplantation of HSPCs. Finally, we also demonstrate that the loxP-flanked reporter allows for specific GFP labeling of different hematopoietic cell subsets when crossed to various Cre reporter lines. By crossing Vav-GFP mice to Flk2-Cre mice, we obtained robust and highly selective GFP expression in hematopoietic stem cells (HSCs). These data describe a new mouse model capable of directing GFP labeling exclusively of hematopoietic cells or exclusively of HSCs. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  14. HEMATOPOIETIC PROGENITOR CELL CONTENT OF VERTEBRAL BODY MARROW USED FOR COMBINED SOLID ORGAN AND BONE MARROW TRANSPLANTATION

    PubMed Central

    Rybka, Witold B.; Fontes, Paulo A.; Rao, Abdul S.; Winkelstein, Alan; Ricordi, Camillo; Ball, Edward D.; Starzl, Thomas E.

    2010-01-01

    While cadaveric vertebral bodies (VB) have long been proposed as a suitable source of bone marrow (BM) for transplantation (BMT), they have rarely been used for this purpose. We have infused VB BM immediately following whole organ (WO) transplantation to augment donor cell chimerism. We quantified the hematopoietic progenitor cell (HPC) content of VB BM as well as BM obtained from the iliac crests (IC) of normal allogeneic donors (ALLO) and from patients with malignancy undergoing autologous marrow harvest (AUTO). Patients undergoing WOIBM transplantation also had AUTO BM harvested in the event that subsequent lymphohematopoietic reconstitution was required. Twenty-four VB BM, 24 IC BM-ALLO, 31 IC AUTO, and 24 IC WO-AUTO were harvested. VB BM was tested 12 to 72 hr after procurement and infused after completion ofWO grafting. IC BM was tested and then used or cryopreserved immediately. HPC were quantified by clonal assay measuring CFU-GM, BFU-E, and CFU-GEMM, and by flow cytometry for CD34+ progenitor cells. On an average, 9 VB were processed during each harvest, and despite an extended processing time the number of viable nucleated cells obtained was significantly higher than that from IC. Furthermore, by HPC content, VB BM was equivalent to IC BM, which is routinely used for BMT. We conclude that VB BM is a clinically valuable source of BM for allogeneic transplantation. PMID:7701582

  15. Sources of Hematopoietic Stem and Progenitor Cells and Methods to Optimize Yields for Clinical Cell Therapy.

    PubMed

    Panch, Sandhya R; Szymanski, James; Savani, Bipin N; Stroncek, David F

    2017-08-01

    Bone marrow (BM) aspirates, mobilized peripheral blood, and umbilical cord blood (UCB) have developed as graft sources for hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation and other cellular therapeutics. Individualized techniques are necessary to enhance graft HSPC yields and cell quality from each graft source. BM aspirates yield adequate CD34 + cells but can result in relative delays in engraftment. Granulocyte colony-stimulating factor (G-CSF)-primed BM HSPCs may facilitate faster engraftment while minimizing graft-versus-host disease in certain patient subsets. The levels of circulating HSPCs are enhanced using mobilizing agents, such as G-CSF and/or plerixafor, which act via the stromal cell-derived factor 1/C-X-C chemokine receptor type 4 axis. Alternate niche pathway mediators, including very late antigen-4/vascular cell adhesion molecule-1, heparan sulfate proteoglycans, parathyroid hormone, and coagulation cascade intermediates, may offer promising alternatives for graft enhancement. UCB grafts have been expanded ex vivo with cytokines, notch-ligand, or mesenchymal stromal cells, and most studies demonstrated greater quantities of CD34 + cells ex vivo and improved short-term engraftment. No significant changes were observed in long-term repopulating potential or in patient survival. Early phase clinical trials using nicotinamide and StemReginin1 may offer improved short- and long-term repopulating ability. Breakthroughs in genome editing and stem cell reprogramming technologies may hasten the generation of pooled, third-party HSPC grafts. This review elucidates past, present, and potential future approaches to HSPC graft optimization. Published by Elsevier Inc.

  16. Exercise increases the frequency of circulating hematopoietic progenitor cells, but reduces hematopoietic colony-forming capacity.

    PubMed

    Kroepfl, Julia Maria; Pekovits, Karin; Stelzer, Ingeborg; Fuchs, Robert; Zelzer, Sieglinde; Hofmann, Peter; Sedlmayr, Peter; Dohr, Gottfried; Wallner-Liebmann, Sandra; Domej, Wolfgang; Mueller, Wolfram

    2012-11-01

    Circulating hematopoietic progenitor cells (CPCs) may be triggered by physical exercise and/or normobaric hypoxia from the bone marrow. The aim of the study was to investigate the influence of physical exercise and normobaric hypoxia on CPC number and functionality in the peripheral blood as well as the involvement of oxidative stress parameters as possibly active agents. Ten healthy male subjects (25.3±4.4 years) underwent a standardized cycle incremental exercise test protocol (40 W+20 W/min) under either normoxic (FiO2 ∼0.21) or hypoxic conditions (FiO2<0.15, equals 3,500 m, 3 h xposure) within a time span of at least 1 week. Blood was drawn from the cubital vein before and 10, 30, 60, and 120 min after exercise. The number of CPCs in the peripheral blood was analyzed by flow cytometry (CD34/CD45-positive cells). The functionality of cells present was addressed by secondary colony-forming unit-granulocyte macrophage (CFU-GM) assays. To determine a possible correlation between the mobilization of CPCs and reactive oxygen species, parameters for oxidative stress such as malondialdehyde (MDA) and myeloperoxidase (MPO) were obtained. Data showed a significant increase of CPC release under normoxic as well as hypoxic conditions after 10 min of recovery (P<0.01). Most interestingly, although CD34+/CD45dim cells increased in number, the proliferative capacity of CPCs decreased significantly 10 min after cessation of exercise (P<0.05). A positive correlation between CPCs and MDA/MPO levels turned out to be significant for both normoxic and hypoxic conditions (P<0.05/P<0.01). Hypoxia did not provoke an additional effect. Although the CPC frequency increased, the functionality of CPCs decreased significantly after exercise, possibly due to the influence of increased oxidative stress levels.

  17. Inhibition of T Cell Protein Tyrosine Phosphatase Enhances Interleukin-18-Dependent Hematopoietic Stem Cell Expansion

    PubMed Central

    Bourdeau, Annie; Trop, Sébastien; Doody, Karen M; Dumont, Daniel J; Tremblayef, Michel L

    2013-01-01

    The clinical application of hematopoietic progenitor cell-based therapies for the treatment of hematological diseases is hindered by current protocols, which are cumbersome and have limited efficacy to augment the progenitor cell pool. We report that inhibition of T-cell protein tyrosine phosphatase (TC-PTP), an enzyme involved in the regulation of cytokine signaling, through gene knockout results in a ninefold increase in the number of hematopoietic progenitors in murine bone marrow (BM). This effect could be reproduced using a short (48 hours) treatment with a pharmacological inhibitor of TC-PTP in murine BM, as well as in human BM, peripheral blood, and cord blood. We also demonstrate that the ex vivo use of TC-PTP inhibitor only provides a temporary effect on stem cells and did not alter their capacity to reconstitute all hematopoietic components in vivo. We establish that one of the mechanisms whereby inhibition of TC-PTP mediates its effects involves the interleukin-18 (IL-18) signaling pathway, leading to increased production of IL-12 and interferon-gamma by progenitor cells. Together, our results reveal a previously unrecognized role for IL-18 in contributing to the augmentation of the stem cell pool and provide a novel and simple method to rapidly expand progenitor cells from a variety of sources using a pharmacological compound. Stem Cells 2013;31:293–304 PMID:23135963

  18. Identification of three molecular and functional subtypes in canine hemangiosarcoma through gene expression profiling and progenitor cell characterization.

    PubMed

    Gorden, Brandi H; Kim, Jong-Hyuk; Sarver, Aaron L; Frantz, Aric M; Breen, Matthew; Lindblad-Toh, Kerstin; O'Brien, Timothy D; Sharkey, Leslie C; Modiano, Jaime F; Dickerson, Erin B

    2014-04-01

    Canine hemangiosarcomas have been ascribed to an endothelial origin based on histologic appearance; however, recent findings suggest that these tumors may arise instead from hematopoietic progenitor cells. To clarify this ontogenetic dilemma, we used genome-wide expression profiling of primary hemangiosarcomas and identified three distinct tumor subtypes associated with angiogenesis (group 1), inflammation (group 2), and adipogenesis (group 3). Based on these findings, we hypothesized that a common progenitor may differentiate into the three tumor subtypes observed in our gene profiling experiment. To investigate this possibility, we cultured hemangiosarcoma cell lines under normal and sphere-forming culture conditions to enrich for tumor cell progenitors. Cells from sphere-forming cultures displayed a robust self-renewal capacity and exhibited genotypic, phenotypic, and functional properties consistent with each of the three molecular subtypes seen in primary tumors, including expression of endothelial progenitor cell (CD133 and CD34) and endothelial cell (CD105, CD146, and αvβ3 integrin) markers, expression of early hematopoietic (CD133, CD117, and CD34) and myeloid (CD115 and CD14) differentiation markers in parallel with increased phagocytic capacity, and acquisition of adipogenic potential. Collectively, these results suggest that canine hemangiosarcomas arise from multipotent progenitors that differentiate into distinct subtypes. Improved understanding of the mechanisms that determine the molecular and phenotypic differentiation of tumor cells in vivo could change paradigms regarding the origin and progression of endothelial sarcomas. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  19. [Protective effects of WR2721 on early bone marrow hematopoietic function in mice exposed to 6.5 Gy of (60)Co γ-rays].

    PubMed

    Deng, Zi-Liang; Zhang, Liu-Zhen; Cong, Yue; Liu, Xiao-Lan; Yu, Zu-Ying; Shan, Ya-Jun; Cui, Yu; Wang, Li-Mei; Xing, Shuang; Cong, Yu-Wen; Luo, Qing-Liang

    2014-06-01

    The aim of this study was to investigate the effect of WR2721(amifostine) against bone marrow hematopoietic damage of mice exposed to 6.5 Gy of (60)Co-γ ray. A total of 60 C57/BL6J mice was divided into 3 groups:normal group (mice were injected with physiological salt solution), irradiation group (mice were injected with physiologic salt solution before irradiation) and WR2721 group (mice were injected with WR2721 before irradiation). The WBC, neutrophil (Neut), Plt and RBC levels in peripheral blood of 3 group mice were counted within 60 days after irradiation; the bone marrow nuclear cells (BMNC) were counted at 2 and 24 hours after irradiation; the hematopoietic stem/progenitor cell (LK/LSK) level and colony formation capability were detected by flow cytometry at 2 and 24 hours after irradiation. The results indicated that the counts of WBC and neut at 4 and 18 days, Plt at 7-18 days and RBC at 10-30 day after irradiation in WR2721 group were higher than those in irradiation group (P < 0.05); the BMNC, LSK and LK levels obviously increased at 24 hours after irradiation (P < 0.05), the CFU-GEMM, CFU-GM, CFU-MK BFU-E and CFU-E all significantly increased at 2 and 24 hours after irradiation (P < 0.01), as compared with irradiation group. It is concluded that WR2721 can effectively alleviate early hematopoietic damage and promote the fast recovery of peripheral blood cells in mice exposed to γ-ray, suggesting that the WR2721 has significant radioprotective effect on hematopoietic system.

  20. Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

    PubMed

    Seet, Christopher S; He, Chongbin; Bethune, Michael T; Li, Suwen; Chick, Brent; Gschweng, Eric H; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B; Baltimore, David; Crooks, Gay M; Montel-Hagen, Amélie

    2017-05-01

    Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3 + TCR-αβ + single-positive CD8 + or CD4 + cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

  1. Breaking the rules? X-ray examination of hematopoietic stem cell grafts at international airports.

    PubMed

    Petzer, Andreas L; Speth, Hans-Georg; Hoflehner, Elisabeth; Clausen, Johannes; Nachbaur, David; Gastl, Günther; Gunsilius, Eberhard

    2002-06-15

    Hematopoietic stem cell grafts from unrelated donors are commonly transported by aircraft. They must not be subjected to x-rays during security checks, which may cause inconvenient discussions between the courier and the airport security staff. We exposed hematopoietic stem cells from mobilized peripheral blood to a widely used x-ray hand-luggage control system. Cell viability as well as growth in vitro of mature progenitor cells (colony-forming cells), primitive progenitor cells (long-term culture-initiating cells), and lymphocytes were not altered even after 10 passages through the hand-luggage control system. Thus, repeated exposure to the low radiation dose of hand-luggage control systems (1.5 +/- 0.6 microSv per exposure) seems to be harmless for hematopoietic stem cells, which should simplify the international transport of stem cell grafts.

  2. Regulation of HDL on hematopoietic stem/progenitor cells in atherosclerosis requires SR-BI expression

    PubMed Central

    Gao, Mingming; Zhao, Dong; Schouteden, Sarah; Sorci-Thomas, Mary G.; Van Veldhoven, Paul P.; Eggermont, Kristel; Liu, George; Verfaillie, Catherine M.; Feng, Yingmei

    2014-01-01

    Objective Recently we demonstrated that scavenger receptor type BI (SR-BI), a HDL receptor, was expressed on murine hematopoietic stem/progenitor cells (HSPC) and infusion of reconstituted HDL and purified human apoA-I suppressed HSPC proliferation. We hypothesized that SR-B1 expression is required for the observed anti-proliferative effects of HDL on HSPC. Approach and Results SR-BI deficient (SR-BI−/−) mice and wild type (WT) controls were fed on chow or HFD (HFD) for 8–10 weeks. Under chow diet, a significant increase in Lin-Sca1+cKit+ cells (LSK cells, so called HSPC) was found in the BM of SR-BI−/− mice compared with WT mice. HFD induced a further expansion of CD150+CD48− LSK cells (HSCs), HSPCs, and granulocyte monocyte progenitors (GMPs) in SR-BI−/− mice. Injection of reactive oxygen species (ROS) inhibitor N-acetylcysteine attenuated HFD-induced HSPC expansion, leukocytosis and atherosclerosis in SR-BI−/− mice. ApoA-I infusion inhibited HSPC cell proliferation, Akt phosphorylation and ROS production in HSPC and plaque progression in low density lipoprotein receptor knockout (LDLr−/−) apoA-I−/− mice on HFD but had no effect on SR-BI−/− mice on HFD. Transplantation of SR-BI−/− BM cells into irradiated LDLr−/− recipients resulted in enhanced white blood cells (WBC) reconstitution, inflammatory cell production and plaque development. In patients with coronary heart disease, HDL levels were negatively correlated with WBC count and HSPC frequency in the peripheral blood. By flow cytometry, SR-BI expression was detected on human HSPC. Conclusions SR-BI plays a critical role in the HDL-mediated regulation HSPC proliferation and differentiation which is associated with atherosclerosis progression. PMID:24969774

  3. Long-active granulocyte colony-stimulating factor for peripheral blood hematopoietic progenitor cell mobilization.

    PubMed

    Martino, Massimo; Laszlo, Daniele; Lanza, Francesco

    2014-06-01

    Peg-filgrastim (PEG-FIL), a polyethylene glycol-conjugated form of granulocyte colony-stimulating factor (G-CSF), has been introduced in clinical practice and is effective in shortening the time of neutropenia after cytotoxic chemotherapy. G-CSF has emerged as the preferred cytokine for hematopoietic progenitor cells' (HPC) mobilization. Nevertheless, data on the ability of PEG-FIL in this field have been published. We review publications in the field with the goal of providing an overview of this approach. PEG-FIL may be able to mobilize CD34(+) cells in a more timely fashion than G-CSF, with the advantages of only a single-dose administration, an earlier start and a reduction in the number of apheresis procedures. The main controversies concern the dosage of the drug and the optimal dose. In the context of chemo-mobilization, a single dose of 6 mg PEG-FIL seems effective in terms of HPC's mobilization and there is no increase in this effect if the dose is doubled to 12 mg. Steady-state mobilization requires higher doses of PEG-FIL and this approach is not cost-effective when compared with G-CSF. The experiences with PEG-FIL in the healthy donor setting are very limited.

  4. Growth Factor-Activated Stem Cell Circuits and Stromal Signals Cooperatively Accelerate Non-Integrated iPSC Reprogramming of Human Myeloid Progenitors

    PubMed Central

    Park, Tea Soon; Huo, Jeffrey S.; Peters, Ann; Talbot, C. Conover; Verma, Karan; Zimmerlin, Ludovic; Kaplan, Ian M.; Zambidis, Elias T.

    2012-01-01

    Nonviral conversion of skin or blood cells into clinically useful human induced pluripotent stem cells (hiPSC) occurs in only rare fractions (∼0.001%–0.5%) of donor cells transfected with non-integrating reprogramming factors. Pluripotency induction of developmentally immature stem-progenitors is generally more efficient than differentiated somatic cell targets. However, the nature of augmented progenitor reprogramming remains obscure, and its potential has not been fully explored for improving the extremely slow pace of non-integrated reprogramming. Here, we report highly optimized four-factor reprogramming of lineage-committed cord blood (CB) myeloid progenitors with bulk efficiencies of ∼50% in purified episome-expressing cells. Lineage-committed CD33+CD45+CD34− myeloid cells and not primitive hematopoietic stem-progenitors were the main targets of a rapid and nearly complete non-integrated reprogramming. The efficient conversion of mature myeloid populations into NANOG+TRA-1-81+ hiPSC was mediated by synergies between hematopoietic growth factor (GF), stromal activation signals, and episomal Yamanaka factor expression. Using a modular bioinformatics approach, we demonstrated that efficient myeloid reprogramming correlated not to increased proliferation or endogenous Core factor expressions, but to poised expression of GF-activated transcriptional circuits that commonly regulate plasticity in both hematopoietic progenitors and embryonic stem cells (ESC). Factor-driven conversion of myeloid progenitors to a high-fidelity pluripotent state was further accelerated by soluble and contact-dependent stromal signals that included an implied and unexpected role for Toll receptor-NFκB signaling. These data provide a paradigm for understanding the augmented reprogramming capacity of somatic progenitors, and reveal that efficient induced pluripotency in other cell types may also require extrinsic activation of a molecular framework that commonly regulates self

  5. Self-Renewal of Single Mouse Hematopoietic Stem Cells Is Reduced by JAK2V617F Without Compromising Progenitor Cell Expansion

    PubMed Central

    Kent, David G.; Li, Juan; Tanna, Hinal; Fink, Juergen; Kirschner, Kristina; Pask, Dean C.; Silber, Yvonne; Hamilton, Tina L.; Sneade, Rachel; Simons, Benjamin D.; Green, Anthony R.

    2013-01-01

    Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F

  6. Osteoclast Progenitors Reside in the Peroxisome Proliferator-Activated Receptor γ-Expressing Bone Marrow Cell Population ▿

    PubMed Central

    Wei, Wei; Zeve, Daniel; Wang, Xueqian; Du, Yang; Tang, Wei; Dechow, Paul C.; Graff, Jonathan M.; Wan, Yihong

    2011-01-01

    Osteoclasts are bone-resorbing cells essential for skeletal development, homeostasis, and regeneration. They derive from hematopoietic progenitors in the monocyte/macrophage lineage and differentiate in response to RANKL. However, the precise nature of osteoclast progenitors is a longstanding and important question. Using inducible peroxisome proliferator-activated receptor γ (PPARγ)-tTA TRE-GFP (green fluorescent protein) reporter mice, we show that osteoclast progenitors reside specifically in the PPARγ-expressing hematopoietic bone marrow population and identify the quiescent PPARγ+ cells as osteoclast progenitors. Importantly, two PPARγ-tTA TRE-Cre-controlled genetic models provide compelling functional evidence. First, Notch activation in PPARγ+ cells causes high bone mass due to impaired osteoclast precursor proliferation. Second, selective ablation of PPARγ+ cells by diphtheria toxin also causes high bone mass due to decreased osteoclast numbers. Furthermore, PPARγ+ cells respond to both pathological and pharmacological resorption-enhancing stimuli. Mechanistically, PPARγ promotes osteoclast progenitors by activating GATA2 transcription. These findings not only identify the long-sought-after osteoclast progenitors but also establish unprecedented tools for their visualization, isolation, characterization, and genetic manipulation. PMID:21947280

  7. Aging-associated inflammation promotes selection for adaptive oncogenic events in B cell progenitors.

    PubMed

    Henry, Curtis J; Casás-Selves, Matias; Kim, Jihye; Zaberezhnyy, Vadym; Aghili, Leila; Daniel, Ashley E; Jimenez, Linda; Azam, Tania; McNamee, Eoin N; Clambey, Eric T; Klawitter, Jelena; Serkova, Natalie J; Tan, Aik Choon; Dinarello, Charles A; DeGregori, James

    2015-12-01

    The incidence of cancer is higher in the elderly; however, many of the underlying mechanisms for this association remain unexplored. Here, we have shown that B cell progenitors in old mice exhibit marked signaling, gene expression, and metabolic defects. Moreover, B cell progenitors that developed from hematopoietic stem cells (HSCs) transferred from young mice into aged animals exhibited similar fitness defects. We further demonstrated that ectopic expression of the oncogenes BCR-ABL, NRAS(V12), or Myc restored B cell progenitor fitness, leading to selection for oncogenically initiated cells and leukemogenesis specifically in the context of an aged hematopoietic system. Aging was associated with increased inflammation in the BM microenvironment, and induction of inflammation in young mice phenocopied aging-associated B lymphopoiesis. Conversely, a reduction of inflammation in aged mice via transgenic expression of α-1-antitrypsin or IL-37 preserved the function of B cell progenitors and prevented NRAS(V12)-mediated oncogenesis. We conclude that chronic inflammatory microenvironments in old age lead to reductions in the fitness of B cell progenitor populations. This reduced progenitor pool fitness engenders selection for cells harboring oncogenic mutations, in part due to their ability to correct aging-associated functional defects. Thus, modulation of inflammation--a common feature of aging--has the potential to limit aging-associated oncogenesis.

  8. MERP1: a mammalian ependymin-related protein gene differentially expressed in hematopoietic cells.

    PubMed

    Gregorio-King, Claudia C; McLeod, Janet L; Collier, Fiona McL; Collier, Gregory R; Bolton, Karyn A; Van Der Meer, Gavin J; Apostolopoulos, Jim; Kirkland, Mark A

    2002-03-20

    We have utilized differential display polymerase chain reaction to investigate the gene expression of hematopoietic progenitor cells from adult bone marrow and umbilical cord blood. A differentially expressed gene was identified in CD34+ hematopoietic progenitor cells, with low expression in CD34- cells. We have obtained the full coding sequence of this gene which we designated human mammalian ependymin-related protein 1 (MERP1). Expression of MERP1 was found in a variety of normal human tissues, and is 4- and 10-fold higher in adult bone marrow and umbilical cord blood CD34+ cells, respectively, compared to CD34- cells. Additionally, MERP1 expression in a hematopoietic stem cell enriched population was down-regulated with proliferation and differentiation. Conceptual translation of the MERP1 open reading frame reveals significant homology to two families of glycoprotein calcium-dependant cell adhesion molecules: ependymins and protocadherins.

  9. The actin polymerization regulator WAVE2 is required for early bone marrow repopulation by hematopoietic stem cells.

    PubMed

    Ogaeri, Takunori; Eto, Koji; Otsu, Makoto; Ema, Hideo; Nakauchi, Hiromitsu

    2009-05-01

    The Rho GTPase family members play essential roles in hematopoiesis. Of these, Rac1 is thought to be required for the appropriate spatial localization of hematopoietic stem and/or progenitor cells (HSPCs) within the bone marrow (BM), whereas Rac2 likely plays a role in BM retention of HSPCs. To elucidate the molecular mechanisms underlying Rac-mediated functions in hematopoietic stem cells (HSCs), we studied Wiskott-Aldrich syndrome protein family verprolin-homologous proteins (WAVEs), the specific effectors downstream of the Rac GTPases in actin polymerization. We here showed that CD34(-/low)c-Kit(+)Sca-1(+)lineage(-) HSCs (CD34(-)KSL HSCs) express WAVE2 but neither WAVE1 nor WAVE3. Because WAVE2 knockout mice are embryonic-lethal, we utilized HSCs in which the expression of WAVE2 was reduced by small interfering RNA. We found that knockdown (KD) of WAVE2 in HSCs affected neither in vitro colony formation nor cell proliferation but did impair in vivo long-term reconstitution. Interestingly, WAVE2 KD HSCs exhibited unaltered homing but showed poor BM repopulation detected as early as day 5 after transplantation. The mechanistic studies on WAVE2 KD HSCs revealed modest but significant impairment in both cobblestone-like area-forming on stromal layers and actin polymerization upon integrin ligation by fibronectin. These results suggested that WAVE2-mediated actin polymerization, potentially downstream of Rac1, plays an important role in intramarrow mobilization and proliferation of HSCs, which are believed to be crucial steps for long-term marrow reconstitution after transplantation.

  10. Early Lineage Priming by Trisomy of Erg Leads to Myeloproliferation in a Down Syndrome Model

    PubMed Central

    Ng, Ashley P.; Hu, Yifang; Metcalf, Donald; Hyland, Craig D.; Ierino, Helen; Phipson, Belinda; Wu, Di; Baldwin, Tracey M.; Kauppi, Maria; Kiu, Hiu; Di Rago, Ladina; Hilton, Douglas J.; Smyth, Gordon K.; Alexander, Warren S.

    2015-01-01

    Down syndrome (DS), with trisomy of chromosome 21 (HSA21), is the commonest human aneuploidy. Pre-leukemic myeloproliferative changes in DS foetal livers precede the acquisition of GATA1 mutations, transient myeloproliferative disorder (DS-TMD) and acute megakaryocytic leukemia (DS-AMKL). Trisomy of the Erg gene is required for myeloproliferation in the Ts(1716)65Dn DS mouse model. We demonstrate here that genetic changes specifically attributable to trisomy of Erg lead to lineage priming of primitive and early multipotential progenitor cells in Ts(1716)65Dn mice, excess megakaryocyte-erythroid progenitors, and malignant myeloproliferation. Gene expression changes dependent on trisomy of Erg in Ts(1716)65Dn multilineage progenitor cells were correlated with those associated with trisomy of HSA21 in human DS hematopoietic stem and primitive progenitor cells. These data suggest a role for ERG as a regulator of hematopoietic lineage potential, and that trisomy of ERG in the context of DS foetal liver hemopoiesis drives the pre-leukemic changes that predispose to subsequent DS-TMD and DS-AMKL. PMID:25973911

  11. BMS 493 Modulates Retinoic Acid-Induced Differentiation During Expansion of Human Hematopoietic Progenitor Cells for Islet Regeneration.

    PubMed

    Elgamal, Ruth M; Bell, Gillian I; Krause, Sarah C T; Hess, David A

    2018-06-06

    Cellular therapies are emerging as a novel treatment strategy for diabetes. Thus, the induction of endogenous islet regeneration in situ represents a feasible goal for diabetes therapy. Umbilical cord blood-derived hematopoietic progenitor cells (HPCs), isolated by high aldehyde dehydrogenase activity (ALDH hi ), have previously been shown to reduce hyperglycemia after intrapancreatic (iPan) transplantation into streptozotocin (STZ)-treated nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. However, these cells are rare and require ex vivo expansion to reach clinically applicable numbers for human therapy. Therefore, we investigated whether BMS 493, an inverse retinoic acid receptor agonist, could prevent retinoic acid-induced differentiation and preserve islet regenerative functions during expansion. After 6-day expansion, BMS 493-treated cells showed a twofold increase in the number of ALDH hi cells available for transplantation compared with untreated controls. Newly expanded ALDH hi cells showed increased numbers of CD34 and CD133-positive cells, as well as a reduction in CD38 expression, a marker of hematopoietic cell differentiation. BMS 493-treated cells showed similar hematopoietic colony-forming capacity compared with untreated cells, with ALDH hi subpopulations producing more colonies than low aldehyde dehydrogenase activity subpopulations for expanded cells. To determine if the secreted proteins of these cells could augment the survival and/or proliferation of β-cells in vitro, conditioned media (CM) from cells expanded with or without BMS 493 was added to human islet cultures. The total number of proliferating β-cells was increased after 3- or 7-day culture with CM generated from BMS 493-treated cells. In contrast to freshly isolated ALDH hi cells, 6-day expansion with or without BMS 493 generated progeny that were unable to reduce hyperglycemia after iPan transplantation into STZ-treated NOD/SCID mice. Further strategies to reduce

  12. Modification of Hematopoietic Stem/Progenitor Cells with CD19-Specific Chimeric Antigen Receptors as a Novel Approach for Cancer Immunotherapy

    PubMed Central

    Ryan, Christine; Giannoni, Francesca; Hardee, Cinnamon L.; Tremcinska, Irena; Katebian, Behrod; Wherley, Jennifer; Sahaghian, Arineh; Tu, Andy; Grogan, Tristan; Elashoff, David; Cooper, Laurence J.N.; Hollis, Roger P.; Kohn, Donald B.

    2013-01-01

    Abstract Chimeric antigen receptors (CARs) against CD19 have been shown to direct T-cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. However, in some cases, there has been insufficient persistence of effector cells, limiting clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to deliver the CD19-specific CAR, with potential for ensuring persistent production of effector cells of multiple lineages targeting B-lineage malignant cells. Assessments were performed using in vitro myeloid or natural killer (NK) cell differentiation of human HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. Gene transfer did not impair hematopoietic differentiation and cell proliferation when transduced at 1–2 copies/cell. CAR-bearing myeloid and NK cells specifically lysed CD19-positive cells, with second-generation CAR including CD28 domains being more efficient in NK cells. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a strategy for generating multiple lineages of effector cells for immunotherapy against B-lineage malignancies to augment graft-versus-leukemia activity. PMID:23978226

  13. Cutting the brakes on hematopoietic regeneration by blocking TGFβ to limit chemotherapy-induced myelosuppression

    PubMed Central

    Brenet, Fabienne; Scandura, Joseph M

    2015-01-01

    Hematopoietic stressors such as infection, bleeding, or toxic injury trigger a hematopoietic adaptation that sacrifices hematopoietic stem and progenitor cell (HSPC) quiescence to meet an urgent need for new blood cell production. Once the hematopoietic demands are adequately met, homeostasis must be restored. Transforming growth factor β (TGFβ) signaling is a central mediator mandating the return of HSPCs to quiescence after stress. Blockade of TGFβ signaling after hematopoietic stress delays the return of cycling HSPCs to quiescence and in so doing promotes hematopoietic stem cell (HSC) self-renewal and accelerates hematopoietic reconstitution. These findings open the door to new therapeutics that modulate the hematopoietic adaptation to stress. In this review, we will discuss the complex context-dependent activities of TGFβ in hematopoiesis and the potential benefits and limitations of using TGFβ pathway inhibitors to promote multilineage hematopoietic reconstitution after myelosuppressive chemotherapy. PMID:27308454

  14. The ISWI ATPase Smarca5 (Snf2h) Is Required for Proliferation and Differentiation of Hematopoietic Stem and Progenitor Cells.

    PubMed

    Kokavec, Juraj; Zikmund, Tomas; Savvulidi, Filipp; Kulvait, Vojtech; Edelmann, Winfried; Skoultchi, Arthur I; Stopka, Tomas

    2017-06-01

    The imitation switch nuclear ATPase Smarca5 (Snf2h) is one of the most conserved chromatin remodeling factors. It exists in a variety of oligosubunit complexes that move DNA with respect to the histone octamer to generate regularly spaced nucleosomal arrays. Smarca5 interacts with different accessory proteins and represents a molecular motor for DNA replication, repair, and transcription. We deleted Smarca5 at the onset of definitive hematopoiesis (Vav1-iCre) and observed that animals die during late fetal development due to anemia. Hematopoietic stem and progenitor cells accumulated but their maturation toward erythroid and myeloid lineages was inhibited. Proerythroblasts were dysplastic while basophilic erythroblasts were blocked in G2/M and depleted. Smarca5 deficiency led to increased p53 levels, its activation at two residues, one associated with DNA damage (S15 Ph ° s ) second with CBP/p300 (K376 Ac ), and finally activation of the p53 targets. We also deleted Smarca5 in committed erythroid cells (Epor-iCre) and observed that animals were anemic postnatally. Furthermore, 4-hydroxytamoxifen-mediated deletion of Smarca5 in the ex vivo cultures confirmed its requirement for erythroid cell proliferation. Thus, Smarca5 plays indispensable roles during early hematopoiesis and erythropoiesis. Stem Cells 2017;35:1614-1623. © 2017 AlphaMed Press.

  15. Supernova 2012ec: identification of the progenitor and early monitoring with PESSTO

    NASA Astrophysics Data System (ADS)

    Maund, J. R.; Fraser, M.; Smartt, S. J.; Botticella, M. T.; Barbarino, C.; Childress, M.; Gal-Yam, A.; Inserra, C.; Pignata, G.; Reichart, D.; Schmidt, B.; Sollerman, J.; Taddia, F.; Tomasella, L.; Valenti, S.; Yaron, O.

    2013-04-01

    We present the identification of the progenitor of the Type IIP SN 2012ec in archival pre-explosion Hubble Space Telescope Wide Field Planetary Camera 2 (WFPC2) and Advanced Camera for Surveys Wide Field Channel F814W images. The properties of the progenitor are further constrained by non-detections in pre-explosion WFPC2 F450W and F606W images. We report a series of early photometric and spectroscopic observations of SN 2012ec. The r'-band light curve shows a plateau with M_{r^' }}=-17.0. The early spectrum is similar to the Type IIP SN 1999em, with the expansion velocity measured at Hα absorption minimum of -11 700 km s-1 (at 1 d post-discovery). The photometric and spectroscopic evolution of SN 2012ec shows it to be a Type IIP SN, discovered only a few days post-explosion (<6 d). We derive a luminosity for the progenitor, in comparison with MARCS model spectral energy distributions, of log {L/L}_{⊙} = 5.15± 0.19, from which we infer an initial mass range of 14-22 M⊙. This is the first SN with an identified progenitor to be followed by the Public ESO Spectroscopic Survey of Transient Objects (PESSTO).

  16. Identification of MS4A3 as a reliable marker for early myeloid differentiation in human hematopoiesis.

    PubMed

    Ishibashi, Tomohiko; Yokota, Takafumi; Satoh, Yusuke; Ichii, Michiko; Sudo, Takao; Doi, Yukiko; Ueda, Tomoaki; Nagate, Yasuhiro; Hamanaka, Yuri; Tanimura, Akira; Ezoe, Sachiko; Shibayama, Hirohiko; Oritani, Kenji; Kanakura, Yuzuru

    2018-01-15

    Information of myeloid lineage-related antigen on hematopoietic stem/progenitor cells (HSPCs) is important to clarify the mechanisms regulating hematopoiesis, as well as for the diagnosis and treatment of myeloid malignancies. We previously reported that special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, promotes lymphoid differentiation from HSPCs. To search a novel cell surface molecule discriminating early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSPCs with mock-transduced HSPCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (Ms4a3) as the most downregulated molecule in HSPCs with forced overexpression of SATB1. Ms4a3 expression was undetectable in hematopoietic stem cells, but showed a concomitant increase with progressive myeloid differentiation, whereas not only lymphoid but also megakaryocytic-erythrocytic progenitors were entirely devoid of Ms4a3 expression. Further analysis revealed that a subset of CD34 + CD38 + CD33 + progenitor population in human adult bone marrow expressed MS4A3, and those MS4A3 + progenitors only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Fanca-/- hematopoietic stem cells demonstrate a mobilization defect which can be overcome by administration of the Rac inhibitor NSC23766.

    PubMed

    Milsom, Michael D; Lee, Andrew W; Zheng, Yi; Cancelas, Jose A

    2009-07-01

    Fanconi anemia is a severe bone marrow failure syndrome resulting from inactivating mutations of Fanconi anemia pathway genes. Gene and cell therapy trials using hematopoietic stem cells and progenitors have been hampered by poor mobilization of HSC to peripheral blood in response to G-CSF. Using a murine model of Fanconi anemia (Fanca(-/-) mice), we found that the Fanca deficiency was associated with a profound defect in hematopoietic stem cells and progenitors mobilization in response to G-CSF in absence of bone marrow failure, which correlates with the findings of clinical trials in Fanconi anemia patients. This mobilization defect was overcome by co-administration of the Rac inhibitor NSC23766, suggesting that Rac signaling is implicated in the retention of Fanca(-/-) hematopoietic stem cells and progenitors in the bone marrow. In view of these data, we propose that targeting Rac signaling may enhance G-CSF-induced HSC mobilization in Fanconi anemia.

  18. Mitigation of radiation-induced hematopoietic injury by the polyphenolic acetate 7, 8-diacetoxy-4-methylthiocoumarin in mice

    PubMed Central

    Venkateswaran, Kavya; Shrivastava, Anju; Agrawala, Paban K.; Prasad, Ashok; Kalra, Namita; Pandey, Parvat R.; Manda, Kailash; Raj, Hanumantharao G.; Parmar, Virinder S.; Dwarakanath, Bilikere S.

    2016-01-01

    Protection of the hematopoietic system from radiation damage, and/or mitigation of hematopoietic injury are the two major strategies for developing medical countermeasure agents (MCM) to combat radiation-induced lethality. In the present study, we investigated the potential of 7, 8-diacetoxy-4-methylthiocoumarin (DAMTC) to ameliorate radiation-induced hematopoietic damage and the associated mortality following total body irradiation (TBI) in C57BL/6 mice. Administration of DAMTC 24 hours post TBI alleviated TBI-induced myelo-suppression and pancytopenia, by augmenting lymphocytes and WBCs in the peripheral blood of mice, while bone marrow (BM) cellularity was restored through enhanced proliferation of the stem cells. It stimulated multi-lineage expansion and differentiation of myeloid progenitors in the BM and induced proliferation of splenic progenitors thereby, facilitating hematopoietic re-population. DAMTC reduced the radiation-induced apoptotic and mitotic death in the hematopoietic compartment. Recruitment of pro-inflammatory M1 macrophages in spleen contributed to the immune-protection linked to the mitigation of hematopoietic injury. Recovery of the hematopoietic compartment correlated well with mitigation of mortality at a lethal dose of 9 Gy, leading to 80% animal survival. Present study establishes the potential of DAMTC to mitigate radiation-induced injury to the hematopoietic system by stimulating the re-population of stem cells from multiple lineages. PMID:27849061

  19. Potential role of immunoablation and hematopoietic cell transplantation in the treatment of early diabetes type 1.

    PubMed

    Snarski, Emilian; Milczarczyk, Alicja; Franek, Edward; Jedrzejczak, Wieslaw

    2010-01-01

    Immunoablation with autologous hematopoietic cell transplantation has shown some effectiveness in the treatment of autoimmune diseases as diverse as aplastic anemia, systemic lupus erythematosus, multiple sclerosis and Crohn's disease. It has been recently shown that this treatment might prevent or delay development of diabetes type 1. The majority of more than 30 patients with early diabetes type 1 who underwent immunoablation and hematopoietic cell transplantation in various centers in the world achieved durable remission of diabetes and independence of exogenous insulin. This review summarizes advantages and risks of this treatment of early diabetes type 1.

  20. TGFβ restores hematopoietic homeostasis after myelosuppressive chemotherapy

    PubMed Central

    Brenet, Fabienne; Kermani, Pouneh; Spektor, Roman; Rafii, Shahin

    2013-01-01

    Myelosuppression is a life-threatening complication of antineoplastic therapy, but treatment is restricted to a few cytokines with unilineage hematopoietic activity. Although hematopoietic stem cells (HSCs) are predominantly quiescent during homeostasis, they are rapidly recruited into cell cycle by stresses, including myelosuppressive chemotherapy. Factors that induce HSCs to proliferate during stress have been characterized, but it is not known how HSC quiescence is then reestablished. In this study, we show that TGFβ signaling is transiently activated in hematopoietic stem and progenitor cells (HSPCs) during hematopoietic regeneration. Blockade of TGFβ signaling after chemotherapy accelerates hematopoietic reconstitution and delays the return of cycling HSCs to quiescence. In contrast, TGFβ blockade during homeostasis fails to induce cycling of HSPCs. We identified the cyclin-dependent kinase inhibitor Cdkn1c (p57) as a key downstream mediator of TGFβ during regeneration because the recovery of chimeric mice, incapable of expressing p57 in HSPCs, phenocopies blockade of TGFβ signaling after chemotherapy. This study demonstrates that context-dependent activation of TGFβ signaling is central to an unrecognized counterregulatory mechanism that promotes homeostasis once hematopoiesis has sufficiently recovered from myelosuppressive chemotherapy. These results open the door to new, potentially superior, approaches to promote multilineage hematopoietic recovery by blocking the TGFβ signaling that dampens regeneration. PMID:23440043

  1. Sumoylation of CCAAT/enhancer-binding protein α is implicated in hematopoietic stem/progenitor cell development through regulating runx1 in zebrafish.

    PubMed

    Yuan, Hao; Zhang, Tao; Liu, Xiaohui; Deng, Min; Zhang, Wenqing; Wen, Zilong; Chen, Saijuan; Chen, Zhu; de The, Hugues; Zhou, Jun; Zhu, Jun

    2015-03-11

    The small ubiquitin-related modifier (SUMO) participates in various cellular processes, including maintenance of genome integrity, nuclear transport, transcription and signal transduction. However, the biological function of sumoylation in hematopoiesis has not been fully explored. We show here that definitive hematopoietic stem/progenitor cells (HSPCs) are depleted in SUMO-deficient zebrafish embryos. Impairment of sumoylation attenuates HSPC generation and proliferation. The hyposumoylation triggered HSPC defects are CCAAT/enhancer-binding protein α (C/ebpα) dependent. Critically, a SUMO-C/ebpα fusion rescues the defective hematopoiesis in SUMO-deficient embryos, at least in part through restored runx1 expression. While C/ebpα-dependent transcription is involved in myeloid differentiation, our studies here reveal that C/ebpα sumoylation is essential for HSPC development during definitive hematopoiesis.

  2. The Differential Expression of Adhesion Molecule and Extracellular Matrix Genes in Mesenchymal Stromal Cells after Interaction with Cord Blood Hematopoietic Progenitors.

    PubMed

    Buravkova, L B; Andreeva, E R; Lobanova, M V; Cotnezova, E V; Grigoriev, A I

    2018-03-01

    The dynamics of the expression of genes encoding adhesion molecules, molecules of the connective tissue matrix, and its remodeling enzymes was studied in multipotent mesenchymal stromal cells (MSCs) from human adipose tissue after interaction with cord blood hematopoietic progenitors (HSPCs). An upregulation of ICAM1 and VCAM1, directly proportional to the coculture time (24-72 h), was found. After 72 h of culturing, a downregulation of the genes encoding the majority of matrix molecules (SPP1; COL6A2,7A1; MMP1,3; TIMP1,3; and HAS1) and cell-matrix adhesion molecules (ITGs) was revealed. The detected changes may ensure the realization of the stromal MSC function due to improvement of adhesion and transmigration of HSPCs into the subcellular space.

  3. Lack of Phenotypical and Morphological Evidences of Endothelial to Hematopoietic Transition in the Murine Embryonic Head during Hematopoietic Stem Cell Emergence.

    PubMed

    Iizuka, Kazuhide; Yokomizo, Tomomasa; Watanabe, Naoki; Tanaka, Yosuke; Osato, Motomi; Takaku, Tomoiku; Komatsu, Norio

    2016-01-01

    During mouse ontogeny, hematopoietic cells arise from specialized endothelial cells, i.e., the hemogenic endothelium, and form clusters in the lumen of arterial vessels. Hemogenic endothelial cells have been observed in several embryonic tissues, such as the dorsal aorta, the placenta and the yolk sac. Recent work suggests that the mouse embryonic head also produces hematopoietic stem cells (HSCs)/progenitors. However, a histological basis for HSC generation in the head has not yet been determined because the hematopoietic clusters and hemogenic endothelium in the head region have not been well characterized. In this study, we used whole-mount immunostaining and 3D confocal reconstruction techniques to analyze both c-Kit+ hematopoietic clusters and Runx1+ hemogenic endothelium in the whole-head vasculature. The number of c-Kit+ hematopoietic cells was 20-fold less in the head arteries than in the dorsal aorta. In addition, apparent nascent hematopoietic cells, which are characterized by a "budding" structure and a Runx1+ hemogenic endothelium, were not observed in the head. These results suggest that head HSCs may not be or are rarely generated from the endothelium in the same manner as aortic HSCs.

  4. Lack of Phenotypical and Morphological Evidences of Endothelial to Hematopoietic Transition in the Murine Embryonic Head during Hematopoietic Stem Cell Emergence

    PubMed Central

    Iizuka, Kazuhide; Yokomizo, Tomomasa; Watanabe, Naoki; Tanaka, Yosuke; Osato, Motomi; Takaku, Tomoiku; Komatsu, Norio

    2016-01-01

    During mouse ontogeny, hematopoietic cells arise from specialized endothelial cells, i.e., the hemogenic endothelium, and form clusters in the lumen of arterial vessels. Hemogenic endothelial cells have been observed in several embryonic tissues, such as the dorsal aorta, the placenta and the yolk sac. Recent work suggests that the mouse embryonic head also produces hematopoietic stem cells (HSCs)/progenitors. However, a histological basis for HSC generation in the head has not yet been determined because the hematopoietic clusters and hemogenic endothelium in the head region have not been well characterized. In this study, we used whole-mount immunostaining and 3D confocal reconstruction techniques to analyze both c-Kit+ hematopoietic clusters and Runx1+ hemogenic endothelium in the whole-head vasculature. The number of c-Kit+ hematopoietic cells was 20-fold less in the head arteries than in the dorsal aorta. In addition, apparent nascent hematopoietic cells, which are characterized by a “budding” structure and a Runx1+ hemogenic endothelium, were not observed in the head. These results suggest that head HSCs may not be or are rarely generated from the endothelium in the same manner as aortic HSCs. PMID:27227884

  5. Pituitary adenylate cyclase-activating polypeptide (PACAP) contributes to the proliferation of hematopoietic progenitor cells in murine bone marrow via PACAP-specific receptor

    PubMed Central

    Xu, Zhifang; Ohtaki, Hirokazu; Watanabe, Jun; Miyamoto, Kazuyuki; Murai, Norimitsu; Sasaki, Shun; Matsumoto, Minako; Hashimoto, Hitoshi; Hiraizumi, Yutaka; Numazawa, Satoshi; Shioda, Seiji

    2016-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP, encoded by adcyap1) plays an important role in ectodermal development. However, the involvement of PACAP in the development of other germ layers is still unclear. This study assessed the expression of a PACAP-specific receptor (PAC1) gene and protein in mouse bone marrow (BM). Cells strongly expressing PAC1+ were large in size, had oval nuclei, and merged with CD34+ cells, suggesting that the former were hematopoietic progenitor cells (HPCs). Compared with wild-type mice, adcyap1−/− mice exhibited lower multiple potential progenitor cell populations and cell frequency in the S-phase of the cell cycle. Exogenous PACAP38 significantly increased the numbers of colony forming unit-granulocyte/macrophage progenitor cells (CFU-GM) with two peaks in semi-solid culture. PACAP also increased the expression of cyclinD1 and Ki67 mRNAs. These increases were completely and partially inhibited by the PACAP receptor antagonists, PACAP6-38 and VIP6-28, respectively. Little or no adcyap1 was expressed in BM and the number of CFU-GM colonies was similar in adcyap1−/− and wild-type mice. However, PACAP mRNA and protein were expressed in paravertebral sympathetic ganglia, which innervate tibial BM, and in the sympathetic fibers of BM cavity. These results suggested that sympathetic nerve innervation may be responsible for PACAP-regulated hematopoiesis in BM, mainly via PAC1. PMID:26925806

  6. Autologous hematopoietic progenitor cell mobilization and collection in adult patients presenting with multiple myeloma and lymphoma: A position-statement from the Turkish Society of Apheresis (TSA).

    PubMed

    Tekgündüz, Emre; Arat, Mutlu; Göker, Hakan; Özdoğu, Hakan; Kaynar, Leylagül; Çağırgan, Seçkin; Erkurt, Mehmet Ali; Vural, Filiz; Kiki, İlhami; Altuntaş, Fevzi; Demirkan, Fatih

    2017-12-01

    Autologous hematopoietic cell transplantation (AHCT) is a routinely used procedure in the treatment of adult patients presenting with multiple myeloma (MM), Hodgkin lymphoma (HL) and various subtypes of non-Hodgkin lymphoma (NHL) in upfront and relapsed/refractory settings. Successful hematopoietic progenitor cell mobilization (HPCM) and collection are the rate limiting first steps for application of AHCT. In 2015, almost 1700 AHCT procedures have been performed for MM, HL and NHL in Turkey. Although there are recently published consensus guidelines addressing critical issues regarding autologous HPCM, there is a tremendous heterogeneity in terms of mobilization strategies of transplant centers across the world. In order to pave the way to a more standardized HPCM approach in Turkey, Turkish Society of Apheresis (TSA) assembled a working group consisting of experts in the field. Here we report the position statement of TSA regarding autologous HPCM mobilization strategies in adult patients presenting with MM and lymphoma. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Enforced expression of KDR receptor promotes proliferation, survival and megakaryocytic differentiation of TF1 progenitor cell line.

    PubMed

    Coppola, S; Narciso, L; Feccia, T; Bonci, D; Calabrò, L; Morsilli, O; Gabbianelli, M; De Maria, R; Testa, U; Peschle, C

    2006-01-01

    Vascular endothelial growth factor (VEGF) receptor-2/kinase insert domain-containing receptor (KDR) is expressed in primitive hematopoietic cells, in megakaryocytes and platelets. In primitive hematopoiesis KDR mediates cell survival via autocrine VEGF, while its effect on cell growth and differentiation has not been elucidated. We induced enforced KDR expression in the granulocyte macrophage-colony-stimulating factor (GM-CSF)-dependent TF1 progenitor cell line (TF1-KDR), treated the cells with VEGF and analyzed their response. In GM-CSF-deprived cells, VEGF induces cell proliferation and protection against apoptosis, followed by enhanced expression of megakaryocytic (MK) markers. Combined with GM-CSF, VEGF induces a mild proliferative stimulus, followed by cell adherence, accumulation in G0/G1, massive MK differentiation and Fas-mediated apoptosis. Accordingly, we observed that MK-differentiating cells, derived from hematopoietic progenitors, produce VEGF, express KDR, inhibition of which reduces MK differentiation, indicating a key role of KDR in megakaryopoiesis. In conclusion, TF1-KDR cells provide a reliable model to investigate the biochemical and molecular mechanisms underlying hematopoietic progenitor proliferation, survival and MK differentiation.

  8. In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

    PubMed Central

    Soares, P.B.; Jeremias, T.S.; Alvarez-Silva, M.; Licínio, M.A.; Santos-Silva, M.C.; Vituri, C.L.

    2012-01-01

    Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved. PMID:23011404

  9. Hematopoietic responses under protracted exposures to low daily dose gamma irradiation

    NASA Astrophysics Data System (ADS)

    Seed, T. M.; Fritz, T. E.; Tolle, D. V.; Jackson, W. E.

    In attempting to evaluate the possible health consequences of chronic ionizing radiation exposure during extended space travel (e.g., Mars Mission), ground-based experimental studies of the clinical and pathological responses of canines under low daily doses of 60Co gamma irradiation (0.3-26.3 cGy d -1) have been examined. Specific reference was given to responses of the blood forming system. Results suggest that the daily dose rate of 7.5 cGy d -1 represents a threshold below which the hematopoietic system can retain either partial or full trilineal cell-producing capacity (erythropoiesis, myelopoiesis, and megakaryopoiesis) for extended periods of exposure (> 1yr). Trilineal capacity was fully retained for several years of exposure at the lowest dose-rate tested (0.3 cGy d -1) but was completely lost within several hundred days at the highest dose-rate (26.3 cGy d -1). Retention of hematopoietic capacity under chronic exposure has been demonstrated to be mediated by hematopoietic progenitors with acquired radioresistance and repair functions, altered cytogenetics, and cell-cycle characteristics. Radiological, biological, and temporal parameters responsible for these vital acquisitions by hematopoietic progenitors have been partially characterized. These parameters, along with threshold responses, are described and discussed in relation to potential health risks of the space traveler under chronic stress of low-dose irradiation.

  10. Hematopoietic responses under protracted exposures to low daily dose gamma irradiation.

    PubMed

    Seed, T M; Fritz, T E; Tolle, D V; Jackson, W E

    2002-01-01

    In attempting to evaluate the possible health consequences of chronic ionizing radiation exposure during extended space travel (e.g., Mars Mission), ground-based experimental studies of the clinical and pathological responses of canines under low daily doses of 60Co gamma irradiation (0.3-26.3 cGy d-1) have been examined. Specific reference was given to responses of the blood forming system. Results suggest that the daily dose rate of 7.5 cGy d-1 represents a threshold below which the hematopoietic system can retain either partial or full trilineal cell-producing capacity (erythropoiesis, myelopoiesis, and megakaryopoiesis) for extended periods of exposure (>1 yr). Trilineal capacity was fully retained for several years of exposure at the lowest dose-rate tested (0.3 cGy d-1) but was completely lost within several hundred days at the highest dose-rate (26.3 cGy d-1). Retention of hematopoietic capacity under chronic exposure has been demonstrated to be mediated by hematopoietic progenitors with acquired radioresistance and repair functions, altered cytogenetics, and cell-cycle characteristics. Radiological, biological, and temporal parameters responsible for these vital acquisitions by hematopoietic progenitors have been partially characterized. These parameters, along with threshold responses, are described and discussed in relation to potential health risks of the space traveler under chronic stress of low-dose irradiation. Published by Elsevier Science Ltd on behalf of COSPAR.

  11. BIGH3 modulates adhesion and migration of hematopoietic stem and progenitor cells

    PubMed Central

    Klamer, Sofieke E; Kuijk, Carlijn GM; Hordijk, Peter L; van der Schoot, C Ellen; von Lindern, Marieke; van Hennik, Paula B; Voermans, Carlijn

    2013-01-01

    Cell adhesion and migration are important determinants of homing and development of hematopoietic stem and progenitor cells (HSPCs) in bone marrow (BM) niches. The extracellular matrix protein transforming growth factor-β (TGF-β) inducible gene H3 (BIGH3) is involved in adhesion and migration, although the effect of BIGH3 is highly cell type-dependent. BIGH3 is abundantly expressed by mesenchymal stromal cells, while its expression in HSPCs is relatively low unless induced by certain BM stressors. Here, we set out to determine how BIGH3 modulates HSPC adhesion and migration. We show that primary HSPCs adhere to BIGH3-coated substrates, which is, in part, integrin-dependent. Overexpression of BIGH3 in HSPCs and HL60 cells reduced the adhesion to the substrate fibronectin in adhesion assays, which was even more profound in electrical cell-substrate impedance sensing (ECIS) assays. Accordingly, the CXCL12 induced migration over fibronectin-coated surface was reduced in BIGH3-expressing HSPCs. The integrin expression profile of HSPCs was not altered upon BIGH3 expression. Although expression of BIGH3 did not alter actin polymerization in response to CXCL12, it inhibited the PMA-induced activation of the small GTPase RAC1 as well as the phosphorylation and activation of extracellular-regulated kinases (ERKs). Reduced activation of ERK and RAC1 may be responsible for the inhibition of cell adhesion and migration by BIGH3 in HSPCs. Induced BIGH3 expression upon BM stress may contribute to the regulation of BM homeostasis. PMID:24152593

  12. SIRT1 deficiency compromises mouse embryonic stem cell hematopoietic differentiation, and embryonic and adult hematopoiesis in the mouse

    PubMed Central

    Ou, Xuan; Chae, Hee-Don; Wang, Rui-Hong; Shelley, William C.; Cooper, Scott; Taylor, Tammi; Kim, Young-June; Deng, Chu-Xia; Yoder, Mervin C.

    2011-01-01

    SIRT1 is a founding member of a sirtuin family of 7 proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1−/− mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1−/− mouse embryonic stem cells (ESCs) in vitro, and hematopoietic progenitors in SIRT1+/++/−, and −/− mice. SIRT1−/− ESCs formed fewer mature blast cell colonies. Replated SIRT1−/− blast colony-forming cells demonstrated defective hematopoietic potential. Endothelial cell production was unaltered, but there were defects in formation of a primitive vascular network from SIRT1−/−-derived embryoid bodies. Development of primitive and definitive progenitors derived from SIRT1−/− ESCs were also delayed and/or defective. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5 expression, decreased β-H1 globin, β-major globin, and Scl gene expression, and reduced activation of Erk1/2. Ectopic expression of SIRT1 rescued SIRT1−/− ESC differentiation deficiencies. SIRT1−/− yolk sacs manifested fewer primitive erythroid precursors. SIRT1−/− and SIRT1+/− adult marrow had decreased numbers and cycling of hematopoietic progenitors, effects more apparent at 5%, than at 20%, oxygen tension, and these progenitors survived less well in vitro under conditions of delayed growth factor addition. This suggests a role for SIRT1 in ESC differentiation and mouse hematopoiesis. PMID:20966168

  13. Mismatch repair deficient hematopoietic stem cells are preleukemic stem cells

    PubMed Central

    Gerson, Stanton L.

    2017-01-01

    Whereas transformation events in hematopoietic malignancies may occur at different developmental stages, the initial mutation originates in hematopoietic stem cells (HSCs), creating a preleukemic stem cell (PLSC). Subsequent mutations at either stem cell or progenitor cell levels transform the PLSC into lymphoma/leukemia initiating cells (LIC). Thymic lymphomas have been thought to develop from developing thymocytes. T cell progenitors are generated from HSCs in the bone marrow (BM), but maturation and proliferation of T cells as well as T-lymphomagenesis depends on both regulatory mechanisms and microenvironment within the thymus. We studied PLSC linked to thymic lymphomas. In this study, we use MSH2-/- mice as a model to investigate the existence of PLSC and the evolution of PLSC to LIC. Following BM transplantation, we found that MSH2-/- BM cells from young mice are able to fully reconstitute multiple hematopoietic lineages of lethally irradiated wild-type recipients. However, all recipients developed thymic lymphomas within three and four months post transplantation. Transplantation of different fractions of BM cells or thymocytes from young health MSH2-/- mice showed that an HSC enriched fraction always reconstituted hematopoiesis followed by lymphoma development. In addition, lymphomas did not occur in thymectomized recipients of MSH2-/- BM. These results suggest that HSCs with DNA repair defects such as MSH2-/- are PLSCs because they retain hematopoietic function, but also carry an obligate lymphomagenic potential within their T-cell progeny that is dependent on the thymic microenvironment. PMID:28767666

  14. Fanca−/− hematopoietic stem cells demonstrate a mobilization defect which can be overcome by administration of the Rac inhibitor NSC23766

    PubMed Central

    Milsom, Michael D.; Lee, Andrew W.; Zheng, Yi; Cancelas, Jose A.

    2009-01-01

    Fanconi anemia is a severe bone marrow failure syndrome resulting from inactivating mutations of Fanconi anemia pathway genes. Gene and cell therapy trials using hematopoietic stem cells and progenitors have been hampered by poor mobilization of HSC to peripheral blood in response to G-CSF. Using a murine model of Fanconi anemia (Fanca−/− mice), we found that the Fanca deficiency was associated with a profound defect in hematopoietic stem cells and progenitors mobilization in response to G-CSF in absence of bone marrow failure, which correlates with the findings of clinical trials in Fanconi anemia patients. This mobilization defect was overcome by co-administration of the Rac inhibitor NSC23766, suggesting that Rac signaling is implicated in the retention of Fanca−/− hematopoietic stem cells and progenitors in the bone marrow. In view of these data, we propose that targeting Rac signaling may enhance G-CSF-induced HSC mobilization in Fanconi anemia. PMID:19491337

  15. Sumoylation of CCAAT/enhancer-binding protein α is implicated in hematopoietic stem/progenitor cell development through regulating runx1 in zebrafish

    PubMed Central

    Yuan, Hao; Zhang, Tao; Liu, Xiaohui; Deng, Min; Zhang, Wenqing; Wen, Zilong; Chen, Saijuan; Chen, Zhu; de The, Hugues; Zhou, Jun; Zhu, Jun

    2015-01-01

    The small ubiquitin-related modifier (SUMO) participates in various cellular processes, including maintenance of genome integrity, nuclear transport, transcription and signal transduction. However, the biological function of sumoylation in hematopoiesis has not been fully explored. We show here that definitive hematopoietic stem/progenitor cells (HSPCs) are depleted in SUMO-deficient zebrafish embryos. Impairment of sumoylation attenuates HSPC generation and proliferation. The hyposumoylation triggered HSPC defects are CCAAT/enhancer-binding protein α (C/ebpα) dependent. Critically, a SUMO-C/ebpα fusion rescues the defective hematopoiesis in SUMO-deficient embryos, at least in part through restored runx1 expression. While C/ebpα-dependent transcription is involved in myeloid differentiation, our studies here reveal that C/ebpα sumoylation is essential for HSPC development during definitive hematopoiesis. PMID:25757417

  16. Physiological Jak2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells.

    PubMed

    Mullally, Ann; Lane, Steven W; Ball, Brian; Megerdichian, Christine; Okabe, Rachel; Al-Shahrour, Fatima; Paktinat, Mahnaz; Haydu, J Erika; Housman, Elizabeth; Lord, Allegra M; Wernig, Gerlinde; Kharas, Michael G; Mercher, Thomas; Kutok, Jeffery L; Gilliland, D Gary; Ebert, Benjamin L

    2010-06-15

    We report a Jak2V617F knockin mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem cell (HSC) compartment has the unique capacity for disease initiation but does not have a significant selective competitive advantage over wild-type HSCs. In contrast, myeloid progenitor populations are expanded and skewed toward the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to JAK2V617F-positive MPN. Copyright 2010 Elsevier Inc. All rights reserved.

  17. Residual Expression of the Reprogramming Factors Prevents Differentiation of iPSC Generated from Human Fibroblasts and Cord Blood CD34+ Progenitors

    PubMed Central

    Ramos-Mejía, Verónica; Montes, Rosa; Bueno, Clara; Ayllón, Verónica; Real, Pedro J.; Rodríguez, René; Menendez, Pablo

    2012-01-01

    Human induced pluripotent stem cells (hiPSC) have been generated from different tissues, with the age of the donor, tissue source and specific cell type influencing the reprogramming process. Reprogramming hematopoietic progenitors to hiPSC may provide a very useful cellular system for modelling blood diseases. We report the generation and complete characterization of hiPSCs from human neonatal fibroblasts and cord blood (CB)-derived CD34+ hematopoietic progenitors using a single polycistronic lentiviral vector containing an excisable cassette encoding the four reprogramming factors Oct4, Klf4, Sox2 and c-myc (OKSM). The ectopic expression of OKSM was fully silenced upon reprogramming in some hiPSC clones and was not reactivated upon differentiation, whereas other hiPSC clones failed to silence the transgene expression, independently of the cell type/tissue origin. When hiPSC were induced to differentiate towards hematopoietic and neural lineages those hiPSC which had silenced OKSM ectopic expression displayed good hematopoietic and early neuroectoderm differentiation potential. In contrast, those hiPSC which failed to switch off OKSM expression were unable to differentiate towards either lineage, suggesting that the residual expression of the reprogramming factors functions as a developmental brake impairing hiPSC differentiation. Successful adenovirus-based Cre-mediated excision of the provirus OKSM cassette in CB-derived CD34+ hiPSC with residual transgene expression resulted in transgene-free hiPSC clones with significantly improved differentiation capacity. Overall, our findings confirm that residual expression of reprogramming factors impairs hiPSC differentiation. PMID:22545141

  18. Endothelial transplantation rejuvenates aged hematopoietic stem cell function

    PubMed Central

    Poulos, Michael G.; Gutkin, Michael C.; Llanos, Pierre; Gilleran, Katherine; Rabbany, Sina Y.; Butler, Jason M.

    2017-01-01

    Age-related changes in the hematopoietic compartment are primarily attributed to cell-intrinsic alterations in hematopoietic stem cells (HSCs); however, the contribution of the aged microenvironment has not been adequately evaluated. Understanding the role of the bone marrow (BM) microenvironment in supporting HSC function may prove to be beneficial in treating age-related functional hematopoietic decline. Here, we determined that aging of endothelial cells (ECs), a critical component of the BM microenvironment, was sufficient to drive hematopoietic aging phenotypes in young HSCs. We used an ex vivo hematopoietic stem and progenitor cell/EC (HSPC/EC) coculture system as well as in vivo EC infusions following myelosuppressive injury in mice to demonstrate that aged ECs impair the repopulating activity of young HSCs and impart a myeloid bias. Conversely, young ECs restored the repopulating capacity of aged HSCs but were unable to reverse the intrinsic myeloid bias. Infusion of young, HSC-supportive BM ECs enhanced hematopoietic recovery following myelosuppressive injury and restored endogenous HSC function in aged mice. Coinfusion of young ECs augmented aged HSC engraftment and enhanced overall survival in lethally irradiated mice by mitigating damage to the BM vascular microenvironment. These data lay the groundwork for the exploration of EC therapies that can serve as adjuvant modalities to enhance HSC engraftment and accelerate hematopoietic recovery in the elderly population following myelosuppressive regimens. PMID:29035282

  19. PUMILIO/FOXP1 signaling drives expansion of hematopoietic stem/progenitor and leukemia cells

    PubMed Central

    Naudin, Cécile; Hattabi, Aurore; Michelet, Fabio; Miri-Nezhad, Ayda; Benyoucef, Aissa; Pflumio, Françoise; Guillonneau, François; Fichelson, Serge; Vigon, Isabelle; Dusanter-Fourt, Isabelle

    2017-01-01

    RNA-binding proteins (RBPs) have emerged as important regulators of invertebrate adult stem cells, but their activities remain poorly appreciated in mammals. Using a short hairpin RNA strategy, we demonstrate here that the 2 mammalian RBPs, PUMILIO (PUM)1 and PUM2, members of the PUF family of posttranscriptional regulators, are essential for hematopoietic stem/progenitor cell (HSPC) proliferation and survival in vitro and in vivo upon reconstitution assays. Moreover, we found that PUM1/2 sustain myeloid leukemic cell growth. Through a proteomic approach, we identified the FOXP1 transcription factor as a new target of PUM1/2. Contrary to its canonical repressive activity, PUM1/2 rather promote FOXP1 expression by a direct binding to 2 canonical PUM responsive elements present in the FOXP1-3′ untranslated region (UTR). Expression of FOXP1 strongly correlates with PUM1 and PUM2 levels in primary HSPCs and myeloid leukemia cells. We demonstrate that FOXP1 by itself supports HSPC and leukemic cell growth, thus mimicking PUM activities. Mechanistically, FOXP1 represses the expression of the p21−CIP1 and p27−KIP1 cell cycle inhibitors. Enforced FOXP1 expression reverses shPUM antiproliferative and proapoptotic activities. Altogether, our results reveal a novel regulatory pathway, underscoring a previously unknown and interconnected key role of PUM1/2 and FOXP1 in regulating normal HSPC and leukemic cell growth. PMID:28232582

  20. Fancb deficiency impairs hematopoietic stem cell function

    PubMed Central

    Du, Wei; Amarachintha, Surya; Erden, Ozlem; Wilson, Andrew; Meetei, Amom Ruhikanta; Andreassen, Paul R.; Namekawa, Satoshi H.; Pang, Qishen

    2015-01-01

    Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, variable congenital malformations and a predisposition to malignancies. FANCB (also known as FAAP95), is the only X-linked FA gene discovered thus far. In the present study, we investigated hematopoiesis in adult Fancb deficient (Fancb−/y) mice and found that Fancb−/y mice have decreased hematopoietic stem cell (HSC) quiescence accompanied by reduced progenitor activity in vitro and reduced repopulating capacity in vivo. Like other FA mouse models previously reported, the hematopoietic system of Fancb−/y mice is hypersensitive to DNA cross-linking agent mitomycin C (MMC), which induces bone marrow failure in Fancb−/y mice. Furthermore, Fancb−/y BM exhibits slower recovery kinetics and less tolerance to myelotoxic stress induced by 5-fluorouracil than wild-type littermates. RNA-seq analysis reveals altered expression of genes involved in HSC function and cell cycle regulation in Fancb−/y HSC and progenitor cells. Thus, this Fancb−/y mouse model provides a novel approach for studying the critical role of the FA pathway not only in germ cell development but also in the maintenance of HSC function. PMID:26658157

  1. STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells.

    PubMed Central

    Small, D; Levenstein, M; Kim, E; Carow, C; Amin, S; Rockwell, P; Witte, L; Burrow, C; Ratajczak, M Z; Gewirtz, A M

    1994-01-01

    We cloned the cDNA for stem cell tyrosine kinase 1 (STK-1), the human homolog of murine Flk-2/Flt-3, from a CD34+ hematopoietic stem cell-enriched library and investigated its expression in subsets of normal human bone marrow. The cDNA encodes a protein of 993 aa with 85% identity and 92% similarity to Flk-2/Flt-3. STK-1 is a member of the type III receptor tyrosine kinase family that includes KIT (steel factor receptor), FMS (colony-stimulating factor 1R), and platelet-derived growth factor receptor. STK-1 expression in human blood and marrow is restricted to CD34+ cells, a population greatly enriched for stem/progenitor cells. Anti-STK-1 antiserum recognizes polypeptides of 160 and 130 kDa in several STK-1-expressing cell lines and in 3T3 cells transfected with a STK-1 expression vector. Antisense oligonucleotides directed against STK-1 sequences inhibited hematopoietic colony formation, most strongly in long-term bone marrow cultures. These data suggest that STK-1 may function as a growth factor receptor on hematopoietic stem and/or progenitor cells. Images Fig. 2 Fig. 3 Fig. 4 PMID:7507245

  2. Differentiation stage-specific regulation of primitive human hematopoietic progenitor cycling by exogenous and endogenous inhibitors in an in vivo model.

    PubMed

    Cashman, J D; Clark-Lewis, I; Eaves, A C; Eaves, C J

    1999-12-01

    Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice transplanted with human cord blood or adult marrow cells and injected 6 weeks posttransplant with 2 daily doses of transforming growth factor-beta(1) (TGF-beta(1)), monocyte chemoattractant protein-1 (MCP-1), or a nonaggregating form of macrophage inflammatory protein-1alpha (MIP-1alpha) showed unique patterns of inhibition of human progenitor proliferation 1 day later. TGF-beta(1) was active on long-term culture initiating cells (LTC-IC) and on primitive erythroid and granulopoietic colony-forming cells (HPP-CFC), but had no effect on mature CFC. MCP-1 inhibited the cycling of both types of HPP-CFC but not LTC-IC. MIP-1alpha did not inhibit either LTC-IC or granulopoietic HPP-CFC but was active on erythroid HPP-CFC and mature granulopoietic CFC. All of these responses were independent of the source of human cells transplanted. LTC-IC of either human cord blood or adult marrow origin continue to proliferate in NOD/SCID mice for many weeks, although the turnover of all types of human CFC in mice transplanted with adult human marrow (but not cord blood) is downregulated after 6 weeks. Interestingly, administration of either MIP-1beta, an antagonist of both MIP-1alpha and MCP-1 or MCP-1(9-76), an antagonist of MCP-1 (and MCP-2 and MCP-3), into mice in which human marrow-derived CFC had become quiescent, caused the rapid reactivation of these progenitors in vivo. These results provide the first definition of stage-specific inhibitors of human hematopoietic progenitor cell cycling in vivo. In addition they show that endogenous chemokines can contribute to late graft failure, which can be reversed by the administration of specific antagonists.

  3. Ex-vivo expansion of hematopoietic progenitor cells: preliminary results in breast cancer.

    PubMed

    McNiece, I; Jones, R; Cagnoni, P; Bearman, S; Nieto, Y; Shpall, E J

    1999-04-01

    Ex-vivo expanded progenitor cells have been proposed as a source of cells to support high-dose chemotherapy and to decrease or eliminate the period of neutropenia following transplantation. To date, no clinical studies using ex vivo expanded cells, have demonstrated any decrease in the time to neutrophil or platelet recovery, although a number of clinical studies have been performed using a variety of growth factor cocktails and culture conditions. Over the past 6 years we have developed a static culture system that results in optimal expansion of myeloid progenitor cells. We have initiated a clinical study to evaluate this culture system in breast cancer patients receiving peripheral blood progenitor cells (PBPC) to support high-dose chemotherapy. CD34 selected cells were cultured for 10 days in 800 ml of defined media (Amgen Inc.) containing 100 ng/ml each of rhSCF, rhG-CSF and rhMGDF in 1L teflon bags (American Fluoroseal) at 20,000 to 50,000 cells per ml. After culture the cells were washed with 3 volumes of PBS to remove all media and growth factors and reinfused on day 0 of transplant followed by daily administration of rhG-CSF. On day +1 the patients received an unexpanded PBPC product to ensure the durability of the graft. Patients transplanted with expanded PBPC cells recovered neutrophil counts (ANC > 500/microl) as early as day 4 post transplant with a median of 6 days (range 4 to 14 days). In comparison, our historical control group of patients (N=175) had a median time to neutrophil engraftment of 9 days (range 7 to 24 days). A second cohort of patients were transplanted with expanded cells alone and a similar rapid engraftment was obtained. The first patients are now over 70 days post transplant with durable engraftment. No effect on platelet recovery has been observed in any patients to date. These data demonstrate that PBPC expanded under the conditions defined can significantly shorten the time to engraftment of neutrophils.

  4. Effects of Developmental Activation of the Aryl Hydrocarbon Receptor by 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Long-term Self-renewal of Murine Hematopoietic Stem Cells.

    PubMed

    Laiosa, Michael D; Tate, Everett R; Ahrenhoerster, Lori S; Chen, Yuhong; Wang, Demin

    2016-07-01

    Human epidemiological and animal studies suggest that developmental exposure to contaminants that activate the aryl hydrocarbon receptor (AHR) lead to suppression of immune system function throughout life. The persistence of immune deficiency throughout life suggests that the cellular target of AHR activation is a fetal hematopoietic progenitor or stem cell. The aim of this study was to identify the effects of transplacental exposure to an AHR agonist on long-term self-renewal of fetal hematopoietic stem cells. Pregnant C57BL/6 or AHR+/- mice were exposed to the AHR agonist, 2,3,7,8-tetra-​chlorodibenzo-p-dioxin (TCDD). On day 14 of gestation, hematopoietic progenitors from wild-type or AHR-deficient fetuses were placed into in vitro T-lymphocyte differentiation cultures to identify the effects of transplacental TCDD on AHR activation in the fetus. We next analyzed the fetal hematopoietic progenitor cells for changes in reactive oxygen species (ROS). Finally, hematopoietic progenitors from fetuses exposed transplacentally to TCDD were mixed 1:1 with cells from congenic controls and used to reconstitute lethally irradiated recipients for analysis of long-term self-renewal potential. Our findings suggested that the effects of TCDD on the developing hematopoietic system were mediated by direct AHR activation in the fetus. Furthermore, developmental AHR activation by TCDD increased ROS in the fetal hematopoietic stem cells, and the elevated ROS was associated with a reduced capacity of the TCDD-exposed fetal cells to compete with control cells in a mixed competitive irradiation/reconstitution assay. Our findings indicate that AHR activation by TCDD in the fetus during pregnancy leads to impairment of long-term self-renewal of hematopoietic stem cells. Laiosa MD, Tate ER, Ahrenhoerster LS, Chen Y, Wang D. 2016. Effects of developmental activation of the aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-p-dioxin on long-term self-renewal of murine hematopoietic

  5. Clonal type I interferon-producing and dendritic cell precursors are contained in both human lymphoid and myeloid progenitor populations.

    PubMed

    Chicha, Laurie; Jarrossay, David; Manz, Markus G

    2004-12-06

    Because of different cytokine responsiveness, surface receptor, and transcription factor expression, human CD11c(-) natural type I interferon-producing cells (IPCs) and CD11c(+) dendritic cells were thought to derive through lymphoid and myeloid hematopoietic developmental pathways, respectively. To directly test this hypothesis, we used an in vitro assay allowing simultaneous IPC, dendritic cell, and B cell development and we tested lymphoid and myeloid committed hematopoietic progenitor cells for their developmental capacity. Lymphoid and common myeloid and granulocyte/macrophage progenitors were capable of developing into both functional IPCs, expressing gene transcripts thought to be associated with lymphoid lineage development, and into dendritic cells. However, clonal progenitors for both populations were about fivefold more frequent within myeloid committed progenitor cells. Thus, in humans as in mice, natural IPC and dendritic cell development robustly segregates with myeloid differentiation. This would fit with natural interferon type I-producing cell and dendritic cell activity in innate immunity, the evolutionary older arm of the cellular immune system.

  6. Clonal Type I Interferon–producing and Dendritic Cell Precursors Are Contained in Both Human Lymphoid and Myeloid Progenitor Populations

    PubMed Central

    Chicha, Laurie; Jarrossay, David; Manz, Markus G.

    2004-01-01

    Because of different cytokine responsiveness, surface receptor, and transcription factor expression, human CD11c− natural type I interferon–producing cells (IPCs) and CD11c+ dendritic cells were thought to derive through lymphoid and myeloid hematopoietic developmental pathways, respectively. To directly test this hypothesis, we used an in vitro assay allowing simultaneous IPC, dendritic cell, and B cell development and we tested lymphoid and myeloid committed hematopoietic progenitor cells for their developmental capacity. Lymphoid and common myeloid and granulocyte/macrophage progenitors were capable of developing into both functional IPCs, expressing gene transcripts thought to be associated with lymphoid lineage development, and into dendritic cells. However, clonal progenitors for both populations were about fivefold more frequent within myeloid committed progenitor cells. Thus, in humans as in mice, natural IPC and dendritic cell development robustly segregates with myeloid differentiation. This would fit with natural interferon type I–producing cell and dendritic cell activity in innate immunity, the evolutionary older arm of the cellular immune system. PMID:15557348

  7. Nucleic Acid Encoding A Lectin-Derived Progenitor Cell Preservation Factor

    DOEpatents

    Colucci, M. Gabriella; Chrispeels, Maarten J.; Moore, Jeffrey G.

    2001-10-30

    The invention relates to an isolated nucleic acid molecule that encodes a protein that is effective to preserve progenitor cells, such as hematopoietic progenitor cells. The nucleic acid comprises a sequence defined by SEQ ID NO:1, a homolog thereof, or a fragment thereof. The encoded protein has an amino acid sequence that comprises a sequence defined by SEQ ID NO:2, a homolog thereof, or a fragment thereof that contains an amino acid sequence TNNVLQVT. Methods of using the encoded protein for preserving progenitor cells in vitro, ex vivo, and in vivo are also described. The invention, therefore, include methods such as myeloablation therapies for cancer treatment wherein myeloid reconstitution is facilitated by means of the specified protein. Other therapeutic utilities are also enabled through the invention, for example, expanding progenitor cell populations ex vivo to increase chances of engraftation, improving conditions for transporting and storing progenitor cells, and facilitating gene therapy to treat and cure a broad range of life-threatening hematologic diseases.

  8. Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

    PubMed Central

    Migita, M; Medin, J A; Pawliuk, R; Jacobson, S; Nagle, J W; Anderson, S; Amiri, M; Humphries, R K; Karlsson, S

    1995-01-01

    The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU

  9. Genistein protects hematopoietic stem cells against G-CSF-induced DNA damage.

    PubMed

    Souza, Liliana R; Silva, Erica; Calloway, Elissa; Kucuk, Omer; Rossi, Michael; McLemore, Morgan L

    2014-05-01

    Granulocyte colony-stimulating factor (G-CSF) has been used to treat neutropenia in various clinical settings. Although clearly beneficial, there are concerns that the chronic use of G-CSF in certain conditions increases the risk of myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). The most striking example is in severe congenital neutropenia (SCN). Patients with SCN develop MDS/AML at a high rate that is directly correlated to the cumulative lifetime dosage of G-CSF. Myelodysplastic syndrome and AML that arise in these settings are commonly associated with chromosomal deletions. We have demonstrated in this study that chronic G-CSF treatment in mice results in expansion of the hematopoietic stem cell (HSC) population. In addition, primitive hematopoietic progenitors from G-CSF-treated mice show evidence of DNA damage as demonstrated by an increase in double-strand breaks and recurrent chromosomal deletions. Concurrent treatment with genistein, a natural soy isoflavone, limits DNA damage in this population. The protective effect of genistein seems to be related to its preferential inhibition of G-CSF-induced proliferation of HSCs. Importantly, genistein does not impair G-CSF-induced proliferation of committed hematopoietic progenitors, nor diminishes neutrophil production. The protective effect of genistein was accomplished with plasma levels that are attainable through dietary supplementation.

  10. Hematopoietic stem cells are acutely sensitive to Acd shelterin gene inactivation

    PubMed Central

    Jones, Morgan; Osawa, Gail; Regal, Joshua A.; Weinberg, Daniel N.; Taggart, James; Kocak, Hande; Friedman, Ann; Ferguson, David O.; Keegan, Catherine E.; Maillard, Ivan

    2013-01-01

    The shelterin complex plays dual functions in telomere homeostasis by recruiting telomerase and preventing the activation of a DNA damage response at telomeric ends. Somatic stem cells require telomerase activity, as evidenced by progressive stem cell loss leading to bone marrow failure in hereditary dyskeratosis congenita. Recent work demonstrates that dyskeratosis congenita can also arise from mutations in specific shelterin genes, although little is known about shelterin functions in somatic stem cells. We found that mouse hematopoietic stem cells (HSCs) are acutely sensitive to inactivation of the shelterin gene Acd, encoding TPP1. Homozygosity for a hypomorphic acd allele preserved the emergence and expansion of fetal HSCs but led to profoundly defective function in transplantation assays. Upon complete Acd inactivation, HSCs expressed p53 target genes, underwent cell cycle arrest, and were severely depleted within days, leading to hematopoietic failure. TPP1 loss induced increased telomeric fusion events in bone marrow progenitors. However, unlike in epidermal stem cells, p53 deficiency did not rescue TPP1-deficient HSCs, indicating that shelterin dysfunction has unique effects in different stem cell populations. Because the consequences of telomere shortening are progressive and unsynchronized, acute loss of shelterin function represents an attractive alternative for studying telomere crisis in hematopoietic progenitors. PMID:24316971

  11. A novel procedure to improve functional preservation of hematopoietic stem and progenitor cells in cord blood stored at +4°c before cryopreservation.

    PubMed

    Chevaleyre, Jean; Rodriguez, Laura; Duchez, Pascale; Plainfossé, Marie; Dazey, Bernard; Lapostolle, Véronique; Vlaski, Marija; Brunet de la Grange, Philippe; Delorme, Bruno; Ivanovic, Zoran

    2014-08-01

    During storage and transportation of collected cord blood units (CBUs) to the bank prior to their processing and cryopreservation, it is imperative to preserve the functional capacities of a relatively small amount of cells of interest (stem and progenitor cells) which are critical for graft potency. To improve CBU storage efficiency, we conceived an approach based on the following two principles: (1) to provide a better nutritive and biochemical environment to stem and progenitor cells in CB and (2) to prevent the hyperoxygenation of these cells transferred from a low- (1.1%-4% O2 in the CB) to a high-oxygen (20%-21% O2 in atmosphere) concentration. Our hypothesis is confirmed by the functional assessment of stem cell (hematopoietic reconstitution capacity in immunodeficient mice-scid repopulating cell assay) and committed progenitor activities (capacity of in vitro colony formation and of ex vivo expansion) after the storage period with our medium (HP02) in gas-impermeable bags. This storage procedure maintains the full functional capacity of a CBU graft for 3 days with respect to day 0. Further, using this procedure, a graft stored 3 days at +4°C exhibits better functional capacities than one currently used in routine storage (CBUs stored at +4°C for 1 day in gas-permeable bags and without medium). We provided the proof of principle of our approach, developed a clinical-scale kit and performed a preclinical assay demonstrating the feasibility and efficiency of our CBU preservation protocol through all steps of preparation (volume reduction, freezing, and thawing).

  12. Studies on the organization and regeneration of bone marrow: origin, growth, and differentiation of endocloned hematopoietic colonies

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lambertsen, R.H.; Weiss, L.

    1983-04-01

    Hematopoietic colonies were studied by light microscopy in the marrow of alternate fraction x-irradiated mice (C576J/B1) to investigate the microenvironmental organization of marrow and identify early hematopoietic cell-stromal cell interactions. Undifferentiated colonies (UC) were detected at 3 days postirradiation, showed a marked predilection for bone surfaces, and disappeared as differentiated colonies developed. Some UC occurred along marrow arteries. Neutrophilic granulocyte colonies (GC) occurred in all areas at 3 days but grew rapidly only subosteally. Few eosinophilic colonies (GCe) occurred. Erythrocytic colonies (EC) appeared at 4 days as dispersed populations of motile cells within a localized area of marrow; these tendedmore » to proliferate initially in intermediate and central marrow zones. Macrophage colonies (M phi C) of two ''subtypes'' were detected, peaking in relative frequency at 4 days. These appeared active in stromal repair and monocytopoiesis. Megakaryocyte colonies (MC) originated along bone and differentiated away from bone. These results were interpreted as evidence that in x-irradiated marrow: (1) hematopoietic microenvironments (HMs) for stem-cell proliferation and commitment to differentiation, with the possible exception of HMs determining erythroid differentiation, occur in endosteal and periarterial regions; (2) a proliferative and/or chemotactic stimulus to erythroid progenitors exists in intermediate and central marrow regions; and (3) some subosteal regions may exclude erythropoiesis, or preferentially support nonerythroid differentiation. Elaborate associations occurred between macrophages and early UC, GC, and EC, but not MC hematopoietic cells. UC and GC often associated with osteoclasts. Reticular and other fibroblastic cells associated with the cells of all colony types.« less

  13. Oncogenic Kras initiates leukemia in hematopoietic stem cells.

    PubMed

    Sabnis, Amit J; Cheung, Laurene S; Dail, Monique; Kang, Hio Chung; Santaguida, Marianne; Hermiston, Michelle L; Passegué, Emmanuelle; Shannon, Kevin; Braun, Benjamin S

    2009-03-17

    How oncogenes modulate the self-renewal properties of cancer-initiating cells is incompletely understood. Activating KRAS and NRAS mutations are among the most common oncogenic lesions detected in human cancer, and occur in myeloproliferative disorders (MPDs) and leukemias. We investigated the effects of expressing oncogenic Kras(G12D) from its endogenous locus on the proliferation and tumor-initiating properties of murine hematopoietic stem and progenitor cells. MPD could be initiated by Kras(G12D) expression in a highly restricted population enriched for hematopoietic stem cells (HSCs), but not in common myeloid progenitors. Kras(G12D) HSCs demonstrated a marked in vivo competitive advantage over wild-type cells. Kras(G12D) expression also increased the fraction of proliferating HSCs and reduced the overall size of this compartment. Transplanted Kras(G12D) HSCs efficiently initiated acute T-lineage leukemia/lymphoma, which was associated with secondary Notch1 mutations in thymocytes. We conclude that MPD-initiating activity is restricted to the HSC compartment in Kras(G12D) mice, and that distinct self-renewing populations with cooperating mutations emerge during cancer progression.

  14. The SKI proto-oncogene enhances the in vivo repopulation of hematopoietic stem cells and causes myeloproliferative disease.

    PubMed

    Singbrant, Sofie; Wall, Meaghan; Moody, Jennifer; Karlsson, Göran; Chalk, Alistair M; Liddicoat, Brian; Russell, Megan R; Walkley, Carl R; Karlsson, Stefan

    2014-04-01

    The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced a gene signature associated with hematopoietic stem cells and myeloid differentiation, as well as hepatocyte growth factor signaling. Here we demonstrate that, in contrast to what has generally been assumed, the significant impact of SKI on hematopoiesis is independent of its ability to inhibit TGF-beta signaling. Instead, myeloid progenitors expressing SKI are partially dependent on functional hepatocyte growth factor signaling. Collectively our results demonstrate that SKI is an important regulator of hematopoietic stem cell activity and its overexpression leads to myeloproliferative disease.

  15. The SKI proto-oncogene enhances the in vivo repopulation of hematopoietic stem cells and causes myeloproliferative disease

    PubMed Central

    Singbrant, Sofie; Wall, Meaghan; Moody, Jennifer; Karlsson, Göran; Chalk, Alistair M.; Liddicoat, Brian; Russell, Megan R.; Walkley, Carl R.; Karlsson, Stefan

    2014-01-01

    The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced a gene signature associated with hematopoietic stem cells and myeloid differentiation, as well as hepatocyte growth factor signaling. Here we demonstrate that, in contrast to what has generally been assumed, the significant impact of SKI on hematopoiesis is independent of its ability to inhibit TGF-beta signaling. Instead, myeloid progenitors expressing SKI are partially dependent on functional hepatocyte growth factor signaling. Collectively our results demonstrate that SKI is an important regulator of hematopoietic stem cell activity and its overexpression leads to myeloproliferative disease. PMID:24415629

  16. Update on the pathogenesis of Scleroderma: focus on circulating progenitor cells

    PubMed Central

    Brunasso, Alexandra Maria Giovanna; Massone, Cesare

    2016-01-01

    In systemic sclerosis (SSc), the development of fibrosis seems to be a consequence of the initial ischemic process related to an endothelial injury. The initial trigger event in SSc is still unknown, but circulating progenitor cells (CPCs) might play a key role. Such cells have the ability to traffic into injury sites, exhibiting inflammatory features of macrophages, tissue remodeling properties of fibroblasts, and vasculogenesis functions of endothelial cells. The different subsets of CPCs described thus far in SSc arise from a pool of circulating monocyte precursors (CD14 + cells) and probably correspond to a different degree of differentiation of a single cell of origin. Several subsets of CPCs have been described in patients with SSc, all have a monocytic origin but may or may not express CD14, and all of these cells have the ability to give origin to endothelial cells, or collagen (Col)-producing cells, or both. We were able to identify six subsets of CPCs: pluripotent stem cells (CD14 +, CD45 +, and CD34 +), monocyte-derived multipotential cells (MOMCs) or monocyte-derived mesenchymal progenitors (CD14 +, CD45 +, CD34 +, Col I +, CD11b +, CD68 +, CD105 +, and VEGFR1 +), early endothelial progenitor cells (EPCs) or monocytic pro-angiogenic hematopoietic cells or circulating hematopoietic cells (CD14 +, CD45 +, CD34 low/−, VEGFR2 +/−, CXCR4 +, c-kit +, and DC117 +), late EPCs (CD14 −, CD133 +, VEGFR2 +, CD144 + [VE-cadherin +], and CD146 +), fibroblast-like cells (FLCs)/circulating Col-producing monocytes (CD14 +, CD45 +, CD34 +/−, and Col I +), and fibrocytes (CD14 −, CD45 +, CD34 +, Col I +, and CXCR4 +). It has been demonstrated that circulating CD14 + monocytes with an activated phenotype are increased in patients with SSc when compared with normal subjects. CD14 +, CD34 +, and Col I + spindle-shaped cells have been found in increased numbers in lungs of SSc patients with interstitial lung disease. Elevated blood amounts of early EPCs have been

  17. Update on the pathogenesis of Scleroderma: focus on circulating progenitor cells.

    PubMed

    Brunasso, Alexandra Maria Giovanna; Massone, Cesare

    2016-01-01

    In systemic sclerosis (SSc), the development of fibrosis seems to be a consequence of the initial ischemic process related to an endothelial injury. The initial trigger event in SSc is still unknown, but circulating progenitor cells (CPCs) might play a key role. Such cells have the ability to traffic into injury sites, exhibiting inflammatory features of macrophages, tissue remodeling properties of fibroblasts, and vasculogenesis functions of endothelial cells. The different subsets of CPCs described thus far in SSc arise from a pool of circulating monocyte precursors (CD14 (+) cells) and probably correspond to a different degree of differentiation of a single cell of origin. Several subsets of CPCs have been described in patients with SSc, all have a monocytic origin but may or may not express CD14, and all of these cells have the ability to give origin to endothelial cells, or collagen (Col)-producing cells, or both. We were able to identify six subsets of CPCs: pluripotent stem cells (CD14 (+), CD45 (+), and CD34 (+)), monocyte-derived multipotential cells (MOMCs) or monocyte-derived mesenchymal progenitors (CD14 (+), CD45 (+), CD34 (+), Col I (+), CD11b (+), CD68 (+), CD105 (+), and VEGFR1 (+)), early endothelial progenitor cells (EPCs) or monocytic pro-angiogenic hematopoietic cells or circulating hematopoietic cells (CD14 (+), CD45 (+), CD34 (low/-), VEGFR2 (+/-), CXCR4 (+), c-kit (+), and DC117 (+)), late EPCs (CD14 (-), CD133 (+), VEGFR2 (+), CD144 (+) [VE-cadherin (+)], and CD146 (+)), fibroblast-like cells (FLCs)/circulating Col-producing monocytes (CD14 (+), CD45 (+), CD34 (+/-), and Col I (+)), and fibrocytes (CD14 (-), CD45 (+), CD34 (+), Col I (+), and CXCR4 (+)). It has been demonstrated that circulating CD14 (+) monocytes with an activated phenotype are increased in patients with SSc when compared with normal subjects. CD14 (+), CD34 (+), and Col I (+) spindle-shaped cells have been found in increased numbers in lungs of SSc patients with

  18. KDR (VEGFR2) identifies a conserved human and murine hepatic progenitor and instructs early liver development

    PubMed Central

    Goldman, Orit; Han, Songyan; Sourrisseau, Marion; Dziedzic, Noelle; Hamou, Wissam; Corneo, Barbara; D’Souza, Sunita; Sato, Thomas; Kotton, Darrell N.; Bissig, Karl-Dimiter; Kalir, Tamara; Jacobs, Adam; Evans, Todd; Evans, Matthew J.; Gouon-Evans, Valerie

    2013-01-01

    SUMMARY Understanding the fetal hepatic niche is essential for optimizing the generation of functional hepatocyte-like (hepatic) cells from human embryonic stem cells (hESCs). Here, we show that KDR (VEGFR2), previously assumed to be mostly restricted to mesodermal lineages, marks a hESC-derived hepatic progenitor. hESC-derived endoderm cells do not express KDR, but when cultured in media supporting hepatic differentiation, generate KDR+ hepatic progenitors and KDR- hepatic cells. KDR+ progenitors require active KDR signaling both to instruct their own differentiation into hepatic cells, and to support non-cell-autonomously the functional maturation of co-cultured KDR- hepatic cells. Analysis of human fetal livers suggests that similar progenitors are present in human livers. Lineage tracing in mice provides in vivo evidence of a KDR+ hepatic progenitor for fetal hepatoblasts and subsequently adult hepatocytes and cholangiocytes. Altogether, our findings reveal that KDR is a conserved marker for endoderm-derived hepatic progenitors, and a functional receptor instructing early liver development. PMID:23746980

  19. Comparison of two apheresis systems during hematopoietic progenitor stem cell collections at a tertiary medical center.

    PubMed

    Li, Yanchun; Li, Jie; Reeves, Hollie M; Reyes, Ramil; Maitta, Robert W

    2016-11-01

    The Spectra Optia is a newer apheresis system developed based on the COBE Spectra platform. COBE Spectra requires more manual control, while Spectra Optia offers greater automation. The purpose of this study was to compare the two systems during hematopoietic progenitor stem cell (HPSC) collections. A retrospective review of 41 collections performed in 26 subjects at a tertiary medical center between June 1, 2013, and December 31, 2013, was conducted, 11 with the Spectra Optia and 30 with the COBE Spectra. Six patients underwent two consecutive daily collections, first on the Spectra Optia followed by the COBE Spectra. Procedure run time with the Spectra Optia was considerably longer than with the COBE Spectra (283 ± 11 min vs. 217 ± 2 min, respectively; p < 0.01). Mean CD34+ cell yields with the Spectra Optia were comparable with those of the COBE Spectra. Products collected with the Spectra Optia had less red blood cell contamination. However, platelet (PLT) attrition was greater with the Spectra Optia. Similar results were obtained in patients who were collected on consecutive days in both systems. Collections with the Spectra Optia take longer and lead to greater PLT losses during HPSC collections. © 2016 AABB.

  20. PRC2 inhibition counteracts the culture-associated loss of engraftment potential of human cord blood-derived hematopoietic stem and progenitor cells.

    PubMed

    Varagnolo, Linda; Lin, Qiong; Obier, Nadine; Plass, Christoph; Dietl, Johannes; Zenke, Martin; Claus, Rainer; Müller, Albrecht M

    2015-07-22

    Cord blood hematopoietic stem cells (CB-HSCs) are an outstanding source for transplantation approaches. However, the amount of cells per donor is limited and culture expansion of CB-HSCs is accompanied by a loss of engraftment potential. In order to analyze the molecular mechanisms leading to this impaired potential we profiled global and local epigenotypes during the expansion of human CB hematopoietic stem and progenitor cells (HPSCs). Human CB-derived CD34+ cells were cultured in serum-free medium together with SCF, TPO, FGF, with or without Igfbp2 and Angptl5 (STF/STFIA cocktails). As compared to the STF cocktail, the STFIA cocktail maintains in vivo repopulation capacity of cultured CD34+ cells. Upon expansion, CD34+ cells genome-wide remodel their epigenotype and depending on the cytokine cocktail, cells show different H3K4me3 and H3K27me3 levels. Expanding cells without Igfbp2 and Angptl5 leads to higher global H3K27me3 levels. ChIPseq analyses reveal a cytokine cocktail-dependent redistribution of H3K27me3 profiles. Inhibition of the PRC2 component EZH2 counteracts the culture-associated loss of NOD scid gamma (NSG) engraftment potential. Collectively, our data reveal chromatin dynamics that underlie the culture-associated loss of engraftment potential. We identify PRC2 component EZH2 as being involved in the loss of engraftment potential during the in vitro expansion of HPSCs.

  1. NOTCH1 Is Aberrantly Activated in Chronic Lymphocytic Leukemia Hematopoietic Stem Cells.

    PubMed

    Di Ianni, Mauro; Baldoni, Stefano; Del Papa, Beatrice; Aureli, Patrizia; Dorillo, Erica; De Falco, Filomena; Albi, Elisa; Varasano, Emanuela; Di Tommaso, Ambra; Giancola, Raffaella; Accorsi, Patrizia; Rotta, Gianluca; Rompietti, Chiara; Silva Barcelos, Estevão Carlos; Campese, Antonio Francesco; Di Bartolomeo, Paolo; Screpanti, Isabella; Rosati, Emanuela; Falzetti, Franca; Sportoletti, Paolo

    2018-01-01

    To investigate chronic lymphocytic leukemia (CLL)-initiating cells, we assessed NOTCH1 mutation/expression in hematopoietic stem cells (HSCs). In NOTCH1- mutated CLL, we detected subclonal mutations in 57% CD34+/CD38- HSCs. NOTCH1 mutation was present in 66% CD34+/CD38+ progenitor cells displaying an increased mutational burden compared to HSCs. Flow cytometric analysis revealed significantly higher NOTCH1 activation in CD34+/CD38- and CD34+/CD38+ cells from CLL patients, regardless NOTCH1 mutation compared to healthy donors. Activated NOTCH1 resulted in overexpression of the NOTCH1 target c-MYC. We conclude that activated NOTCH1 is an early event in CLL that may contribute to aberrant HSCs in this disease.

  2. WDR62 Regulates Early Neural and Glial Progenitor Specification of Human Pluripotent Stem Cells

    PubMed Central

    Alshawaf, Abdullah J.; Antonic, Ana; Skafidas, Efstratios

    2017-01-01

    Mutations in WD40-repeat protein 62 (WDR62) are commonly associated with primary microcephaly and other developmental cortical malformations. We used human pluripotent stem cells (hPSC) to examine WDR62 function during human neural differentiation and model early stages of human corticogenesis. Neurospheres lacking WDR62 expression showed decreased expression of intermediate progenitor marker, TBR2, and also glial marker, S100β. In contrast, inhibition of c-Jun N-terminal kinase (JNK) signalling during hPSC neural differentiation induced upregulation of WDR62 with a corresponding increase in neural and glial progenitor markers, PAX6 and EAAT1, respectively. These findings may signify a role of WDR62 in specifying intermediate neural and glial progenitors during human pluripotent stem cell differentiation. PMID:28690640

  3. Mitophagy in hematopoietic stem cells

    PubMed Central

    Joshi, Aashish; Kundu, Mondira

    2013-01-01

    Hematopoietic stem cells (HSCs) are inherently quiescent and self-renewing, yet can differentiate and commit to multiple blood cell types. Intracellular mitochondrial content is dynamic, and there is an increase in mitochondrial content during differentiation and lineage commitment in HSCs. HSCs reside in a hypoxic niche within the bone marrow and rely heavily on glycolysis, while differentiated and committed progenitors rely on oxidative phosphorylation. Increased oxidative phosphorylation during differentiation and commitment is not only due to increased mitochondrial content but also due to changes in mitochondrial cytosolic distribution and efficiency. These changes in the intracellular mitochondrial landscape contribute signals toward regulating differentiation and commitment. Thus, a functional relationship exists between the mitochondria in HSCs and the state of the HSCs (i.e., stemness vs. differentiated). This review focuses on how autophagy-mediated mitochondrial clearance (i.e., mitophagy) may affect HSC mitochondrial content, thereby influencing the fate of HSCs and maintenance of hematopoietic homeostasis. PMID:24135495

  4. Immunohistochemical Markers of Neural Progenitor Cells in the Early Embryonic Human Cerebral Cortex

    PubMed Central

    Vinci, L.; Ravarino, A.; Fanos, V.; Naccarato, A.G.; Senes, G.; Gerosa, C.; Bevilacqua, G.; Faa, G.; Ambu, R.

    2016-01-01

    The development of the human central nervous system represents a delicate moment of embryogenesis. The purpose of this study was to analyze the expression of multiple immunohistochemical markers in the stem/progenitor cells in the human cerebral cortex during the early phases of development. To this end, samples from cerebral cortex were obtained from 4 human embryos of 11 weeks of gestation. Each sample was formalin-fixed, paraffin embedded and immunostained with several markers including GFAP, WT1, Nestin, Vimentin, CD117, S100B, Sox2, PAX2, PAX5, Tβ4, Neurofilament, CD44, CD133, Synaptophysin and Cyclin D1. Our study shows the ability of the different immunohistochemical markers to evidence different zones of the developing human cerebral cortex, allowing the identification of the multiple stages of differentiation of neuronal and glial precursors. Three important markers of radial glial cells are evidenced in this early gestational age: Vimentin, Nestin and WT1. Sox2 was expressed by the stem/progenitor cells of the ventricular zone, whereas the postmitotic neurons of the cortical plate were immunostained by PAX2 and NSE. Future studies are needed to test other important stem/progenitor cells markers and to better analyze differences in the immunohistochemical expression of these markers during gestation. PMID:26972711

  5. Assessment of Benzene-Induced Hematotoxicity Using a Human-Like Hematopoietic Lineage in NOD/Shi-scid/IL-2Rγnull Mice

    PubMed Central

    Takahashi, Masayuki; Tsujimura, Noriyuki; Yoshino, Tomoko; Hosokawa, Masahito; Otsuka, Kensuke; Matsunaga, Tadashi; Nakasono, Satoshi

    2012-01-01

    Despite recent advancements, it is still difficult to evaluate in vivo responses to toxicants in humans. Development of a system that can mimic the in vivo responses of human cells will enable more accurate health risk assessments. A surrogate human hematopoietic lineage can be established in NOD/Shi-scid/IL-2Rγnull (NOG) mice by transplanting human hematopoietic stem/progenitor cells (Hu-NOG mice). Here, we first evaluated the toxic response of human-like hematopoietic lineage in NOG mice to a representative toxic agent, benzene. Flow cytometric analysis showed that benzene caused a significant decrease in the number of human hematopoietic stem/progenitor cells in the bone marrow and the number of human leukocytes in the peripheral blood and hematopoietic organs. Next, we established chimeric mice by transplanting C57BL/6 mouse-derived bone marrow cells into NOG mice (Mo-NOG mice). A comparison of the degree of benzene-induced hematotoxicity in donor-derived hematopoietic lineage cells within Mo-NOG mice indicated that the toxic response of Hu-NOG mice reflected interspecies differences in susceptibilities to benzene. Responses to the toxic effects of benzene were greater in lymphoid cells than in myeloid cells in Mo-NOG and Hu-NOG mice. These findings suggested that Hu-NOG mice may be a powerful in vivo tool for assessing hematotoxicity in humans, while accounting for interspecies differences. PMID:23226520

  6. CD34(+) Liver Cancer Stem Cells Were Formed by Fusion of Hepatobiliary Stem/Progenitor Cells with Hematopoietic Precursor-Derived Myeloid Intermediates.

    PubMed

    Zeng, Changjun; Zhang, Yanling; Park, Su Cheol; Eun, Jong Ryeol; Nguyen, Ngoc Tue; Tschudy-Seney, Benjamin; Jung, Yong Jin; Theise, Neil D; Zern, Mark A; Duan, Yuyou

    2015-11-01

    A large number of cancer stem cells (CSCs) were identified and characterized; however, the origins and formation of CSCs remain elusive. In this study, we examined the origination of the newly identified CD34(+) liver CSC (LCSC). We found that CD34(+) LCSC coexpressed liver stem cell and myelomonocytic cell markers, showing a mixed phenotype, a combination of hepatobiliary stem/progenitor cells (HSPCs) and myelomonocytic cells. Moreover, human xenografts produced by CD34(+) LCSCs and the parental cells, which CD34(+) LCSC was isolated from, coexpressed liver cancer and myelomonocytic markers, also demonstrating mixed phenotypes. The xenografts and the parental cells secreted albumin demonstrating their hepatocyte origin and also expressed cytokines [interleukin (IL)-1b, IL-6, IL-12A, IL-18, tumor necrosis factor-alpha (TNF-α), and CSF1] and chemokines (IL-8, CCL2, and CCL5). Expression of these cytokines and chemokines responded to the stimuli [interferon-γ (INF-γ), IL-4, and lipopolysaccharide (LPS)]. Furthermore, human xenografts and the parental cells phagocytized Escherichia coli. CD34(+) LCSC coexpressed CD45, demonstrating that its origin appears to be from a hematopoietic precursor. The percentage of cells positive for OV6, CD34, and CD31, presenting the markers of HSPC, hematopoietic, and myelomonocytic cells, increased under treatment of CD34(+) LCSC with a drug. Cytogenetic analysis showed that CD34(+) LCSC contained a greater number of chromosomes. HBV DNA integrations and mutations in CD34(+) LCSC and the parental cells were identical to those in the literature or the database. Thus, these results demonstrated that CD34(+) LCSCs were formed by fusion of HSPC with CD34(+) hematopoietic precursor-derived myeloid intermediates; it appears that this is the first report that human CSCs have been formed by the fusion. Therefore, it represents a significant step toward better understanding of the formation of human CSC and the diverse origins of liver

  7. Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells

    PubMed Central

    Hess, David A.; Wirthlin, Louisa; Craft, Timothy P.; Herrbrich, Phillip E.; Hohm, Sarah A.; Lahey, Ryan; Eades, William C.; Creer, Michael H.; Nolta, Jan A.

    2006-01-01

    The development of novel cell-based therapies requires understanding of distinct human hematopoietic stem and progenitor cell populations. We recently isolated reconstituting hematopoietic stem cells (HSCs) by lineage depletion and purification based on high aldehyde dehydrogenase activity (ALDHhiLin- cells). Here, we further dissected the ALDHhi-Lin- population by selection for CD133, a surface molecule expressed on progenitors from hematopoietic, endothelial, and neural lineages. ALDHhiCD133+Lin- cells were primarily CD34+, but also included CD34-CD38-CD133+ cells, a phenotype previously associated with repopulating function. Both ALDHhiCD133-Lin- and ALDHhiCD133+Lin- cells demonstrated distinct clonogenic progenitor function in vitro, whereas only the ALDHhiCD133+Lin- population seeded the murine bone marrow 48 hours after transplantation. Significant human cell repopulation was observed only in NOD/SCID and NOD/SCID β2M-null mice that received transplants of ALDHhiCD133+Lin- cells. Limiting dilution analysis demonstrated a 10-fold increase in the frequency of NOD/SCID repopulating cells compared with CD133+Lin- cells, suggesting that high ALDH activity further purified cells with repopulating function. Transplanted ALDHhiCD133+Lin- cells also maintained primitive hematopoietic phenotypes (CD34+CD38-) and demonstrated enhanced repopulating function in recipients of serial, secondary transplants. Cell selection based on ALDH activity and CD133 expression provides a novel purification of HSCs with long-term repopulating function and may be considered an alternative to CD34 cell selection for stem cell therapies. PMID:16269619

  8. Therapeutic gene editing in CD34+ hematopoietic progenitors from Fanconi anemia patients.

    PubMed

    Diez, Begoña; Genovese, Pietro; Roman-Rodriguez, Francisco J; Alvarez, Lara; Schiroli, Giulia; Ugalde, Laura; Rodriguez-Perales, Sandra; Sevilla, Julian; Diaz de Heredia, Cristina; Holmes, Michael C; Lombardo, Angelo; Naldini, Luigi; Bueren, Juan Antonio; Rio, Paula

    2017-11-01

    Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)-mediated insertion of a non-therapeutic EGFP-reporter donor in the AAVS1 "safe harbor" locus of FA-A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic FANCA donors, an efficient phenotypic correction of FA-A LCLs was obtained. Using primary cord blood CD34 + cells from healthy donors, gene targeting was confirmed not only in in vitro cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34 + cells from FA-A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  9. Expansion on Stromal Cells Preserves the Undifferentiated State of Human Hematopoietic Stem Cells Despite Compromised Reconstitution Ability

    PubMed Central

    Magnusson, Mattias; Sierra, Maria I.; Sasidharan, Rajkumar; Prashad, Sacha L.; Romero, Melissa; Saarikoski, Pamela; Van Handel, Ben; Huang, Andy; Li, Xinmin; Mikkola, Hanna K. A.

    2013-01-01

    Lack of HLA-matched hematopoietic stem cells (HSC) limits the number of patients with life-threatening blood disorders that can be treated by HSC transplantation. So far, insufficient understanding of the regulatory mechanisms governing human HSC has precluded the development of effective protocols for culturing HSC for therapeutic use and molecular studies. We defined a culture system using OP9M2 mesenchymal stem cell (MSC) stroma that protects human hematopoietic stem/progenitor cells (HSPC) from differentiation and apoptosis. In addition, it facilitates a dramatic expansion of multipotent progenitors that retain the immunophenotype (CD34+CD38−CD90+) characteristic of human HSPC and proliferative potential over several weeks in culture. In contrast, transplantable HSC could be maintained, but not significantly expanded, during 2-week culture. Temporal analysis of the transcriptome of the ex vivo expanded CD34+CD38−CD90+ cells documented remarkable stability of most transcriptional regulators known to govern the undifferentiated HSC state. Nevertheless, it revealed dynamic fluctuations in transcriptional programs that associate with HSC behavior and may compromise HSC function, such as dysregulation of PBX1 regulated genetic networks. This culture system serves now as a platform for modeling human multilineage hematopoietic stem/progenitor cell hierarchy and studying the complex regulation of HSC identity and function required for successful ex vivo expansion of transplantable HSC. PMID:23342037

  10. Reduced survival in patients with early-stage non-small-cell lung cancer is associated with high pleural endothelial progenitor cell levels.

    PubMed

    Pirro, Matteo; Cagini, Lucio; Mannarino, Massimo R; Andolfi, Marco; Potenza, Rossella; Paciullo, Francesco; Bianconi, Vanessa; Frangione, Maria Rosaria; Bagaglia, Francesco; Puma, Francesco; Mannarino, Elmo

    2016-12-01

    Endothelial progenitor cells are capable of contributing to neovascularization in tumours. In patients with either malignant or transudative pleural effusion, we tested the presence of pleural endothelial progenitor cells. We also measured the number of endothelial progenitor cells in post-surgery pleural drainage of either patients with early non-small-cell lung cancer or control patients with benign lung disease undergoing pulmonary resection. The prospective influence of post-surgery pleural-drainage endothelial progenitor cells on cancer recurrence/survival was investigated. Pleural endothelial progenitor cell levels were quantified by fluorescence-activated cell sorting analysis in pleural effusion of 15 patients with late-stage non-small-cell lung cancer with pleural involvement and in 15 control patients with congestive heart failure. Also, pleural-drainage endothelial progenitor cells were measured in pleural-drainage fluid 48 h after surgery in 64 patients with early-stage non-small-cell lung cancer and 20 benign lung disease patients undergoing pulmonary resection. Cancer recurrence and survival was evaluated in patients with high pleural-drainage endothelial progenitor cell levels. The number of pleural endothelial progenitor cells was higher in non-small-cell lung cancer pleural effusion than in transudative pleural effusion. Also, pleural-drainage endothelial progenitor cell levels were higher in patients with non-small-cell lung cancer than in patients with benign lung disease undergoing pulmonary resection (P < 0.05). Non-small-cell lung cancer patients with high pleural-drainage endothelial progenitor cell levels had a significantly 4.9 higher rate of cancer recurrence/death than patients with lower pleural-drainage endothelial progenitor cell levels, irrespective of confounders. Endothelial progenitor cells are present in the pleural effusion and are higher in patients with late-stage non-small-cell lung cancer with pleural involvement than in

  11. Hematopoietic Stem Cell Regeneration Enhanced by Ectopic Expression of ROS-detoxifying Enzymes in Transplant Mice

    PubMed Central

    Miao, Weimin; XuFeng, Richard; Park, Moo-Rim; Gu, Haihui; Hu, Linping; Kang, Jin Wook; Ma, Shihui; Liang, Paulina H; Li, Yanxin; Cheng, Haizi; Yu, Hui; Epperly, Michael; Greenberger, Joel; Cheng, Tao

    2013-01-01

    High levels of reactive oxygen species (ROS) can exhaust hematopoietic stem cells (HSCs). Thus, maintaining a low state of redox in HSCs by modulating ROS-detoxifying enzymes may augment the regeneration potential of HSCs. Our results show that basal expression of manganese superoxide dismutase (MnSOD) and catalase were at low levels in long-term and short-term repopulating HSCs, and administration of a MnSOD plasmid and lipofectin complex (MnSOD-PL) conferred radiation protection on irradiated recipient mice. To assess the intrinsic role of elevated MnSOD or catalase in HSCs and hematopoietic progenitor cells, the MnSOD or catalase gene was overexpressed in mouse hematopoietic cells via retroviral transduction. The impact of MnSOD and catalase on hematopoietic progenitor cells was mild, as measured by colony-forming units (CFUs). However, overexpressed catalase had a significant beneficial effect on long-term engraftment of transplanted HSCs, and this effect was further enhanced after an insult of low-dose γ-irradiation in the transplant mice. In contrast, overexpressed MnSOD exhibited an insignificant effect on long-term engraftment of transplanted HSCs, but had a significant beneficial effect after an insult of sublethal irradiation. Taken together, these results demonstrate that HSC function can be enhanced by ectopic expression of ROS-detoxifying enzymes, especially after radiation exposure in vivo. PMID:23295952

  12. Repeated hematopoietic stem and progenitor cell mobilization without depletion of the bone marrow stem and progenitor cell pool in mice after repeated administration of recombinant murine G-CSF.

    PubMed

    de Kruijf, Evert-Jan F M; van Pel, Melissa; Hagoort, Henny; Kruysdijk, Donnée; Molineux, Graham; Willemze, Roel; Fibbe, Willem E

    2007-05-01

    Administration of recombinant-human G-CSF (rhG-CSF) is highly efficient in mobilizing hematopoietic stem and progenitor cells (HSC/HPC) from the bone marrow (BM) toward the peripheral blood. This study was designed to investigate whether repeated G-CSF-induced HSC/HPC mobilization in mice could lead to a depletion of the bone marrow HSC/HPC pool with subsequent loss of mobilizing capacity. To test this hypothesis Balb/c mice were treated with a maximum of 12 repeated 5-day cycles of either 10 microg rhG-CSF/day or 0.25 microg rmG-CSF/day. Repeated administration of rhG-CSF lead to strong inhibition of HSC/HPC mobilization toward the peripheral blood and spleen after >4 cycles because of the induction of anti-rhG-CSF antibodies. In contrast, after repeated administration of rmG-CSF, HSC/HPC mobilizing capacity remained intact for up to 12 cycles. The number of CFU-GM per femur did not significantly change for up to 12 cycles. We conclude that repeated administration of G-CSF does not lead to depletion of the bone marrow HSC/HPC pool.

  13. Asymmetric segregation and self-renewal of hematopoietic stem and progenitor cells with endocytic Ap2a2.

    PubMed

    Ting, Stephen B; Deneault, Eric; Hope, Kristin; Cellot, Sonia; Chagraoui, Jalila; Mayotte, Nadine; Dorn, Jonas F; Laverdure, Jean-Philippe; Harvey, Michael; Hawkins, Edwin D; Russell, Sarah M; Maddox, Paul S; Iscove, Norman N; Sauvageau, Guy

    2012-03-15

    The stem cell-intrinsic model of self-renewal via asymmetric cell division (ACD) posits that fate determinants be partitioned unequally between daughter cells to either activate or suppress the stemness state. ACD is a purported mechanism by which hematopoietic stem cells (HSCs) self-renew, but definitive evidence for this cellular process remains open to conjecture. To address this issue, we chose 73 candidate genes that function within the cell polarity network to identify potential determinants that may concomitantly alter HSC fate while also exhibiting asymmetric segregation at cell division. Initial gene-expression profiles of polarity candidates showed high and differential expression in both HSCs and leukemia stem cells. Altered HSC fate was assessed by our established in vitro to in vivo screen on a subcohort of candidate polarity genes, which revealed 6 novel positive regulators of HSC function: Ap2a2, Gpsm2, Tmod1, Kif3a, Racgap1, and Ccnb1. Interestingly, live-cell videomicroscopy of the endocytic protein AP2A2 shows instances of asymmetric segregation during HSC/progenitor cell cytokinesis. These results contribute further evidence that ACD is functional in HSC self-renewal, suggest a role for Ap2a2 in HSC activity, and provide a unique opportunity to prospectively analyze progeny from HSC asymmetric divisions.

  14. Continuous blockade of CXCR4 results in dramatic mobilization and expansion of hematopoietic stem and progenitor cells.

    PubMed

    Karpova, Darja; Ritchey, Julie K; Holt, Matthew S; Abou-Ezzi, Grazia; Monlish, Darlene; Batoon, Lena; Millard, Susan; Spohn, Gabriele; Wiercinska, Eliza; Chendamarai, Ezhil; Yang, Wei; Christ, Stephanie; Gehrs, Leah; Schuettpelz, Laura G; Dembowsky, Klaus; Pettit, Allison R; Rettig, Michael P; Bonig, Halvard; DiPersio, John F

    2017-05-25

    Interaction between the chemokine receptor CXCR4 and its chief ligand CXCL12 plays a critical role in the retention and migration of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) microenvironment. In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of the CXCR4/CXCL12 axis on the HSPC compartment were investigated by using 3 structurally unrelated small molecule CXCR4 antagonists. A >10-fold increase in mobilization efficiency was achieved by administering the antagonists as a subcutaneous continuous infusion for 2 weeks compared to a single bolus injection. A concurrent increase in self-renewing proliferation leading to a twofold to fourfold expansion of the HSPC pool in the BM was observed. The expanded BM showed a distinct repopulating advantage when tested in serial competitive transplantation experiments. Furthermore, major changes within the HSPC niche associated with previously described HSPC expansion strategies were not detected in bones treated with a CXCR4 antagonist infusion. Our data suggest that prolonged but reversible pharmacologic blockade of the CXCR4/CXCL12 axis represents an approach that releases HSPC with efficiency superior to any other known mobilization strategy and may also serve as an effective method to expand the BM HSPC pool. © 2017 by The American Society of Hematology.

  15. Continuous blockade of CXCR4 results in dramatic mobilization and expansion of hematopoietic stem and progenitor cells

    PubMed Central

    Karpova, Darja; Ritchey, Julie K.; Holt, Matthew S.; Abou-Ezzi, Grazia; Monlish, Darlene; Batoon, Lena; Millard, Susan; Spohn, Gabriele; Wiercinska, Eliza; Chendamarai, Ezhil; Yang, Wei; Christ, Stephanie; Gehrs, Leah; Schuettpelz, Laura G.; Dembowsky, Klaus; Pettit, Allison R.; Rettig, Michael P.; Bonig, Halvard

    2017-01-01

    Interaction between the chemokine receptor CXCR4 and its chief ligand CXCL12 plays a critical role in the retention and migration of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) microenvironment. In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of the CXCR4/CXCL12 axis on the HSPC compartment were investigated by using 3 structurally unrelated small molecule CXCR4 antagonists. A >10-fold increase in mobilization efficiency was achieved by administering the antagonists as a subcutaneous continuous infusion for 2 weeks compared to a single bolus injection. A concurrent increase in self-renewing proliferation leading to a twofold to fourfold expansion of the HSPC pool in the BM was observed. The expanded BM showed a distinct repopulating advantage when tested in serial competitive transplantation experiments. Furthermore, major changes within the HSPC niche associated with previously described HSPC expansion strategies were not detected in bones treated with a CXCR4 antagonist infusion. Our data suggest that prolonged but reversible pharmacologic blockade of the CXCR4/CXCL12 axis represents an approach that releases HSPC with efficiency superior to any other known mobilization strategy and may also serve as an effective method to expand the BM HSPC pool. PMID:28400375

  16. Loss of β-catenin triggers oxidative stress and impairs hematopoietic regeneration

    PubMed Central

    Lento, William; Ito, Takahiro; Zhao, Chen; Harris, Jeffrey R.; Huang, Wei; Jiang, Chen; Owzar, Kouros; Piryani, Sadhna; Racioppi, Luigi; Chao, Nelson; Reya, Tannishtha

    2014-01-01

    Accidental or deliberate ionizing radiation exposure can be fatal due to widespread hematopoietic destruction. However, little is known about either the course of injury or the molecular pathways that regulate the subsequent regenerative response. Here we show that the Wnt signaling pathway is critically important for regeneration after radiation-induced injury. Using Wnt reporter mice, we show that radiation triggers activation of Wnt signaling in hematopoietic stem and progenitor cells. β-Catenin-deficient mice, which lack the ability to activate canonical Wnt signaling, exhibited impaired hematopoietic stem cell regeneration and bone marrow recovery after radiation. We found that, as part of the mechanism, hematopoietic stem cells lacking β-catenin fail to suppress the generation of reactive oxygen species and cannot resolve DNA double-strand breaks after radiation. Consistent with the impaired response to radiation, β-catenin-deficient mice are also unable to recover effectively after chemotherapy. Collectively, these data indicate that regenerative responses to distinct hematopoietic injuries share a genetic dependence on β-catenin and raise the possibility that modulation of Wnt signaling may be a path to improving bone marrow recovery after damage. PMID:24788518

  17. BCAP inhibits proliferation and differentiation of myeloid progenitors in the steady state and during demand situations.

    PubMed

    Duggan, Jeffrey M; Buechler, Matthew B; Olson, Rebecca M; Hohl, Tobias M; Hamerman, Jessica A

    2017-03-16

    B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) is a signaling adaptor expressed in mature hematopoietic cells, including monocytes and neutrophils. Here we investigated the role of BCAP in the homeostasis and development of these myeloid lineages. BCAP -/- mice had more bone marrow (BM) monocytes than wild-type (WT) mice, and in mixed WT:BCAP -/- BM chimeras, monocytes and neutrophils skewed toward BCAP -/- origin, showing a competitive advantage for BCAP -/- myeloid cells. BCAP was expressed in BM hematopoietic progenitors, including lineage - Sca-1 + c-kit + (LSK), common myeloid progenitor, and granulocyte/macrophage progenitor (GMP) cells. At the steady state, BCAP -/- GMP cells expressed more IRF8 and less C/EBPα than did WT GMP cells, which correlated with an increase in monocyte progenitors and a decrease in granulocyte progenitors among GMP cells. Strikingly, BCAP -/- progenitors proliferated and produced more myeloid cells of both neutrophil and monocyte/macrophage lineages than did WT progenitors in myeloid colony-forming unit assays, supporting a cell-intrinsic role of BCAP in inhibiting myeloid proliferation and differentiation. Consistent with these findings, during cyclophosphamide-induced myeloablation or specific monocyte depletion, BCAP -/- mice replenished circulating monocytes and neutrophils earlier than WT mice. During myeloid replenishment after cyclophosphamide-induced myeloablation, BCAP -/- mice had increased LSK proliferation and increased numbers of LSK and GMP cells compared with WT mice. Furthermore, BCAP -/- mice accumulated more monocytes and neutrophils in the spleen than did WT mice during Listeria monocytogenes infection. Together, these data identify BCAP as a novel inhibitor of myelopoiesis in the steady state and of emergency myelopoiesis during demand conditions. © 2017 by The American Society of Hematology.

  18. Hematopoiesis and hematopoietic organs in arthropods.

    PubMed

    Grigorian, Melina; Hartenstein, Volker

    2013-03-01

    Hemocytes (blood cells) are motile cells that move throughout the extracellular space and that exist in all clades of the animal kingdom. Hemocytes play an important role in shaping the extracellular environment and in the immune response. Developmentally, hemocytes are closely related to the epithelial cells lining the vascular system (endothelia) and the body cavity (mesothelia). In vertebrates and insects, common progenitors, called hemangioblasts, give rise to the endothelia and blood cells. In the adult animal, many differentiated hemocytes seem to retain the ability to proliferate; however, in most cases investigated closely, the bulk of hemocyte proliferation takes place in specialized hematopoietic organs. Hematopoietic organs provide an environment where undifferentiated blood stem cells are able to self-renew, and at the same time generate offspring that differentiate into different blood cell types. Hematopoiesis in vertebrates, taking place in the bone marrow, has been subject to intensive research by immunologists and stem cell biologists. Much less is known about blood cell formation in invertebrate animals. In this review, we will survey structural and functional properties of invertebrate hematopoietic organs, with a main focus on insects and other arthropod taxa. We will then discuss similarities, at the molecular and structural level, that are apparent when comparing the development of blood cells in hematopoietic organs of vertebrates and arthropods. Our comparative review is intended to elucidate aspects of the biology of blood stem cells that are more easily missed when focusing on one or a few model species.

  19. Hematopoietic-to-mesenchymal transition of adipose tissue macrophages is regulated by integrin β1 and fabricated fibrin matrices

    PubMed Central

    Majka, Susan M.; Kohrt, Wendy M.; Miller, Heidi L.; Sullivan, Timothy M.; Klemm, Dwight J.

    2017-01-01

    ABSTRACT Some bona fide adult adipocytes arise de novo from a bone marrow-derived myeloid lineage. These studies further demonstrate that adipose tissue stroma contains a resident population of myeloid cells capable of adipocyte and multilineage mesenchymal differentiation. These resident myeloid cells lack hematopoietic markers and express mesenchymal and progenitor cell markers. Because bone marrow mesenchymal progenitor cells have not been shown to enter the circulation, we hypothesized that myeloid cells acquire mesenchymal differentiation capacity in adipose tissue. We fabricated a 3-dimensional fibrin matrix culture system to define the adipose differentiation potential of adipose tissue-resident myeloid subpopulations, including macrophages, granulocytes and dendritic cells. Our data show that multilineage mesenchymal potential was limited to adipose tissue macrophages, characterized by the acquisition of adipocyte, osteoblast, chondrocyte and skeletal muscle myocyte phenotypes. Fibrin hydrogel matrices stimulated macrophage loss of hematopoietic cell lineage determinants and the expression of mesenchymal and progenitor cell markers, including integrin β1. Ablation of integrin β1 in macrophages inhibited adipocyte specification. Therefore, some bona fide adipocytes are specifically derived from adipose tissue-resident macrophages via an integrin β1-dependent hematopoietic-to-mesenchymal transition, whereby they become capable of multipotent mesenchymal differentiation. The requirement for integrin β1 highlights this molecule as a potential target for controlling the production of marrow-derived adipocytes and their contribution to adipose tissue development and function. PMID:28441086

  20. Ineffective vaccination against solid tumors can be enhanced by hematopoietic cell transplantation.

    PubMed

    Filatenkov, Alexander; Müller, Antonia M S; Tseng, William Wei-Lin; Dejbakhsh-Jones, Sussan; Winer, Daniel; Luong, Richard; Shizuru, Judith A; Engleman, Edgar G; Strober, Samuel

    2009-12-01

    Vaccination with tumor Ags has not been an effective treatment for solid tumors. The goal of the current study was to determine whether a combination of vaccination and hematopoietic cell transplantation (HCT) can effectively treat primary, disseminated, or metastatic CT26 and MC38 murine colon tumors. Vaccination of tumor-bearing mice with irradiated tumor cells and CpG adjuvant failed to alter progressive tumor growth. However, mice bearing primary, disseminated lung, or metastatic liver tumors were uniformly cured after administration of total body irradiation, followed by the transplantation of hematopoietic progenitor cells and T cells from syngeneic, but not allogeneic vaccinated donors. Requirements for effective treatment of tumors included irradiation of hosts, vaccination of donors with both tumor cells and CpG, transfer of both CD4(+) and CD8(+) T cells along with progenitor cells, and ability of donor cells to produce IFN-gamma. Irradiation markedly increased the infiltration of donor T cells into the tumors, and the combined irradiation and HCT altered the balance of tumor-infiltrating cells to favor CD8(+) effector memory T cells as compared with CD4(+)CD25(+)FoxP3(+) T regulatory cells. The combination of vaccination and autologous hematopoietic cell transplantation was also effective in treating tumors. In conclusion, these findings show that otherwise ineffective vaccination to solid nonhematologic tumors can be dramatically enhanced by HCT.

  1. Bone marrow adipocytes as negative regulators of the hematopoietic microenvironment

    PubMed Central

    Naveiras, Olaia; Nardi, Valentina; Wenzel, Pamela L.; Fahey, Frederic; Daley, George Q.

    2009-01-01

    Osteoblasts and endothelium constitute functional niches that support hematopoietic stem cells (HSC) in mammalian bone marrow (BM) 1,2,3 . Adult BM also contains adipocytes, whose numbers correlate inversely with the hematopoietic activity of the marrow. Fatty infiltration of hematopoietic red marrow follows irradiation or chemotherapy and is a diagnostic feature in biopsies from patients with marrow aplasia 4. To explore whether adipocytes influence hematopoiesis or simply fill marrow space, we compared the hematopoietic activity of distinct regions of the mouse skeleton that differ in adiposity. By flow cytometry, colony forming activity, and competitive repopulation assay, HSCs and short-term progenitors are reduced in frequency in the adipocyte-rich vertebrae of the mouse tail relative to the adipocyte-free vertebrae of the thorax. In lipoatrophic A-ZIP/F1 “fatless” mice, which are genetically incapable of forming adipocytes8, and in mice treated with the PPARγ inhibitor Bisphenol-A-DiGlycidyl-Ether (BADGE), which inhibits adipogenesis9, post-irradiation marrow engraftment is accelerated relative to wild type or untreated mice. These data implicate adipocytes as predominantly negative regulators of the bone marrow microenvironment, and suggest that antagonizingmarrow adipogenesis may enhance hematopoietic recovery in clinical bone marrow transplantation. PMID:19516257

  2. In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

    PubMed Central

    Malide, Daniela; Métais, Jean-Yves; Dunbar, Cynthia E.

    2014-01-01

    We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner. PMID:25145579

  3. Multilineage hematopoietic recovery by a single injection of pegylated recombinant human megakaryocyte growth and development factor in myelosuppressed mice.

    PubMed

    Shibuya, K; Akahori, H; Takahashi, K; Tahara, E; Kato, T; Miyazaki, H

    1998-01-01

    that a single injection of PEG-rHuMGDF at an early time after irradiation is able to effectively improve thrombocytopenia, anemia, and leukopenia with concomitant accelerated recovery of both primitive and committed hematopoietic progenitors in irradiated mice. Our data also show that compared with the rhG-CSF shown to exert multilineage effects on hematopoiesis, PEG-rHuMGDF has more wide-ranging effects on peripheral blood cell recovery.

  4. Ex Vivo Expansion of CD34+CD90+CD49f+ Hematopoietic Stem and Progenitor Cells from Non‐Enriched Umbilical Cord Blood with Azole Compounds

    PubMed Central

    Bari, Sudipto; Zhong, Qixing; Fan, Xiubo; Poon, Zhiyong; Lim, Alvin Soon Tiong; Lim, Tse Hui; Dighe, Niraja; Li, Shang; Chiu, Gigi Ngar Chee; Chai, Christina Li Lin

    2018-01-01

    Abstract Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study, we aimed to perform ex vivo expansion of UCB HSPC from non‐enriched mononucleated cells (MNC) using novel azole‐based small molecules. Freshly‐thawed UCB–MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of >50 small molecules were developed using structure‐activity‐relationship studies of SB203580, a known p38‐MAPK inhibitor. A particular analog, C7, resulted in 1,554.1 ± 27.8‐fold increase of absolute viable CD45+CD34+CD38–CD45RA– progenitors which was at least 3.7‐fold higher than control cultures (p < .001). In depth phenotypic analysis revealed >600‐fold expansion of CD34+/CD90+/CD49f+ rare HSPCs coupled with significant (p < .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p < .001) higher engraftment of human CD45+ and CD45+CD34+ cells in the PB and BM by day 21 compared to non‐expanded and cytokine expanded grafts. The C7 expanded grafts maintained long‐term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre‐culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376–393 PMID:29392885

  5. Standard sub-thermoneutral caging temperature influences radiosensitivity of hematopoietic stem and progenitor cells.

    PubMed

    Povinelli, Benjamin J; Kokolus, Kathleen M; Eng, Jason W-L; Dougher, Christopher W; Curtin, Leslie; Capitano, Maegan L; Sailsbury-Ruf, Christi T; Repasky, Elizabeth A; Nemeth, Michael J

    2015-01-01

    The production of new blood cells relies on a hierarchical network of hematopoietic stem and progenitor cells (HSPCs). To maintain lifelong hematopoiesis, HSPCs must be protected from ionizing radiation or other cytotoxic agents. For many years, murine models have been a valuable source of information regarding factors that either enhance or reduce the survival of HSPCs after exposure of marrow to ionizing radiation. In a recent series of studies, however, it has become clear that housing-related factors such as the cool room temperature required for laboratory mice can exert a surprising influence on the outcome of experiments. Here we report that the mild, but chronic cold-stress endured by mice housed under these conditions exerts a protective effect on HSPCs after both non-lethal and lethal doses of total body irradiation (TBI). Alleviation of this cold-stress by housing mice at a thermoneutral temperature (30°C) resulted in significantly greater baseline radiosensitivity to a lethal dose of TBI with more HSPCs from mice housed at thermoneutral temperature undergoing apoptosis following non-lethal TBI. Cold-stressed mice have elevated levels of norepinephrine, a key molecule of the sympathetic nervous system that binds to β-adrenergic receptors. We show that blocking this signaling pathway in vivo through use of the β-blocker propanolol completely mitigates the protective effect of cold-stress on HSPC apoptosis. Collectively this study demonstrates that chronic stress endured by the standard housing conditions of laboratory mice increases the resistance of HSPCs to TBI-induced apoptosis through a mechanism that depends upon β-adrenergic signaling. Since β-blockers are commonly prescribed to a wide variety of patients, this information could be important when predicting the clinical impact of HSPC sensitivity to TBI.

  6. Standard Sub-Thermoneutral Caging Temperature Influences Radiosensitivity of Hematopoietic Stem and Progenitor Cells

    PubMed Central

    Eng, Jason W.-L.; Dougher, Christopher W.; Curtin, Leslie; Capitano, Maegan L.; Sailsbury-Ruf, Christi T.; Repasky, Elizabeth A.; Nemeth, Michael J.

    2015-01-01

    The production of new blood cells relies on a hierarchical network of hematopoietic stem and progenitor cells (HSPCs). To maintain lifelong hematopoiesis, HSPCs must be protected from ionizing radiation or other cytotoxic agents. For many years, murine models have been a valuable source of information regarding factors that either enhance or reduce the survival of HSPCs after exposure of marrow to ionizing radiation. In a recent series of studies, however, it has become clear that housing-related factors such as the cool room temperature required for laboratory mice can exert a surprising influence on the outcome of experiments. Here we report that the mild, but chronic cold-stress endured by mice housed under these conditions exerts a protective effect on HSPCs after both non-lethal and lethal doses of total body irradiation (TBI). Alleviation of this cold-stress by housing mice at a thermoneutral temperature (30°C) resulted in significantly greater baseline radiosensitivity to a lethal dose of TBI with more HSPCs from mice housed at thermoneutral temperature undergoing apoptosis following non-lethal TBI. Cold-stressed mice have elevated levels of norepinephrine, a key molecule of the sympathetic nervous system that binds to β-adrenergic receptors. We show that blocking this signaling pathway in vivo through use of the β-blocker propanolol completely mitigates the protective effect of cold-stress on HSPC apoptosis. Collectively this study demonstrates that chronic stress endured by the standard housing conditions of laboratory mice increases the resistance of HSPCs to TBI-induced apoptosis through a mechanism that depends upon β-adrenergic signaling. Since β-blockers are commonly prescribed to a wide variety of patients, this information could be important when predicting the clinical impact of HSPC sensitivity to TBI. PMID:25793392

  7. The TGFβ pathway is a key player for the endothelial-to-hematopoietic transition in the embryonic aorta.

    PubMed

    Lempereur, A; Canto, P Y; Richard, C; Martin, S; Thalgott, J; Raymond, K; Lebrin, F; Drevon, C; Jaffredo, T

    2018-02-15

    The embryonic aorta produces hematopoietic stem and progenitor cells from a hemogenic endothelium localized in the aortic floor through an endothelial to hematopoietic transition. It has been long proposed that the Bone Morphogenetic Protein (BMP)/Transforming Growth Factor ß (TGFß) signaling pathway was implicated in aortic hematopoiesis but the very nature of the signal was unknown. Here, using thorough expression analysis of the BMP/TGFß signaling pathway members in the endothelial and hematopoietic compartments of the aorta at pre-hematopoietic and hematopoietic stages, we show that the TGFß pathway is preferentially balanced with a prominent role of Alk1/TgfßR2/Smad1 and 5 on both chicken and mouse species. Functional analysis using embryonic stem cells mutated for Acvrl1 revealed an enhanced propensity to produce hematopoietic cells. Collectively, we reveal that TGFß through the Alk1/TgfßR2 receptor axis is acting on endothelial cells to produce hematopoiesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Hematopoietic Stem/Progenitor Cell Proliferation and Differentiation Is Differentially Regulated by High-Density and Low-Density Lipoproteins in Mice

    PubMed Central

    Feng, Yingmei; Schouteden, Sarah; Geenens, Rachel; Van Duppen, Vik; Herijgers, Paul; Holvoet, Paul; Van Veldhoven, Paul P.; Verfaillie, Catherine M.

    2012-01-01

    Rationale Hematopoietic stem/progenitor cells (HSPC) are responsible for maintaining the blood system as a result of their self-renewal and multilineage differentiation capacity. Recently, studies have suggested that HDL cholesterol may inhibit and impaired cholesterol efflux may increase HSPC proliferation and differentiation. Objectives We hypothesized that LDL may enhance HSPC proliferation and differentiation while HDL might have the opposing effect which might influence the size of the pool of inflammatory cells. Methods and Results HSPC number and function were studied in hypercholesterolemic LDL receptor knockout (LDLr−/−) mice on high fat diet. Hypercholesterolemia was associated with increased frequency of HSPC, monocytes and granulocytes in the peripheral blood (PB). In addition, an increased proportion of BM HSPC was in G2M of the cell cycle, and the percentage of HSPC and granulocyte-macrophage progenitors (GMP) increased in BM of LDLr−/− mice. When BM Lin-Sca-1+cKit+ (i.e. “LSK”) cells were cultured in the presence of LDL in vitro we also found enhanced differentiation towards monocytes and granulocytes. Furthermore, LDL promoted lineage negative (Lin−) cells motility. The modulation by LDL on HSPC differentiation into granulocytes and motility was inhibited by inhibiting ERK phosphorylation. By contrast, when mice were infused with human apoA-I (the major apolipoprotein of HDL) or reconstituted HDL (rHDL), the frequency and proliferation of HSPC was reduced in BM in vivo. HDL also reversed the LDL-induced monocyte and granulocyte differentiation in vitro. Conclusion Our data suggest that LDL and HDL have opposing effects on HSPC proliferation and differentiation. It will be of interest to determine if breakdown of HSPC homeostasis by hypercholesterolemia contributes to inflammation and atherosclerosis progression. PMID:23144813

  9. Inflamm-Aging of Hematopoiesis, Hematopoietic Stem Cells, and the Bone Marrow Microenvironment

    PubMed Central

    Kovtonyuk, Larisa V.; Fritsch, Kristin; Feng, Xiaomin; Manz, Markus G.; Takizawa, Hitoshi

    2016-01-01

    All hematopoietic and immune cells are continuously generated by hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) through highly organized process of stepwise lineage commitment. In the steady state, HSCs are mostly quiescent, while HPCs are actively proliferating and contributing to daily hematopoiesis. In response to hematopoietic challenges, e.g., life-threatening blood loss, infection, and inflammation, HSCs can be activated to proliferate and engage in blood formation. The HSC activation induced by hematopoietic demand is mediated by direct or indirect sensing mechanisms involving pattern recognition receptors or cytokine/chemokine receptors. In contrast to the hematopoietic challenges with obvious clinical symptoms, how the aging process, which involves low-grade chronic inflammation, impacts hematopoiesis remains undefined. Herein, we summarize recent findings pertaining to functional alternations of hematopoiesis, HSCs, and the bone marrow (BM) microenvironment during the processes of aging and inflammation and highlight some common cellular and molecular changes during the processes that influence hematopoiesis and its cells of origin, HSCs and HPCs, as well as the BM microenvironment. We also discuss how age-dependent alterations of the immune system lead to subclinical inflammatory states and how inflammatory signaling might be involved in hematopoietic aging. Our aim is to present evidence supporting the concept of “Inflamm-Aging,” or inflammation-associated aging of hematopoiesis. PMID:27895645

  10. Sirt1 Protects Stressed Adult Hematopoietic Stem Cells | Center for Cancer Research

    Cancer.gov

    The immune system relies on a stable pool of hematopoietic stem and progenitor cells (HSPCs) to respond properly to injury or stress. Maintaining genomic integrity and appropriate gene expression is essential for HSPC homeostasis, and dysregulation can result in myeloproliferative disorders or loss of immune function. Sirt1 is a histone deacetylase that can protect embryonic

  11. Dynamic equilibrium of heterogeneous and interconvertible multipotent hematopoietic cell subsets.

    PubMed

    Weston, Wendy; Zayas, Jennifer; Perez, Ruben; George, John; Jurecic, Roland

    2014-06-06

    Populations of hematopoietic stem cells and progenitors are quite heterogeneous and consist of multiple cell subsets with distinct phenotypic and functional characteristics. Some of these subsets also appear to be interconvertible and oscillate between functionally distinct states. The multipotent hematopoietic cell line EML has emerged as a unique model to study the heterogeneity and interconvertibility of multipotent hematopoietic cells. Here we describe extensive phenotypic and functional heterogeneity of EML cells which stems from the coexistence of multiple cell subsets. Each of these subsets is phenotypically and functionally heterogeneous, and displays distinct multilineage differentiation potential, cell cycle profile, proliferation kinetics, and expression pattern of HSC markers and some of the key lineage-associated transcription factors. Analysis of their maintenance revealed that on a population level all EML cell subsets exhibit cell-autonomous interconvertible properties, with the capacity to generate all other subsets and re-establish complete parental EML cell population. Moreover, all EML cell subsets generated during multiple cell generations maintain their distinct phenotypic and functional signatures and interconvertible properties. The model of EML cell line suggests that interconvertible multipotent hematopoietic cell subsets coexist in a homeostatically maintained dynamic equilibrium which is regulated by currently unknown cell-intrinsic mechanisms.

  12. Evaluating Interaction of Cord Blood Hematopoietic Stem/Progenitor Cells with Functionally Integrated Three-Dimensional Microenvironments.

    PubMed

    Mokhtari, Saloomeh; Baptista, Pedro M; Vyas, Dipen A; Freeman, Charles Jordan; Moran, Emma; Brovold, Matthew; Llamazares, Guillermo A; Lamar, Zanneta; Porada, Christopher D; Soker, Shay; Almeida-Porada, Graça

    2018-03-01

    Despite advances in ex vivo expansion of cord blood-derived hematopoietic stem/progenitor cells (CB-HSPC), challenges still remain regarding the ability to obtain, from a single unit, sufficient numbers of cells to treat an adolescent or adult patient. We and others have shown that CB-HSPC can be expanded ex vivo in two-dimensional (2D) cultures, but the absolute percentage of the more primitive stem cells decreases with time. During development, the fetal liver is the main site of HSPC expansion. Therefore, here we investigated, in vitro, the outcome of interactions of primitive HSPC with surrogate fetal liver environments. We compared bioengineered liver constructs made from a natural three-dimensional-liver-extracellular-matrix (3D-ECM) seeded with hepatoblasts, fetal liver-derived (LvSt), or bone marrow-derived stromal cells, to their respective 2D culture counterparts. We showed that the inclusion of cellular components within the 3D-ECM scaffolds was necessary for maintenance of HSPC viability in culture, and that irrespective of the microenvironment used, the 3D-ECM structures led to the maintenance of a more primitive subpopulation of HSPC, as determined by flow cytometry and colony forming assays. In addition, we showed that the timing and extent of expansion depends upon the biological component used, with LvSt providing the optimal balance between preservation of primitive CB HSPC and cellular differentiation. Stem Cells Translational Medicine 2018;7:271-282. © 2018 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  13. Mobilization of primitive and committed hematopoietic progenitors in nonhuman primates treated with defibrotide and recombinant human granulocyte colony-stimulating factor.

    PubMed

    Carlo-Stella, Carmelo; Di Nicola, Massimo; Longoni, Paolo; Milani, Raffaella; Milanesi, Marco; Guidetti, Anna; Haanstra, Krista; Jonker, Margaret; Cleris, Loredana; Magni, Michele; Formelli, Franca; Gianni, Alesssandro M

    2004-01-01

    The aim of this study was to evaluate the capacity of defibrotide in enhancing cytokine-induced hematopoietic mobilization in rhesus monkeys. Animals received recombinant human granulocyte colony-stimulating factor (rhG-CSF, 100 microg/kg/day SC for 5 days) and, after a 4- to 6-week washout period, were remobilized with defibrotide (15 mg/kg/hour continuous intravenous for 5 days) plus rhG-CSF. Hematopoietic mobilization was evaluated by complete blood counts, differential counts, as well as frequency and absolute numbers of colony-forming cells (CFCs), high-proliferative potential CFCs (HPP-CFCs), and long-term culture-initiating cells (LTC-ICs). Compared to baseline values, rhG-CSF increased circulating CFCs, HPP-CFCs, and LTC-ICs by 158-, 125-, and 67-fold, respectively; the same figures for defibrotide/rhG-CSF were 299-, 1452-, and 295-fold, respectively. Defibrotide/rhG-CSF treatment compared to rhG-CSF alone increased CFCs, HPP-CFCs, and LTC-ICs by 1.4- (35,089 vs 25,825, p< or =0.02), 6- (4358 vs 748, p< or =0.02), and 5-fold (884 vs 168, p< or =0.04), respectively. We then evaluated the effects of a 2-day defibrotide treatment associated with a 5-day rhG-CSF treatment. Compared to rhG-CSF, defibrotide/rhG-CSF increased the mobilization of CFCs, HPP-CFCs, and LTC-ICs by 2- (31,128 vs 15,527, p< or =0.05), 8- (5361 vs 660, p< or =0.01), and 8-fold (954 vs 119, p< or =0.01), respectively. Our data demonstrate that in nonhuman primates: 1) defibrotide enhances rhG-CSF-elicited mobilization of primitive and committed progenitors; and 2) a 2-day defibrotide injection is as effective as a 5-day injection.

  14. Developmental exposure to 2,3,7,8 tetrachlorodibenzo-p-dioxin attenuates capacity of hematopoietic stem cells to undergo lymphocyte differentiation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ahrenhoerster, Lori S.; Tate, Everett R.; Lakatos, Peter A.

    The process of hematopoiesis, characterized by long-term self-renewal and multi-potent lineage differentiation, has been shown to be regulated in part by the ligand-activated transcription factor known as the aryl hydrocarbon receptor (AHR). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a ubiquitous contaminant and the most potent AHR agonist, also modulates regulation of adult hematopoietic stem and progenitor cell (HSC/HPC) homeostasis. However, the effect of developmental TCDD exposure on early life hematopoiesis has not been fully explored. Given the inhibitory effects of TCDD on hematopoiesis and lymphocyte development, we hypothesized that in utero exposure to TCDD would alter the functional capacity of fetal HSC/HPCs to completemore » lymphocyte differentiation. To test this hypothesis, we employed a co-culture system designed to facilitate the maturation of progenitor cells to either B or T lymphocytes. Furthermore, we utilized an innovative limiting dilution assay to precisely quantify differences in lymphocyte differentiation between HSC/HPCs obtained from fetuses of dams exposed to 3 μg/kg TCDD or control. We found that the AHR is transcribed in yolk sac hematopoietic cells and is transcriptionally active as early as gestational day (GD) 7.5. Furthermore, the number of HSC/HPCs present in the fetal liver on GD 14.5 was significantly increased in fetuses whose mothers were exposed to TCDD throughout pregnancy. Despite this increase in HSC/HPC cell number, B and T lymphocyte differentiation is decreased by approximately 2.5 fold. These findings demonstrate that inappropriate developmental AHR activation in HSC/HPCs adversely impacts lymphocyte differentiation and may have consequences for lymphocyte development in the bone marrow and thymus later in life.« less

  15. Formaldehyde and co-exposure with benzene induce compensation of bone marrow and hematopoietic stem/progenitor cells in BALB/c mice during post-exposure period

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wei, Chenxi

    Formaldehyde (FA) is a human leukemogen. Since there is a latency period between initial FA exposure and the development of leukemia, the subsequent impact of FA on hematopoietic stem or progenitor cells (HSCs/HPCs) in post-exposure stage is crucial for a deep understanding of FA-induced hematotoxicity. BALB/c mice were exposed to 3 mg/m{sup 3} FA for 2 weeks, mimicking occupational exposure, and were monitored for another 7 days post-exposure. Meanwhile, we included benzene (BZ) as a positive control, separately and together with FA because co-exposure occurs frequently. After 7-day recovery, colonies of progenitors for CFU-GM and BFU-E, and nucleated bone marrowmore » cells in FA-exposed mice were comparable to controls, although they were significantly reduced during exposure. Levels of reactive oxygen species (ROS) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in CFU-GM and BFU-E from FA-exposed mice were higher than controls, although the increase in 8-OHdG was not significant. Granulocyte-macrophage colony stimulating factor (GM-CSF) level in the FA group was lower than controls, but the expression level for the receptor was not upregulated. It suggests that HSCs/HPCs in FA-exposed mice respond to a small amount of GM-CSF and proliferate rapidly, which may cause a possible risk of expansion of abnormal stem/progenitor cell clones. FA co-exposure with BZ was more potent for promoting CFU-GM formation and inducing ROS in BFU-E and 8-OHdG in CFU-GM during the post-exposure period. The compensation of myeloid progenitors with elevated ROS and 8-OHdG may lead to a risk of transforming normal HSCs/HPCs to leukemic stem/progenitor cells. Thus, co-exposure may pose a greater leukemia risk. - Highlights: • Nucleated bone marrow cell count recovered after 7 days post-FA and/or BZ exposure. • CFU-GM showed an increase in colonies and 8-OHdG after 7 days post-FA + BZ exposure. • Levels of ROS in CFU-GM and BFU-E were increased by FA or FA + BZ during recovery.

  16. Genetic modification of hematopoietic stem cells with nonviral systems: past progress and future prospects.

    PubMed

    Papapetrou, E P; Zoumbos, N C; Athanassiadou, A

    2005-10-01

    Serious unwanted complications provoked by retroviral gene transfer into hematopoietic stem cells (HSCs) have recently raised the need for the development and assessment of alternative gene transfer vectors. Within this context, nonviral gene transfer systems are attracting increasing interest. Their main advantages include low cost, ease of handling and large-scale production, large packaging capacity and, most importantly, biosafety. While nonviral gene transfer into HSCs has been restricted in the past by poor transfection efficiency and transient maintenance, in recent years, biotechnological developments are converting nonviral transfer into a realistic approach for genetic modification of cells of hematopoietic origin. Herein we provide an overview of past accomplishments in the field of nonviral gene transfer into hematopoietic progenitor/stem cells and we point at future challenges. We argue that episomally maintained self-replicating vectors combined with physical methods of delivery show the greatest promise among nonviral gene transfer strategies for the treatment of disorders of the hematopoietic system.

  17. Effect of Calcium-Infiltrated Hydroxyapatite Scaffolds on the Hematopoietic Fate of Human Umbilical Vein Endothelial Cells.

    PubMed

    Zhang, Qinghao; Gerlach, Jörg C; Schmelzer, Eva; Nettleship, Ian

    2017-01-01

    Foamed hydroxyapatite offers a three-dimensional scaffold for the development of bone constructs, mimicking perfectly the in vivo bone structure. In vivo, calcium release at the surface is assumed to provide a locally increased gradient supporting the maintenance of the hematopoietic stem cells niche. We fabricated hydroxyapatite scaffolds with high surface calcium concentration by infiltration, and used human umbilical vein endothelial cells (HUVECs) as a model to study the effects on hematopoietic lineage direction. HUVECs are umbilical vein-derived and thus possess progenitor characteristics, with a prospective potential to give rise to hematopoietic lineages. HUVECs were cultured for long term on three-dimensional porous hydroxyapatite scaffolds, which were either infiltrated biphasic foams or untreated. Controls were cultured in two-dimensional dishes. The release of calcium into culture medium was determined, and cells were analyzed for typical hematopoietic and endothelial gene expressions, surface markers by flow cytometry, and hematopoietic potential using colony-forming unit assays. Our results indicate that the biphasic foams promoted a hematopoietic lineage direction of HUVECs, suggesting an improved in vivo-like scaffold for hematopoietic bone tissue engineering. © 2017 S. Karger AG, Basel.

  18. Brief report: ectopic expression of NUP98-HOXA10 augments erythroid differentiation of human embryonic stem cells.

    PubMed

    Ji, Junfeng; Risueño, Ruth M; Hong, Seokho; Allan, David; Rosten, Patty; Humphries, Keith; Bhatia, Mickie

    2011-04-01

    Hox genes encode highly conserved transcription factors that have been implicated in hematopoietic development and self-renewal of hematopoietic stem cells (HSCs) and hematopoietic development. The potency of NUP98-HOXA10hd (NA10) on adult murine bone marrow HSC self-renewal prompted us to examine its effect on specification and proliferation of hematopoietic cells derived from human embryonic stem cells (hESCs). Here, we demonstrate that expression of NA10 in hESCs influences the hematopoietic differentiation program. The specific effect of NA10 is dependent on the developmental stage of hematopoietic emergence from hESCs. Overexpression of NA10 in either undifferentiated hESCs or early hemogenic precursors augmented the frequency of CD45(-) GlycophorinA(+) cells and erythroid progenitors (blast-forming unit-erythrocyte). In contrast, targeted NA10 expression in primitive CD34+ cells committed to the hematopoietic lineage had no effect on erythropoietic capacity but instead increased hematopoietic progenitor proliferation. Our study reveals a novel neomorphic effect of NA10 in early human erythroid development from pluripotent stem cells. Copyright © 2011 AlphaMed Press.

  19. Inhibiting glycogen synthase kinase-3 mitigates the hematopoietic acute radiation syndrome in mice.

    PubMed

    Lee, Chang-Lung; Lento, William E; Castle, Katherine D; Chao, Nelson J; Kirsch, David G

    2014-05-01

    Exposure to a nuclear accident or radiological attack can cause death from acute radiation syndrome (ARS), which results from radiation injury to vital organs such as the hematopoietic system. However, the U.S. Food and Drug Administration (FDA) has not approved any medical countermeasures for this specific purpose. With growing concern over nuclear terrorism, there is an urgent need to develop small molecule deliverables that mitigate mortality from ARS. One emerging modulator of hematopoietic stem/progenitor cell (HSPC) activity is glycogen synthase kinase-3 (GSK-3). The inhibition of GSK-3 has been shown to augment hematopoietic repopulation in mouse models of bone marrow transplantation. In this study, we performed an in vitro screen using irradiated bone marrow mononuclear cells (BM-MNCs) to test the effects of four GSK-3 inhibitors: CHIR99021; 6-Bromoindirubin-3'-oxime (BIO); SB415286; and SB216763. This screen showed that SB216763 significantly increased the frequency of c-Kit(+) Lin(-) Sca1(+) (KLS) cells and hematopoietic colony-forming cells in irradiated BM-MNCs. Importantly, administration of a single dose of SB216763 to C57BL/6J mice by subcutaneous injection 24 h after total-body irradiation significantly improved hematopoietic recovery and mitigated hematopoietic ARS. Collectively, our results demonstrate that the GSK-3 inhibitor SB216763 is an effective medical countermeasure against acute radiation injury of the hematopoietic system.

  20. Transformation of human mesenchymal cells and skin fibroblasts into hematopoietic cells.

    PubMed

    Harris, David M; Hazan-Haley, Inbal; Coombes, Kevin; Bueso-Ramos, Carlos; Liu, Jie; Liu, Zhiming; Li, Ping; Ravoori, Murali; Abruzzo, Lynne; Han, Lin; Singh, Sheela; Sun, Michael; Kundra, Vikas; Kurzrock, Razelle; Estrov, Zeev

    2011-01-01

    Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs) and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza) and the growth factors (GF) granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy.

  1. Transformation of Human Mesenchymal Cells and Skin Fibroblasts into Hematopoietic Cells

    PubMed Central

    Harris, David M.; Hazan-Haley, Inbal; Coombes, Kevin; Bueso-Ramos, Carlos; Liu, Jie; Liu, Zhiming; Li, Ping; Ravoori, Murali; Abruzzo, Lynne; Han, Lin; Singh, Sheela; Sun, Michael; Kundra, Vikas; Kurzrock, Razelle; Estrov, Zeev

    2011-01-01

    Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs) and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza) and the growth factors (GF) granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy. PMID:21731684

  2. Thymus-autonomous T cell development in the absence of progenitor import.

    PubMed

    Martins, Vera C; Ruggiero, Eliana; Schlenner, Susan M; Madan, Vikas; Schmidt, Manfred; Fink, Pamela J; von Kalle, Christof; Rodewald, Hans-Reimer

    2012-07-30

    Thymus function is thought to depend on a steady supply of T cell progenitors from the bone marrow. The notion that the thymus lacks progenitors with self-renewal capacity is based on thymus transplantation experiments in which host-derived thymocytes replaced thymus-resident cells within 4 wk. Thymus grafting into T cell-deficient mice resulted in a wave of T cell export from the thymus, followed by colonization of the thymus by host-derived progenitors, and cessation of T cell development. Compound Rag2(-/-)γ(c)(-/-)Kit(W/Wv) mutants lack competitive hematopoietic stem cells (HSCs) and are devoid of T cell progenitors. In this study, using this strain as recipients for wild-type thymus grafts, we noticed thymus-autonomous T cell development lasting several months. However, we found no evidence for export of donor HSCs from thymus to bone marrow. A diverse T cell antigen receptor repertoire in progenitor-deprived thymus grafts implied that many thymocytes were capable of self-renewal. Although the process was most efficient in Rag2(-/-)γ(c)(-/-)Kit(W/Wv) hosts, γ(c)-mediated signals alone played a key role in the competition between thymus-resident and bone marrow-derived progenitors. Hence, the turnover of each generation of thymocytes is not only based on short life span but is also driven via expulsion of resident thymocytes by fresh progenitors entering the thymus.

  3. Coordinated and unique functions of the E-selectin ligand ESL-1 during inflammatory and hematopoietic recruitment in mice

    PubMed Central

    Sreeramkumar, Vinatha; Leiva, Magdalena; Stadtmann, Anika; Pitaval, Christophe; Ortega-Rodríguez, Inés; Wild, Martin K.; Lee, Brendan; Zarbock, Alexander; Hidalgo, Andrés

    2013-01-01

    Beyond its well-established roles in mediating leukocyte rolling, E-selectin is emerging as a multifunctional receptor capable of inducing integrin activation in neutrophils, and of regulating various biological processes in hematopoietic precursors. Although these effects suggest important homeostatic contributions of this selectin in the immune and hematologic systems, the ligands responsible for transducing these effects in different leukocyte lineages are not well defined. We have characterized mice deficient in E-selectin ligand-1 (ESL-1), or in both P-selectin glycoprotein-1 (PSGL-1) and ESL-1, to explore and compare the contributions of these glycoproteins in immune and hematopoietic cell trafficking. In the steady state, ESL-1 deficiency resulted in a moderate myeloid expansion that became more prominent when both glycoproteins were eliminated. During inflammation, PSGL-1 dominated E-selectin binding, rolling, integrin activation, and extravasation of mature neutrophils, but only the combined deficiency in PSGL-1 and ESL-1 completely abrogated leukocyte recruitment. Surprisingly, we find that the levels of ESL-1 were strongly elevated in hematopoietic progenitor cells. These elevations correlated with a prominent function of ESL-1 for E-selectin binding and for migration of hematopoietic progenitor cells into the bone marrow. Our results uncover dominant roles for ESL-1 in the immature compartment, and a functional shift toward PSGL-1 dependence in mature neutrophils. PMID:24106206

  4. Coordinated and unique functions of the E-selectin ligand ESL-1 during inflammatory and hematopoietic recruitment in mice.

    PubMed

    Sreeramkumar, Vinatha; Leiva, Magdalena; Stadtmann, Anika; Pitaval, Christophe; Ortega-Rodríguez, Inés; Wild, Martin K; Lee, Brendan; Zarbock, Alexander; Hidalgo, Andrés

    2013-12-05

    Beyond its well-established roles in mediating leukocyte rolling, E-selectin is emerging as a multifunctional receptor capable of inducing integrin activation in neutrophils, and of regulating various biological processes in hematopoietic precursors. Although these effects suggest important homeostatic contributions of this selectin in the immune and hematologic systems, the ligands responsible for transducing these effects in different leukocyte lineages are not well defined. We have characterized mice deficient in E-selectin ligand-1 (ESL-1), or in both P-selectin glycoprotein-1 (PSGL-1) and ESL-1, to explore and compare the contributions of these glycoproteins in immune and hematopoietic cell trafficking. In the steady state, ESL-1 deficiency resulted in a moderate myeloid expansion that became more prominent when both glycoproteins were eliminated. During inflammation, PSGL-1 dominated E-selectin binding, rolling, integrin activation, and extravasation of mature neutrophils, but only the combined deficiency in PSGL-1 and ESL-1 completely abrogated leukocyte recruitment. Surprisingly, we find that the levels of ESL-1 were strongly elevated in hematopoietic progenitor cells. These elevations correlated with a prominent function of ESL-1 for E-selectin binding and for migration of hematopoietic progenitor cells into the bone marrow. Our results uncover dominant roles for ESL-1 in the immature compartment, and a functional shift toward PSGL-1 dependence in mature neutrophils.

  5. Discovery, Progenitor and Early Evolution of a Stripped Envelope Supernova iPTF13bvn

    NASA Astrophysics Data System (ADS)

    Cao, Yi; Kasliwal, Mansi M.; Arcavi, Iair; Horesh, Assaf; Hancock, Paul; Valenti, Stefano; Cenko, S. Bradley; Kulkarni, S. R.; Gal-Yam, Avishay; Gorbikov, Evgeny; Ofek, Eran O.; Sand, David; Yaron, Ofer; Graham, Melissa; Silverman, Jeffrey M.; Wheeler, J. Craig; Marion, G. H.; Walker, Emma S.; Mazzali, Paolo; Howell, D. Andrew; Li, K. L.; Kong, A. K. H.; Bloom, Joshua S.; Nugent, Peter E.; Surace, Jason; Masci, Frank; Carpenter, John; Degenaar, Nathalie; Gelino, Christopher R.

    2013-09-01

    The intermediate Palomar Transient Factory reports our discovery of a young supernova, iPTF13bvn, in the nearby galaxy, NGC 5806 (22.5 Mpc). Our spectral sequence in the optical and infrared suggests a Type Ib classification. We identify a blue progenitor candidate in deep pre-explosion imaging within a 2σ error circle of 80 mas (8.7 pc). The candidate has an MB luminosity of -5.52 ± 0.39 mag and a B - I color of 0.25 ± 0.25 mag. If confirmed by future observations, this would be the first direct detection for a progenitor of a Type Ib. Fitting a power law to the early light curve, we find an extrapolated explosion date around 0.6 days before our first detection. We see no evidence of shock cooling. The pre-explosion detection limits constrain the radius of the progenitor to be smaller than a few solar radii. iPTF13bvn is also detected in centimeter and millimeter wavelengths. Fitting a synchrotron self-absorption model to our radio data, we find a mass-loading parameter of 1.3×1012 g cm-1. Assuming a wind velocity of 103 km s-1, we derive a progenitor mass-loss rate of 3 × 10-5 M ⊙ yr-1. Our observations, taken as a whole, are consistent with a Wolf-Rayet progenitor of the supernova iPTF13bvn.

  6. Accelerated hematopoietic toxicity by high energy (56)Fe radiation.

    PubMed

    Datta, Kamal; Suman, Shubhankar; Trani, Daniela; Doiron, Kathryn; Rotolo, Jimmy A; Kallakury, Bhaskar V S; Kolesnick, Richard; Cole, Michael F; Fornace, Albert J

    2012-03-01

    There is little information on the relative toxicity of highly charged (Z) high-energy (HZE) radiation in animal models compared to γ or X-rays, and the general assumption based on in vitro studies has been that acute toxicity is substantially greater. C57BL/6J mice were irradiated with (56)Fe ions (1 GeV/nucleon), and acute (within 30 d) toxicity compared to that of γ rays or protons (1 GeV). To assess relative hematopoietic and gastrointestinal toxicity, the effects of (56)Fe ions were compared to γ rays using complete blood count (CBC), bone marrow granulocyte-macrophage colony forming unit (GM-CFU), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis in bone marrow, and intestinal crypt survival. Although onset was more rapid, (56)Fe ions were only slightly more toxic than γ rays or protons with lethal dose (LD)(50/30) (a radiation dose at which 50% lethality occurs at 30-day) values of 5.8, 7.25, and 6.8 Gy, respectively, with relative biologic effectiveness for (56)Fe ions of 1.25 and 1.06 for protons. (56)Fe radiation caused accelerated and more severe hematopoietic toxicity. Early mortality correlated with more profound leukopenia and subsequent sepsis. Results indicate that there is selective enhanced toxicity to bone marrow progenitor cells, which are typically resistant to γ rays, and bone marrow stem cells, because intestinal crypt cells did not show increased HZE toxicity.

  7. Transplantation of wild-type mouse hematopoietic stem and progenitor cells ameliorates deficits in a mouse model of Friedreich’s ataxia

    PubMed Central

    Rocca, Celine J.; Goodman, Spencer M.; Dulin, Jennifer N.; Haquang, Joseph H.; Gertsman, Ilya; Blondelle, Jordan; Smith, Janell L. M.; Heyser, Charles J.; Cherqui, Stephanie

    2017-01-01

    Friedreich’s ataxia (FRDA) is an incurable autosomal recessive neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin due to an intronic GAA-repeat expansion in the FXN gene. We report the therapeutic efficacy of transplanting wild-type mouse hematopoietic stem and progenitor cells (HSPCs) into the YG8R mouse model of FRDA. In the HSPC-transplanted YG8R mice, development of muscle weakness and locomotor deficits was abrogated as was degeneration of large sensory neurons in the dorsal root ganglia (DRGs) and mitochondrial capacity was improved in brain, skeletal muscle, and heart. Transplanted HSPCs engrafted and then differentiated into microglia in the brain and spinal cord and into macrophages in the DRGs, heart, and muscle of YG8R FRDA mice. We observed the transfer of wild-type frataxin and Cox8 mitochondrial proteins from HSPC-derived microglia/macrophages to FRDA mouse neurons and muscle myocytes in vivo. Our results show the HSPC-mediated phenotypic rescue of FRDA in YG8R mice and suggest that this approach should be investigated further as a strategy for treating FRDA. PMID:29070698

  8. JAK2V617F expression in mice amplifies early hematopoietic cells and gives them a competitive advantage that is hampered by IFNα.

    PubMed

    Hasan, Salma; Lacout, Catherine; Marty, Caroline; Cuingnet, Marie; Solary, Eric; Vainchenker, William; Villeval, Jean-Luc

    2013-08-22

    The acquired gain-of-function V617F mutation in the Janus Kinase 2 (JAK2(V617F)) is the main mutation involved in BCR/ABL-negative myeloproliferative neoplasms (MPNs), but its effect on hematopoietic stem cells as a driver of disease emergence has been questioned. Therefore, we reinvestigated the role of endogenous expression of JAK2(V617F) on early steps of hematopoiesis as well as the effect of interferon-α (IFNα), which may target the JAK2(V617F) clone in humans by using knock-in mice with conditional expression of JAK2(V617F) in hematopoietic cells. These mice develop a MPN mimicking polycythemia vera with large amplification of myeloid mature and precursor cells, displaying erythroid endogenous growth and progressing to myelofibrosis. Interestingly, early hematopoietic compartments [Lin-, LSK, and SLAM (LSK/CD48-/CD150+)] increased with the age. Competitive repopulation assays demonstrated disease appearance and progressive overgrowth of myeloid, Lin-, LSK, and SLAM cells, but not lymphocytes, from a low number of engrafted JAK2(V617F) SLAM cells. Finally, IFNα treatment prevented disease development by specifically inhibiting JAK2(V617F) cells at an early stage of differentiation and eradicating disease-initiating cells. This study shows that JAK2(V617F) in mice amplifies not only late but also early hematopoietic cells, giving them a proliferative advantage through high cell cycling and low apoptosis that may sustain MPN emergence but is lost upon IFNα treatment.

  9. Sirt1 Protects Stressed Adult Hematopoietic Stem Cells | Center for Cancer Research

    Cancer.gov

    The immune system relies on a stable pool of hematopoietic stem and progenitor cells (HSPCs) to respond properly to injury or stress. Maintaining genomic integrity and appropriate gene expression is essential for HSPC homeostasis, and dysregulation can result in myeloproliferative disorders or loss of immune function. Sirt1 is a histone deacetylase that can protect embryonic stem (ES) cells from accumulating DNA damage and has been linked to hematopoietic differentiation of ES cells. Satyendra Singh, Ph.D., a postdoctoral fellow working with Philipp Oberdoerffer, Ph.D., in CCR’s Laboratory of Receptor Biology and Gene Expression, and their colleagues set out to determine whether Sirt1 could play a similar protective role in adult HSPCs.

  10. Vascular wall progenitor cells in health and disease.

    PubMed

    Psaltis, Peter J; Simari, Robert D

    2015-04-10

    The vasculature plays an indispensible role in organ development and maintenance of tissue homeostasis, such that disturbances to it impact greatly on developmental and postnatal health. Although cell turnover in healthy blood vessels is low, it increases considerably under pathological conditions. The principle sources for this phenomenon have long been considered to be the recruitment of cells from the peripheral circulation and the re-entry of mature cells in the vessel wall back into cell cycle. However, recent discoveries have also uncovered the presence of a range of multipotent and lineage-restricted progenitor cells in the mural layers of postnatal blood vessels, possessing high proliferative capacity and potential to generate endothelial, smooth muscle, hematopoietic or mesenchymal cell progeny. In particular, the tunica adventitia has emerged as a progenitor-rich compartment with niche-like characteristics that support and regulate vascular wall progenitor cells. Preliminary data indicate the involvement of some of these vascular wall progenitor cells in vascular disease states, adding weight to the notion that the adventitia is integral to vascular wall pathogenesis, and raising potential implications for clinical therapies. This review discusses the current body of evidence for the existence of vascular wall progenitor cell subpopulations from development to adulthood and addresses the gains made and significant challenges that lie ahead in trying to accurately delineate their identities, origins, regulatory pathways, and relevance to normal vascular structure and function, as well as disease. © 2015 American Heart Association, Inc.

  11. Identification of the Niche and Phenotype of the First Human Hematopoietic Stem Cells

    PubMed Central

    Ivanovs, Andrejs; Rybtsov, Stanislav; Anderson, Richard A.; Turner, Marc L.; Medvinsky, Alexander

    2014-01-01

    Summary In various vertebrate species, the dorsal aorta (Ao) is the site of specification of adult hematopoietic stem cells (HSCs). It has been observed that the upregulation of essential hematopoietic transcription factors and the formation of specific intra-aortic hematopoietic cell clusters occur predominantly in the ventral domain of the Ao (AoV). In the mouse, the first HSCs emerge in the AoV. Here, we demonstrate that in the human embryo the first definitive HSCs also emerge asymmetrically and are localized to the AoV, which thus identifies a functional niche for developing human HSCs. Using magnetic cell separation and xenotransplantations, we show that the first human HSCs are CD34+VE-cadherin+CD45+C-KIT+THY-1+Endoglin+RUNX1+CD38−/loCD45RA−. This population harbors practically all committed hematopoietic progenitors and is underrepresented in the dorsal domain of the Ao (AoD) and urogenital ridges (UGRs). The present study provides a foundation for analysis of molecular mechanisms underpinning embryonic specification of human HSCs. PMID:24749070

  12. Dynamic equilibrium of heterogeneous and interconvertible multipotent hematopoietic cell subsets

    PubMed Central

    Weston, Wendy; Zayas, Jennifer; Perez, Ruben; George, John; Jurecic, Roland

    2014-01-01

    Populations of hematopoietic stem cells and progenitors are quite heterogeneous and consist of multiple cell subsets with distinct phenotypic and functional characteristics. Some of these subsets also appear to be interconvertible and oscillate between functionally distinct states. The multipotent hematopoietic cell line EML has emerged as a unique model to study the heterogeneity and interconvertibility of multipotent hematopoietic cells. Here we describe extensive phenotypic and functional heterogeneity of EML cells which stems from the coexistence of multiple cell subsets. Each of these subsets is phenotypically and functionally heterogeneous, and displays distinct multilineage differentiation potential, cell cycle profile, proliferation kinetics, and expression pattern of HSC markers and some of the key lineage-associated transcription factors. Analysis of their maintenance revealed that on a population level all EML cell subsets exhibit cell-autonomous interconvertible properties, with the capacity to generate all other subsets and re-establish complete parental EML cell population. Moreover, all EML cell subsets generated during multiple cell generations maintain their distinct phenotypic and functional signatures and interconvertible properties. The model of EML cell line suggests that interconvertible multipotent hematopoietic cell subsets coexist in a homeostatically maintained dynamic equilibrium which is regulated by currently unknown cell-intrinsic mechanisms. PMID:24903657

  13. Quantification of Acute Lymphoblastic Leukemia Clonotypes in Leukapheresed Peripheral Blood Progenitor Cells Predicts Relapse Risk after Autologous Hematopoietic Stem Cell Transplantation.

    PubMed

    Mannis, Gabriel N; Martin, Thomas G; Damon, Lloyd E; Andreadis, Charalambos; Olin, Rebecca L; Kong, Katherine A; Faham, Malek; Hwang, Jimmy; Ai, Weiyun Z; Gaensler, Karin M L; Sayre, Peter H; Wolf, Jeffrey L; Logan, Aaron C

    2016-06-01

    Since the incorporation of tyrosine kinase inhibitors into the treatment of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL), the notion that all patients with "high-risk" ALL uniformly require allogeneic (allo) hematopoietic cell transplantation (HCT) has received increasing scrutiny. Although multiple studies have shown superiority of alloHCT over autologous (auto) hematopoietic cell transplantation for high-risk patients, these findings may be explained, in part, by contamination of the peripheral blood progenitor cell (PBPC) leukapheresis product by residual leukemic cells in patients undergoing autoHCT. We retrospectively evaluated minimal residual disease (MRD) using next-generation sequencing (NGS) in the PBPC leukapheresis product of 32 ALL patients who underwent autoHCT. Twenty-eight patients (88%) had diagnostic samples with quantifiable immunoreceptor rearrangements to follow for MRD. Twelve (38%) patients had Ph+ B-ALL, 12 (38%) had Philadelphia chromosome-negative (Ph-) B-ALL, and 4 (14%) had T cell ALL. With a median follow-up of 41 months (range, 3 to 217), median relapse-free survival (RFS) and overall survival for the entire cohort were 3.2 and 4.2 years, respectively; at 5 years after transplantation, 42% of patients remain alive and relapse free. Using MRD detection at a threshold of ≥ 1 × 10(-6), median RFS for patients with detectable MRD was 6.5 months and was not reached for patients without detectable disease (P = .0005). In multivariate analysis, the only factor significantly associated with relapse was the presence of MRD ≥1 × 10(-6) (odds ratio, 23.8; confidence interval, 1.8 to 312.9; P = .0158). Our findings suggest that NGS for MRD detection can predict long-term RFS in patients undergoing autoHCT for high-risk ALL. Copyright © 2016 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  14. Synergistic Integration of Mesenchymal Stem Cells and Hydrostatic Pressure in the Expansion and Maintenance of Human Hematopoietic/Progenitor Cells

    PubMed Central

    2018-01-01

    Ex vivo expansion of hematopoietic stem/progenitor cell (HSPC) has been investigated to improve the clinical outcome of HSPC transplantation. However, ex vivo expansion of HSPCs still faces a major obstacle in that HPSCs tend to differentiate when proliferating. Here, we cocultured HSPCs with mesenchymal stem cells (MSCs) and divided the HSPCs into two fractions according to whether they came into adherent to MSCs or not. Additionally, we used hydrostatic pressure (HP) to mimic the physical conditions in vivo. Even nonadherent cells expanded to yield a significantly larger number of total nucleated cells (TNCs), adherent cells maintained the HSPC phenotype (CD34+, CD34+CD38−, and CD133+CD38−) to a greater extent than nonadherent cells and had superior clonogenic potential. Moreover, applying HP significantly increased the number of TNCs, the frequency of the immature HSPC phenotype, and the clonogenic potential. Furthermore, the genetic markers for the HSPC niche were significantly increased under HP. Our data suggest that the nonadherent fraction is the predominant site of HSPC expansion, whereas the adherent fraction seems to mimic the HSPC niche for immature cells. Moreover, HP has a synergistic effect on expansion and functional maintenance. This first study utilizing HP has a potential of designing clinically applicable expansion systems. PMID:29681947

  15. Regulation of Drosophila hematopoietic sites by Activin-β from active sensory neurons

    PubMed Central

    Makhijani, Kalpana; Alexander, Brandy; Rao, Deepti; Petraki, Sophia; Herboso, Leire; Kukar, Katelyn; Batool, Itrat; Wachner, Stephanie; Gold, Katrina S.; Wong, Corinna; O’Connor, Michael B.; Brückner, Katja

    2017-01-01

    An outstanding question in animal development, tissue homeostasis and disease is how cell populations adapt to sensory inputs. During Drosophila larval development, hematopoietic sites are in direct contact with sensory neuron clusters of the peripheral nervous system (PNS), and blood cells (hemocytes) require the PNS for their survival and recruitment to these microenvironments, known as Hematopoietic Pockets. Here we report that Activin-β, a TGF-β family ligand, is expressed by sensory neurons of the PNS and regulates the proliferation and adhesion of hemocytes. These hemocyte responses depend on PNS activity, as shown by agonist treatment and transient silencing of sensory neurons. Activin-β has a key role in this regulation, which is apparent from reporter expression and mutant analyses. This mechanism of local sensory neurons controlling blood cell adaptation invites evolutionary parallels with vertebrate hematopoietic progenitors and the independent myeloid system of tissue macrophages, whose regulation by local microenvironments remain undefined. PMID:28748922

  16. Dual role of wingless signaling in stem-like hematopoietic precursor maintenance in Drosophila.

    PubMed

    Sinenko, Sergey A; Mandal, Lolitika; Martinez-Agosto, Julian A; Banerjee, Utpal

    2009-05-01

    In Drosophila, blood development occurs in a specialized larval hematopoietic organ, the lymph gland (LG), within which stem-like hemocyte precursors or prohemocytes differentiate to multiple blood cell types. Here we show that components of the Wingless (Wg) signaling pathway are expressed in prohemocytes. Loss- and gain-of-function analysis indicates that canonical Wg signaling is required for maintenance of prohemocytes and negatively regulates their differentiation. Wg signals locally in a short-range fashion within different compartments of the LG. In addition, Wg signaling positively regulates the proliferation and maintenance of cells that function as a hematopoietic niche in Drosophila, the posterior signaling center (PSC), and in the proliferation of crystal cells. Our studies reveal a conserved function of Wg signaling in the maintenance of stem-like blood progenitors and reveal an involvement of this pathway in the regulation of hemocyte differentiation through its action in the hematopoietic niche.

  17. BCR-ABL enhances differentiation of long-term repopulating hematopoietic stem cells

    PubMed Central

    Schemionek, Mirle; Elling, Christian; Steidl, Ulrich; Bäumer, Nicole; Hamilton, Ashley; Spieker, Tilmann; Göthert, Joachim R.; Stehling, Martin; Wagers, Amy; Huettner, Claudia S.; Tenen, Daniel G.; Tickenbrock, Lara; Berdel, Wolfgang E.; Serve, Hubert; Holyoake, Tessa L.; Müller-Tidow, Carsten

    2010-01-01

    In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL+ Lin−Sca-1+c-kit+ (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin−Sca-1−c-kit+), nor mature granulocytes (CD11b+Gr-1+), nor potential stem cell niche cells (CD45−Ter119−) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL+ LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity. PMID:20053753

  18. Enhanced genetic modification of adult growth factor mobilized peripheral blood hematopoietic stem and progenitor cells with rapamycin.

    PubMed

    Li, Lijing; Torres-Coronado, Mónica; Gu, Angel; Rao, Anitha; Gardner, Agnes M; Epps, Elizabeth W; Gonzalez, Nancy; Tran, Chy-Anh; Wu, Xiwei; Wang, Jin-Hui; DiGiusto, David L

    2014-10-01

    Genetic modification of adult human hematopoietic stem and progenitor cells (HSPCs) with lentiviral vectors leads to long-term gene expression in the progeny of the HSPCs and has been used to successfully treat several monogenic diseases. In some cases, the gene-modified cells have a selective growth advantage over nonmodified cells and eventually are the dominant engrafted population. However, in disease indications for which the gene-modified cells do not have a selective advantage, optimizing transduction of HSPC is paramount to successful stem cell-based gene therapy. We demonstrate here that transduction of adult CD34+ HSPCs with lentiviral vectors in the presence of rapamycin, a widely used mTORC1 inhibitor, results in an approximately threefold increase in stable gene marking with minimal effects on HSPC growth and differentiation. Using this approach, we have demonstrated that we can enhance the frequency of gene-modified HSPCs that give rise to clonogenic progeny in vitro without excessive increases in the number of vector copies per cell or changes in integration pattern. The genetic marking of HSPCs and expression of transgenes is durable, and transplantation of gene-modified HSPCs into immunodeficient mice results in high levels of gene marking of the lymphoid and myeloid progeny in vivo. The prior safe clinical history of rapamycin in other applications supports the use of this compound to generate gene-modified autologous HSPCs for our HIV gene therapy clinical trials. ©AlphaMed Press.

  19. Enhanced Genetic Modification of Adult Growth Factor Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells With Rapamycin

    PubMed Central

    Li, Lijing; Torres-Coronado, Mónica; Gu, Angel; Rao, Anitha; Gardner, Agnes M.; Epps, Elizabeth W.; Gonzalez, Nancy; Tran, Chy-Anh; Wu, Xiwei; Wang, Jin-Hui

    2014-01-01

    Genetic modification of adult human hematopoietic stem and progenitor cells (HSPCs) with lentiviral vectors leads to long-term gene expression in the progeny of the HSPCs and has been used to successfully treat several monogenic diseases. In some cases, the gene-modified cells have a selective growth advantage over nonmodified cells and eventually are the dominant engrafted population. However, in disease indications for which the gene-modified cells do not have a selective advantage, optimizing transduction of HSPC is paramount to successful stem cell-based gene therapy. We demonstrate here that transduction of adult CD34+ HSPCs with lentiviral vectors in the presence of rapamycin, a widely used mTORC1 inhibitor, results in an approximately threefold increase in stable gene marking with minimal effects on HSPC growth and differentiation. Using this approach, we have demonstrated that we can enhance the frequency of gene-modified HSPCs that give rise to clonogenic progeny in vitro without excessive increases in the number of vector copies per cell or changes in integration pattern. The genetic marking of HSPCs and expression of transgenes is durable, and transplantation of gene-modified HSPCs into immunodeficient mice results in high levels of gene marking of the lymphoid and myeloid progeny in vivo. The prior safe clinical history of rapamycin in other applications supports the use of this compound to generate gene-modified autologous HSPCs for our HIV gene therapy clinical trials. PMID:25107584

  20. Cotransplantation of ex vivo expanded progenitors with nonexpanded cord blood cells improves platelet recovery.

    PubMed

    Émond, Hélène; Boyer, Lucie; Roy, Denis-Claude; Pineault, Nicolas

    2012-11-20

    Umbilical cord blood (UCB) transplantation is associated with prolonged periods of cytopenia. Ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs) is currently investigated as a mean to accelerate hematological recovery. Contrary to neutrophils, platelet recovery remains problematic. For this reason, we have developed a culture protocol promoting the expansion of megakaryocyte (Mk) progenitors. The objective of this work was to determine whether the expanded (E) UCB HSPCs could accelerate platelet recovery in vivo using a murine HSPC transplantation model. The thrombopoietic activity of UCB and mobilized peripheral blood CD34(+) cells expanded under mild hyperthermia (MH, ie, 39°C) with the optimized megakaryocyte progenitor cocktail (OMPC) diverged significantly from the nonexpanded (NE) cells of origin; E cells provided rapid platelet release, while NE cells strongly contributed to platelet production past 10 days of transplantation. Consequently, the complementary of both cell sources was investigated. Cotransplantation of NE with E UCB cells significantly improved the recovery of human platelets (hPLTs) in vivo due to their complementary and synergistic thrombopoietic activities. Moreover, short-term human bone marrow (BM) reconstitution was also improved. Finally, we show that early hPLT release is dependent on Mk-primed cells and that E cells do not act as accessory cells, but have a more active role. In conclusion, hPLT recovery and short-term BM engraftment can be efficiently improved by the cotransplantation of Mk-primed UCB cells with NE HSPCs in a murine transplantation model.

  1. Inductive potential of recombinant human granulocyte colony-stimulating factor to mature neutrophils from x-irradiated human peripheral blood hematopoietic progenitor cells.

    PubMed

    Katsumori, Takeo; Yoshino, Hironori; Hayashi, Masako; Takahashi, Kenji; Kashiwakura, Ikuo

    2009-11-01

    Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been used for treatment of neutropenia. Filgrastim, Nartograstim, and Lenograstim are clinically available in Japan. However, the differences in potential benefit for radiation-induced disorder between these types of rhG-CSFs remain unknown. Therefore, the effects of three different types of rhG-CSFs on granulocyte progenitor cells and expansion of neutrophils from nonirradiated or 2 Gy X-irradiated human CD34+ hematopoietic progenitor cells were examined. For analysis of granulocyte colony-forming units (CFU-G) and a surviving fraction of CFU-G, nonirradiated or X-irradiated CD34+ cells were cultured in methylcellulose containing rhG-CSF. These cells were cultured in serum-free medium supplemented with rhG-CSF, and the expansion and characteristics of neutrophils were analyzed. All three types of rhG-CSFs increased the number of CFU-G in a dose-dependent manner; however, Lenograstim is superior to others because of CFU-G-derived colony formation at relatively low doses. The surviving fraction of CFU-G was independent of the types of rhG-CSFs. Expansion of neutrophils by rhG-CSF was largely attenuated by X-irradiation, though no significant difference in neutrophil number was observed between the three types of rhG-CSFs under both nonirradiation and X-irradiation conditions. In terms of functional characteristics of neutrophils, Lenograstim-induced neutrophils produced high levels of reactive oxygen species compared to Filgrastim, when rhG-CSF was applied to nonirradiated CD34(+) cells. In conclusion, different types of rhG-CSFs lead to different effects when rhG-CSF is applied to nonirradiated CD34+ cells, though Filgrastim, Nartograstim, and Lenograstim show equal effects on X-irradiated CD34+ cells.

  2. Short Stat5-Interacting Peptide Derived from Phospholipase C-β3 Inhibits Hematopoietic Cell Proliferation and Myeloid Differentiation

    PubMed Central

    Yasudo, Hiroki; Ando, Tomoaki; Xiao, Wenbin; Kawakami, Yuko; Kawakami, Toshiaki

    2011-01-01

    Constitutive activation of the transcription factor Stat5 in hematopoietic stem/progenitor cells leads to various hematopoietic malignancies including myeloproliferative neoplasm (MPN). Our recent study found that phospholipase C (PLC)-β3 is a novel tumor suppressor involved in MPN, lymphoma and other tumors. Stat5 activity is negatively regulated by the SH2 domain-containing protein phosphatase SHP-1 in a PLC-β3-dependent manner. PLC-β3 can form the multimolecular SPS complex together with SHP-1 and Stat5. The close physical proximity of SHP-1 and Stat5 brought about by interacting with the C-terminal segment of PLC-β3 (PLC-β3-CT) accelerates SHP-1-mediated dephosphorylation of Stat5. Here we identify the minimal sequences within PLC-β3-CT required for its tumor suppressor function. Two of the three Stat5-binding noncontiguous regions, one of which also binds SHP-1, substantially inhibited in vitro proliferation of Ba/F3 cells. Surprisingly, an 11-residue Stat5-binding peptide (residues 988-998) suppressed Stat5 activity in Ba/F3 cells and in vivo proliferation and myeloid differentiation of hematopoietic stem/progenitor cells. Therefore, this study further defines PLC-β3-CT as the Stat5- and SHP-1-binding domain by identifying minimal functional sequences of PLC-β3 for its tumor suppressor function and implies their potential utility in the control of hematopoietic malignancies. PMID:21949826

  3. Amino acid–insensitive mTORC1 regulation enables nutritional stress resilience in hematopoietic stem cells

    PubMed Central

    Kalaitzidis, Demetrios; Efeyan, Alejo; Kfoury, Youmna; Nayyar, Naema; Sykes, David B.; Mercier, Francois E.; Papazian, Ani; Baryawno, Ninib; Victora, Gabriel D.; Sabatini, David M.; Scadden, David T.

    2017-01-01

    The mTOR pathway is a critical determinant of cell persistence and growth wherein mTOR complex 1 (mTORC1) mediates a balance between growth factor stimuli and nutrient availability. Amino acids or glucose facilitates mTORC1 activation by inducing RagA GTPase recruitment of mTORC1 to the lysosomal outer surface, enabling activation of mTOR by the Ras homolog Rheb. Thereby, RagA alters mTORC1-driven growth in times of nutrient abundance or scarcity. Here, we have evaluated differential nutrient-sensing dependence through RagA and mTORC1 in hematopoietic progenitors, which dynamically drive mature cell production, and hematopoietic stem cells (HSC), which provide a quiescent cellular reserve. In nutrient-abundant conditions, RagA-deficient HSC were functionally unimpaired and upregulated mTORC1 via nutrient-insensitive mechanisms. RagA was also dispensable for HSC function under nutritional stress conditions. Similarly, hyperactivation of RagA did not affect HSC function. In contrast, RagA deficiency markedly altered progenitor population function and mature cell output. Therefore, RagA is a molecular mechanism that distinguishes the functional attributes of reactive progenitors from a reserve stem cell pool. The indifference of HSC to nutrient sensing through RagA contributes to their molecular resilience to nutritional stress, a characteristic that is relevant to organismal viability in evolution and in modern HSC transplantation approaches. PMID:28319048

  4. Cell proliferation and differentiation in chemical leukemogenesis

    NASA Technical Reports Server (NTRS)

    Irons, R. D.; Stillman, W. S.; Clarkson, T. W. (Principal Investigator)

    1993-01-01

    In tissues such as bone marrow with normally high rates of cell division, proliferation is tightly coordinated with cell differentiation. Survival, proliferation and differentiation of early hematopoietic progenitor cells depend on the growth factors, interleukin 3 (IL-3) and/or granulocyte-macrophage colony stimulating factor (GM-CSF) and their synergism with other cytokines. We provide evidence that a characteristic shared by a diverse group of compounds with demonstrated leukemogenic potential is the ability to act synergistically with GM-CSF. This results in an increase in recruitment of a resting population of hematopoietic progenitor cells normally unresponsive to the cytokine and a twofold increase in the size of the proliferating cell population normally regarded to be at risk of transformation in leukemogenesis. These findings support the possibility that transient alterations in hematopoietic progenitor cell differentiation may be an important factor in the early stages of development of leukemia secondary to chemical or drug exposure.

  5. Analyses of cell surface molecules on hepatic stem/progenitor cells in mouse fetal liver.

    PubMed

    Kakinuma, Sei; Ohta, Haruhiko; Kamiya, Akihide; Yamazaki, Yuji; Oikawa, Tsunekazu; Okada, Ken; Nakauchi, Hiromitsu

    2009-07-01

    Hepatic stem/progenitor cells possess active proliferative ability and the capacity for differentiation into hepatic and cholangiocytic lineages. Our group and others have shown that a prospectively defined population in mid-gestational fetal liver contains hepatic stem/progenitor cells. However, the phenotypes of such cells are incompletely elucidated. We analyzed the profile of cell-surface molecules on primary hepatic stem/progenitor cells. Expression of cell surface molecules on primary hepatic stem/progenitor cells in mouse mid-gestational fetal liver was analyzed using flow cytometric multicolor analyses and colony-formation assays. The potential of the cells for liver repopulation was examined by transplantation assay. We found that CD13 (aminopeptidase N) was detected on the cells of the previously reported (Dlk/Pref-1(+)) hepatic stem/progenitor fraction. Colony-formation assays revealed that the CD13(+) fraction, compared with the Dlk(+) fraction, of non-hematopoietic cells in fetal liver was enriched in hepatic stem/progenitor cells. Transplantation assay showed the former fraction exhibited repopulating potential in regenerating liver. Moreover, flow cytometric analysis for over 90 antigens demonstrated enrichment of hepatic stem/progenitor cells using several positive selection markers, including (hitherto unknown) CD13, CD73, CD106, and CD133. Our data indicated that CD13 is a positive selection marker for hepatic stem/progenitor cells in mid-gestational fetal liver.

  6. Early Blue Excess from the Type Ia Supernova 2017cbv and Implications for Its Progenitor

    NASA Astrophysics Data System (ADS)

    Hosseinzadeh, Griffin; Sand, David J.; Valenti, Stefano; Brown, Peter; Howell, D. Andrew; McCully, Curtis; Kasen, Daniel; Arcavi, Iair; Azalee Bostroem, K.; Tartaglia, Leonardo; Hsiao, Eric Y.; Davis, Scott; Shahbandeh, Melissa; Stritzinger, Maximilian D.

    2017-08-01

    We present very early, high-cadence photometric observations of the nearby Type Ia SN 2017cbv. The light curve is unique in that it has a blue bump during the first five days of observations in the U, B, and g bands, which is clearly resolved given our photometric cadence of 5.7 hr during that time span. We model the light curve as the combination of early shocking of the supernova ejecta against a nondegenerate companion star plus a standard SN Ia component. Our best-fit model suggests the presence of a subgiant star 56 R ⊙ from the exploding white dwarf, although this number is highly model-dependent. While this model matches the optical light curve well, it overpredicts the observed flux in the ultraviolet bands. This may indicate that the shock is not a blackbody, perhaps because of line blanketing in the UV. Alternatively, it could point to another physical explanation for the optical blue bump, such as interaction with circumstellar material or an unusual nickel distribution. Early optical spectra of SN 2017cbv show strong carbon (C II λ6580) absorption up through day -13 with respect to maximum light, suggesting that the progenitor system contains a significant amount of unburned material. These early results on SN 2017cbv illustrate the power of early discovery and intense follow-up of nearby supernovae to resolve standing questions about the progenitor systems and explosion mechanisms of SNe Ia.

  7. Early Blue Excess from the Type Ia Supernova 2017cbv and Implications for Its Progenitor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hosseinzadeh, Griffin; Howell, D. Andrew; McCully, Curtis

    We present very early, high-cadence photometric observations of the nearby Type Ia SN 2017cbv. The light curve is unique in that it has a blue bump during the first five days of observations in the U , B , and g bands, which is clearly resolved given our photometric cadence of 5.7 hr during that time span. We model the light curve as the combination of early shocking of the supernova ejecta against a nondegenerate companion star plus a standard SN Ia component. Our best-fit model suggests the presence of a subgiant star 56 R {sub ☉} from the explodingmore » white dwarf, although this number is highly model-dependent. While this model matches the optical light curve well, it overpredicts the observed flux in the ultraviolet bands. This may indicate that the shock is not a blackbody, perhaps because of line blanketing in the UV. Alternatively, it could point to another physical explanation for the optical blue bump, such as interaction with circumstellar material or an unusual nickel distribution. Early optical spectra of SN 2017cbv show strong carbon (C ii λ 6580) absorption up through day −13 with respect to maximum light, suggesting that the progenitor system contains a significant amount of unburned material. These early results on SN 2017cbv illustrate the power of early discovery and intense follow-up of nearby supernovae to resolve standing questions about the progenitor systems and explosion mechanisms of SNe Ia.« less

  8. Accelerated Hematopoietic Toxicity by High Energy 56Fe Radiation

    PubMed Central

    Datta, Kamal; Suman, Shubhankar; Trani, Daniela; Doiron, Kathryn; Rotolo, Jimmy A.; Kallakury, Bhaskar V. S.; Kolesnick, Richard; Cole, Michael F.; Fornace, Albert J.

    2013-01-01

    Purpose There is little information on the relative toxicity of highly charged (Z) high-energy (HZE) radiation in animal models compared to γ or x-rays, and the general assumption based on in vitro studies has been that acute toxicity is substantially greater. Methods C57BL/6J mice were irradiated with 56Fe ions (1 GeV/nucleon), and acute (within 30 d) toxicity compared to that of γ rays or protons (1 GeV). To assess relative hematopoietic and gastrointestinal toxicity, the effects of 56Fe ions were compared to γ rays using complete blood count (CBC), bone marrow granulocyte-macrophage colony forming unit (GM-CFU), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis in bone marrow, and intestinal crypt survival. Results Although onset was more rapid, 56Fe ions were only slightly more toxic than γ rays or protons with lethal dose (LD)50/30 (a radiation dose at which 50% lethality occurs at 30-day) values of 5.8, 7.25, and 6.8 Gy respectively with relative biologic effectiveness for 56Fe ions of 1.25 and 1.06 for protons. Conclusions 56Fe radiation caused accelerated and more severe hematopoietic toxicity. Early mortality correlated with more profound leukopenia and subsequent sepsis. Results indicate that there is selective enhanced toxicity to bone marrow progenitor cells, which are typically resistant to γ rays, and bone marrow stem cells, because intestinal crypt cells did not show increased HZE toxicity. PMID:22077279

  9. Preservation of differentiation and clonogenic potential of human hematopoietic stem and progenitor cells during lyophilization and ambient storage.

    PubMed

    Buchanan, Sandhya S; Pyatt, David W; Carpenter, John F

    2010-09-01

    Progenitor cell therapies show great promise, but their potential for clinical applications requires improved storage and transportation. Desiccated cells stored at ambient temperature would provide economic and practical advantages over approaches employing cell freezing and subzero temperature storage. The objectives of this study were to assess a method for loading the stabilizing sugar, trehalose, into hematopoietic stem and progenitor cells (HPC) and to evaluate the effects of subsequent freeze-drying and storage at ambient temperature on differentiation and clonogenic potential. HPC were isolated from human umbilical cord blood and loaded with trehalose using an endogenous cell surface receptor, termed P2Z. Solution containing trehalose-loaded HPC was placed into vials, which were transferred to a tray freeze-dryer and removed during each step of the freeze-drying process to assess differentiation and clonogenic potential. Control groups for these experiments were freshly isolated HPC. Control cells formed 1450+/-230 CFU-GM, 430+/-140 BFU-E, and 50+/-40 CFU-GEMM per 50 microL. Compared to the values for the control cells, there was no statistical difference observed for cells removed at the end of the freezing step or at the end of primary drying. There was a gradual decrease in the number of CFU-GM and BFU-E for cells removed at different temperatures during secondary drying; however, there were no significant differences in the number of CFU-GEMM. To determine storage stability of lyophilized HPC, cells were stored for 4 weeks at 25 degrees C in the dark. Cells reconstituted immediately after lyophilization produced 580+/-90 CFU-GM ( approximately 40%, relative to unprocessed controls p<0.0001), 170+/-70 BFU-E (approximately 40%, p<0.0001), and 41+/-22 CFU-GEMM (approximately 82%, p = 0.4171), and cells reconstituted after 28 days at room temperature produced 513+/-170 CFU-GM (approximately 35%, relative to unprocessed controls, p<0.0001), 112+/-68 BFU

  10. Serum microRNAs are early indicators of survival after radiation-induced hematopoietic injury

    PubMed Central

    Acharya, Sanket S.; Fendler, Wojciech; Watson, Jacqueline; Hamilton, Abigail; Pan, Yunfeng; Gaudiano, Emily; Moskwa, Patryk; Bhanja, Payel; Saha, Subhrajit; Guha, Chandan; Parmar, Kalindi; Chowdhury, Dipanjan

    2015-01-01

    Accidental radiation exposure is a threat to human health that necessitates effective clinical planning and diagnosis. Minimally invasive biomarkers that can predict long-term radiation injury are urgently needed for optimal management after a radiation accident. We have identified serum microRNA (miRNA) signatures that indicate long-term impact of total body irradiation (TBI) in mice when measured within 24 hours of exposure. Impact of TBI on the hematopoietic system was systematically assessed to determine a correlation of residual hematopoietic stem cells (HSCs) with increasing doses of radiation. Serum miRNA signatures distinguished untreated mice from animals exposed to radiation and correlated with the impact of radiation on HSCs. Mice exposed to sublethal (6.5 Gy) and lethal (8 Gy) doses of radiation were indistinguishable for 3 to 4 weeks after exposure. A serum miRNA signature detectable 24 hours after radiation exposure consistently segregated these two cohorts. Furthermore, using either a radioprotective agent before, or radiation mitigation after, lethal radiation, we determined that the serum miRNA signature correlated with the impact of radiation on animal health rather than the radiation dose. Last, using humanized mice that had been engrafted with human CD34+ HSCs, we determined that the serum miRNA signature indicated radiation-induced injury to the human bone marrow cells. Our data suggest that serum miRNAs can serve as functional dosimeters of radiation, representing a potential breakthrough in early assessment of radiation-induced hematopoietic damage and timely use of medical countermeasures to mitigate the long-term impact of radiation. PMID:25972001

  11. Transient detection of beta-galactosidase activity in hematopoietic cells, following reinjection of retrovirally marked autologous blood progenitors in patients with breast or ovarian cancer receiving high-dose chemotherapy.

    PubMed

    Bagnis, Claude; Chabannon, Christian; Gravis, Gwenaelle; Imbert, Anne-Marie; Maroc, Christine; Bardin, Florence; Ladaique, Patrick; Viret, Frédéric; Genre, Dominique; Faucher, Catherine; Stoppa, Anne-Marie; Vey, Norbert; Blaise, Didier; Maraninchi, Dominique; Viens, Patrice; Mannoni, Patrice

    2002-02-01

    The aim of this report is to demonstrate the feasibility and safety of genetically modifying autologous human blood CD34(+) cells in vitro, with a retroviral vector that encodes a marker gene. The fate of genetically modified cells and their progeny was followed in vivo, after reinfusion in patients treated with high-dose chemotherapy for poor-prognosis breast or ovarian carcinomas. Six patients received genetically modified autologous peripheral blood progenitors, together with unmanipulated aphereses, following high-dose chemotherapy. CD34(+) cells were immunoselected from aphereses, and retrovirally transduced by coculture with the retroviral vector producing cell line, to express a nuclear localized version of E. coli beta-galactosidase, encoded by a defective Moloney-murine leukemia virus-derived retroviral vector. Cells were reinfused to the patients after myeloablation, without prior ex vivo selection. Five out of six patients showed the transient presence of low numbers of beta-galactosidase(+) cells, as detected with an immunocytochemical assay, in the peripheral blood, during the first month following infusion. One patient had beta-galactosidase(+) clonogenic progenitors in her marrow at two months after transplantation, including HPP-CFC; intriguingly, this patient had the lowest percentage of X-gal(+) cells in her graft. Patients experienced side effects that are often observed after high-dose chemotherapy. Feasibility and safety of genetic modification of human hematopoietic stem and progenitor cells are demonstrated by this study. Ex vivo or in vivo selection is not mandatory, even in clinical situations where transduced cells have no survival advantage over wild-type cells; however, significant improvements in gene transfer technology are needed to achieve potentially useful levels of expression in such clinical situations.

  12. Visualizing Intrapopulation Hematopoietic Cell Heterogeneity with Self-Organizing Maps of SIMS Data.

    PubMed

    Mirshafiee, Vahid; Harley, Brendan A C; Kraft, Mary L

    2018-05-07

    Characterization of the heterogeneity within stem cell populations, which affects their differentiation potential, is necessary for the design of artificial cultures for stem cell expansion. In this study, we assessed whether self-organizing maps (SOMs) of single-cell time-of-flight secondary ion mass spectrometry (TOF-SIMS) data provide insight into the spectral, and thus the related functional heterogeneity between and within three hematopoietic cell populations. SOMs were created of TOF-SIMS data from individual hematopoietic stem and progenitor cells (HSPCs), lineage-committed common lymphoid progenitors (CLPs), and fully differentiated B cells that had been isolated from murine bone marrow via conventional flow cytometry. The positions of these cells on the SOMs and the spectral variation between adjacent map units, shown on the corresponding unified distance matrix (U-matrix), indicated the CLPs exhibited the highest intrapopulation spectral variation, regardless of the age of the donor mice. SOMs of HSPCs, CLPs, and B cells isolated from young and old mice using the same surface antigen profiles revealed the HSPCs exhibited the most age-related spectral variation, whereas B cells exhibited the least. These results demonstrate that SOMs of single-cell spectra enable characterizing the heterogeneity between and within cell populations that lie along distinct differentiation pathways.

  13. C/EBPδ deficiency sensitizes mice to ionizing radiation-induced hematopoietic and intestinal injury.

    PubMed

    Pawar, Snehalata A; Shao, Lijian; Chang, Jianhui; Wang, Wenze; Pathak, Rupak; Zhu, Xiaoyan; Wang, Junru; Hendrickson, Howard; Boerma, Marjan; Sterneck, Esta; Zhou, Daohong; Hauer-Jensen, Martin

    2014-01-01

    Knowledge of the mechanisms involved in the radiation response is critical for developing interventions to mitigate radiation-induced injury to normal tissues. Exposure to radiation leads to increased oxidative stress, DNA-damage, genomic instability and inflammation. The transcription factor CCAAT/enhancer binding protein delta (Cebpd; C/EBPδ is implicated in regulation of these same processes, but its role in radiation response is not known. We investigated the role of C/EBPδ in radiation-induced hematopoietic and intestinal injury using a Cebpd knockout mouse model. Cebpd-/- mice showed increased lethality at 7.4 and 8.5 Gy total-body irradiation (TBI), compared to Cebpd+/+ mice. Two weeks after a 6 Gy dose of TBI, Cebpd-/- mice showed decreased recovery of white blood cells, neutrophils, platelets, myeloid cells and bone marrow mononuclear cells, decreased colony-forming ability of bone marrow progenitor cells, and increased apoptosis of hematopoietic progenitor and stem cells compared to Cebpd+/+ controls. Cebpd-/- mice exhibited a significant dose-dependent decrease in intestinal crypt survival and in plasma citrulline levels compared to Cebpd+/+ mice after exposure to radiation. This was accompanied by significantly decreased expression of γ-H2AX in Cebpd-/- intestinal crypts and villi at 1 h post-TBI, increased mitotic index at 24 h post-TBI, and increase in apoptosis in intestinal crypts and stromal cells of Cebpd-/- compared to Cebpd+/+ mice at 4 h post-irradiation. This study uncovers a novel biological function for C/EBPδ in promoting the response to radiation-induced DNA-damage and in protecting hematopoietic and intestinal tissues from radiation-induced injury.

  14. Rutin-Enriched Extract from Coriandrum sativum L. Ameliorates Ionizing Radiation-Induced Hematopoietic Injury

    PubMed Central

    Han, Xiaodan; Xue, Xiaolei; Zhao, Yu; Li, Yuan; Liu, Weili; Zhang, Junling; Fan, Saijun

    2017-01-01

    Hematopoietic injury is a major cause of mortality in radiation accidents and a primary side effect in patients undergoing radiotherapy. Ionizing radiation (IR)-induced myelosuppression is largely attributed to the injury of hematopoietic stem and progenitor cells (HSPCs). Coriander is a culinary herb with multiple pharmacological effects and has been widely used in traditional medicine. In this study, flavonoids were identified as the main component of coriander extract with rutin being the leading compound (rutin-enriched coriander extract; RE-CE). We evaluated the radioprotective effect of RE-CE against IR-induced HSPCs injury. Results showed that RE-CE treatment markedly improved survival, ameliorated organ injuries and myelosuppression, elevated HSPCs frequency, and promoted differentiation and proliferation of HSPCs in irradiated mice. The protective role of RE-CE in hematopoietic injury is probably attributed to its anti-apoptotic and anti-DNA damage effect in irradiated HSPCs. Moreover, these changes were associated with reduced reactive oxygen species (ROS) and enhanced antioxidant enzymatic activities in irradiated HSPCs. Collectively, these findings demonstrate that RE-CE is able to ameliorate IR-induced hematopoietic injury partly by reducing IR-induced oxidative stress. PMID:28468251

  15. Age-related mutations associated with clonal hematopoietic expansion and malignancies.

    PubMed

    Xie, Mingchao; Lu, Charles; Wang, Jiayin; McLellan, Michael D; Johnson, Kimberly J; Wendl, Michael C; McMichael, Joshua F; Schmidt, Heather K; Yellapantula, Venkata; Miller, Christopher A; Ozenberger, Bradley A; Welch, John S; Link, Daniel C; Walter, Matthew J; Mardis, Elaine R; Dipersio, John F; Chen, Feng; Wilson, Richard K; Ley, Timothy J; Ding, Li

    2014-12-01

    Several genetic alterations characteristic of leukemia and lymphoma have been detected in the blood of individuals without apparent hematological malignancies. The Cancer Genome Atlas (TCGA) provides a unique resource for comprehensive discovery of mutations and genes in blood that may contribute to the clonal expansion of hematopoietic stem/progenitor cells. Here, we analyzed blood-derived sequence data from 2,728 individuals from TCGA and discovered 77 blood-specific mutations in cancer-associated genes, the majority being associated with advanced age. Remarkably, 83% of these mutations were from 19 leukemia and/or lymphoma-associated genes, and nine were recurrently mutated (DNMT3A, TET2, JAK2, ASXL1, TP53, GNAS, PPM1D, BCORL1 and SF3B1). We identified 14 additional mutations in a very small fraction of blood cells, possibly representing the earliest stages of clonal expansion in hematopoietic stem cells. Comparison of these findings to mutations in hematological malignancies identified several recurrently mutated genes that may be disease initiators. Our analyses show that the blood cells of more than 2% of individuals (5-6% of people older than 70 years) contain mutations that may represent premalignant events that cause clonal hematopoietic expansion.

  16. Cannabinoid receptor signaling in progenitor/stem cell proliferation and differentiation.

    PubMed

    Galve-Roperh, Ismael; Chiurchiù, Valerio; Díaz-Alonso, Javier; Bari, Monica; Guzmán, Manuel; Maccarrone, Mauro

    2013-10-01

    Cannabinoids, the active components of cannabis (Cannabis sativa) extracts, have attracted the attention of human civilizations for centuries, much earlier than the discovery and characterization of their substrate of action, the endocannabinoid system (ECS). The latter is an ensemble of endogenous lipids, their receptors [in particular type-1 (CB1) and type-2 (CB2) cannabinoid receptors] and metabolic enzymes. Cannabinoid signaling regulates cell proliferation, differentiation and survival, with different outcomes depending on the molecular targets and cellular context involved. Cannabinoid receptors are expressed and functional from the very early developmental stages, when they regulate embryonic and trophoblast stem cell survival and differentiation, and thus may affect the formation of manifold adult specialized tissues derived from the three different germ layers (ectoderm, mesoderm and endoderm). In the ectoderm-derived nervous system, both CB1 and CB2 receptors are present in neural progenitor/stem cells and control their self-renewal, proliferation and differentiation. CB1 and CB2 show opposite patterns of expression, the former increasing and the latter decreasing along neuronal differentiation. Recently, endocannabinoid (eCB) signaling has also been shown to regulate proliferation and differentiation of mesoderm-derived hematopoietic and mesenchymal stem cells, with a key role in determining the formation of several cell types in peripheral tissues, including blood cells, adipocytes, osteoblasts/osteoclasts and epithelial cells. Here, we will review these new findings, which unveil the involvement of eCB signaling in the regulation of progenitor/stem cell fate in the nervous system and in the periphery. The developmental regulation of cannabinoid receptor expression and cellular/subcellular localization, together with their role in progenitor/stem cell biology, may have important implications in human health and disease. Copyright © 2013 Elsevier Ltd

  17. The triterpenoid RTA 408 is a robust mitigator of hematopoietic acute radiation syndrome in mice.

    PubMed

    Goldman, Devorah C; Alexeev, Vitali; Lash, Elizabeth; Guha, Chandan; Rodeck, Ulrich; Fleming, William H

    2015-03-01

    Bone marrow suppression due to exposure to ionizing radiation is a significant clinical problem associated with radiation therapy as well as with nonmedical radiation exposure. Currently, there are no small molecule agents available that can enhance hematopoietic regeneration after radiation exposure. Here, we report on the effective mitigation of acute hematopoietic radiation syndrome in mice by the synthetic triterpenoid, RTA 408. The administration of a brief course of RTA 408 treatment, beginning 24 h after lethal doses of radiation to bone marrow, significantly increased overall survival. Importantly, treatment with RTA 408 led to the full recovery of steady state hematopoiesis with normalization of the frequency of hematopoietic stem and progenitor cells. Moreover, hematopoietic stem cells from RTA 408-mitigated mice showed lineage-balanced, long-term, multilineage potential in serial transplantation assays, indicative of their normal self-renewal activity. The potency of RTA 408 in mitigating radiation-induced bone marrow suppression makes it an attractive candidate for potential clinical use in treating both therapy-related and unanticipated radiation exposure.

  18. Cryopreservation of hematopoietic stem and progenitor cells amplified ex vivo from cord blood CD34+ cells.

    PubMed

    Duchez, Pascale; Chevaleyre, Jean; Brunet de la Grange, Philippe; Vlaski, Marija; Boiron, Jean-Michel; Wouters, Guy; Ivanovic, Zoran

    2013-09-01

    Our ex vivo expansion procedure starting from cord blood (CB) CD34+ cells enabled expansion of committed progenitors (CPs) without a negative impact on hematopoietic stem cells (HSCs) exhibiting both short- and long-term repopulating capacity. Upgraded to clinical scale (Macopharma HP01 in the presence of stem cell factor, FLT3-L [100 ng/mL each], granulocyte-colony-stimulating factor [10 ng/mL], and thrombopoietin [20 ng/mL]), it is being used for an ongoing clinical trial (adult allogeneic context) yielding promising preliminary results. Transplantation of ex vivo expanded CB cells is becoming a reality, while the issue of expanded cells' cryopreservation emerges as an option that allows the conservation of the product for transportation and future use. Here, we investigated whether it is possible to maintain the functional HSC and CP properties after freezing and thawing of expanded cells. We compared cryopreservation efficiency of the ex vivo expanded CB cells using the standard protocol (freezing solution human serum albumin (HSA)-dimethyl sulfoxide [DMSO]) with the newly designed protocol based on an enriched freezing solution (HP01-DMSO) with respect to the viability index, number of CD34+ and total cells, and recovery of CPs (colony-forming units) and HSCs (NOG/Scid/gamma-null mice engraftment). Cryopreservation and thawing of expanded CB cells using the "standard" procedure (HSA-DMSO) reduced recovery of the CPs (40%) and HSCs (drastically decreasing engraftment capacity). HP01-based protocol resulted in improvement of preservation of both CPs (>60%) and HSCs (nonaltered engraftment capacities). Functional maintenance of the expanded graft by cryopreservation is feasible in conditions compatible with human cell therapy requirements. © 2012 American Association of Blood Banks.

  19. A novel hematopoietic progenitor cell mobilization regimen, utilizing bortezomib and filgrastim, for patients undergoing autologous transplant.

    PubMed

    Abhyankar, Sunil; Lubanski, Philip; DeJarnette, Shaun; Merkel, Dean; Bunch, Jennifer; Daniels, Kelly; Aljitawi, Omar; Lin, Tara; Ganguly, Sid; McGuirk, Joseph

    2016-12-01

    Adequate hematopoietic progenitor cell (HPC) collection is critical for patients undergoing autologous HPC transplant (AHPCT). Historically, 15 - 30% of patients failed HPC mobilization with granulocyte-colony stimulating factor (G-CSF) alone. Bortezomib, a proteasome inhibitor, has been shown to down regulate very late antigen-4 (VLA-4), an adhesion molecule expressed on HPCs. In this pilot study, bortezomib was administered on days -11 and -8 at a dose of 1.3 mg/m 2 intravenously (IV) or subcutaneously (SQ), followed by G-CSF 10 mcg/kg SQ, on days -4 to -1 prior to HPC collection (Day 1). Nineteen patients, with multiple myeloma (n = 12) or non-Hodgkin lymphoma (n = 7) undergoing AHPCT for the first time, were enrolled. Patients were excluded if they had worse than grade II neuropathy or platelet count less than 100 x 10 9 /L. Bortezomib was well tolerated and all patients had adequate HPC collections with no mobilization failures. One patient (6%) had a CD34 + cell count of 3.9 cells/µL on Day 1 and received plerixafor per institutional algorithm. Eleven patients completed HPC collection in 1 day and eight in 2 days. All patients underwent AHPCT and had timely neutrophil and platelet engraftment. Comparison with a historical control group of 70 MM and lymphoma patients, who were mobilized with G-CSF, showed significantly higher CD 34+ cells/kg collected in the bortezomib mobilization study group. Bortezomib plus G-CSF is an effective HPC mobilizing regimen worth investigating further in subsequent studies. J. Clin. Apheresis 31:559-563, 2016. © 2015 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  20. PDL Progenitor-Mediated PDL Recovery Contributes to Orthodontic Relapse.

    PubMed

    Feng, L; Yang, R; Liu, D; Wang, X; Song, Y; Cao, H; He, D; Gan, Y; Kou, X; Zhou, Y

    2016-08-01

    Periodontal ligament (PDL) is subjected to mechanical force during physiologic activities. PDL stem /: progenitor cells are the main mesenchymal stem cells in PDL. However, how PDL progenitors participate in PDL homeostasis upon and after mechanical force is largely unknown. In this study, force-triggered orthodontic tooth movement and the following relapse were used as models to demonstrate the response of PDL progenitors and their role in PDL remodeling upon and after mechanical force. Upon orthodontic force, PDL collagen on the compression side significantly degraded, showing a broken and disorganized pattern. After force withdrawal, the degraded PDL collagen recovered during the early stage of relapse. Correspondingly, increased CD90(+) PDL progenitors with suppressed expression of type I collagen (Col-I) were observed upon orthodontic force, whereas these cells accumulated at the degradation regions and regained Col-I expression after force withdrawal during early relapse. Our results further showed that compressive force altered cell morphology and repressed collagen expression in cultured PDL progenitors, which both recovered after force withdrawal. Force withdrawal-induced recovery of collagen expression in cultured PDL progenitors could be regulated by transforming growth factor-β (TGF-β), a key molecule for tissue homeostasis and extracellular matrix remodeling. More interesting, inhibiting the regained Col-I expression in CD90(+) PDL progenitors by blocking TGF-β interrupted PDL collagen recovery and partially inhibited the early relapse. These data suggest that PDL progenitors can respond to mechanical force and may process intrinsic stability to recover to original status after force withdrawal. PDL progenitors with intrinsic stability are required for PDL recovery and consequently contribute to early orthodontic relapse, which can be regulated by TGF-β signaling. © International & American Associations for Dental Research 2016.

  1. Altered Hematopoiesis in Mice Lacking DNA Polymerase μ Is Due to Inefficient Double-Strand Break Repair

    PubMed Central

    Lucas, Daniel; Escudero, Beatriz; Ligos, José Manuel; Segovia, Jose Carlos; Estrada, Juan Camilo; Terrados, Gloria; Blanco, Luis; Samper, Enrique; Bernad, Antonio

    2009-01-01

    Polymerase mu (Polμ) is an error-prone, DNA-directed DNA polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polμ deficiency results in impaired Vκ-Jκ recombination and altered somatic hypermutation and centroblast development. In Polμ−/− mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body γ-irradiation revealed that Polμ also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polμ function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues. PMID:19229323

  2. MiR-17/20/93/106 promote hematopoietic cell expansion by targeting sequestosome 1–regulated pathways in mice

    PubMed Central

    Meenhuis, Annemarie; van Veelen, Peter A.; de Looper, Hans; van Boxtel, Nicole; van den Berge, Iris J.; Sun, Su M.; Taskesen, Erdogan; Stern, Patrick; de Ru, Arnoud H.; van Adrichem, Arjan J.; Demmers, Jeroen; Jongen-Lavrencic, Mojca; Löwenberg, Bob; Touw, Ivo P.; Sharp, Phillip A.

    2011-01-01

    MicroRNAs (miRNAs) are pivotal for regulation of hematopoiesis but their critical targets remain largely unknown. Here, we show that ectopic expression of miR-17, -20,-93 and -106, all AAAGUGC seed-containing miRNAs, increases proliferation, colony outgrowth and replating capacity of myeloid progenitors and results in enhanced P-ERK levels. We found that these miRNAs are endogenously and abundantly expressed in myeloid progenitors and down-regulated in mature neutrophils. Quantitative proteomics identified sequestosome 1 (SQSTM1), an ubiquitin-binding protein and regulator of autophagy-mediated protein degradation, as a major target for these miRNAs in myeloid progenitors. In addition, we found increased expression of Sqstm1 transcripts during CSF3-induced neutrophil differentiation of 32D-CSF3R cells and an inverse correlation of SQSTM1 protein levels and miR-106 expression in AML samples. ShRNA-mediated silencing of Sqstm1 phenocopied the effects of ectopic miR-17/20/93/106 expression in hematopoietic progenitors in vitro and in mice. Further, SQSTM1 binds to the ligand-activated colony-stimulating factor 3 receptor (CSF3R) mainly in the late endosomal compartment, but not in LC3 positive autophagosomes. SQSTM1 regulates CSF3R stability and ligand-induced mitogen-activated protein kinase signaling. We demonstrate that AAAGUGC seed-containing miRNAs promote cell expansion, replating capacity and signaling in hematopoietic cells by interference with SQSTM1-regulated pathways. PMID:21628417

  3. Wharton's Jelly Mesenchymal Stromal Cells as a Feeder Layer for the Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells: a Review.

    PubMed

    Lo Iacono, Melania; Anzalone, Rita; La Rocca, Giampiero; Baiamonte, Elena; Maggio, Aurelio; Acuto, Santina

    2017-02-01

    In recent years, umbilical cord blood (UCB) has been widely used as an alternative source to bone marrow (BM) for transplantation of hematopoietic stem and progenitor cells (HSPCs) in a variety of hematological and non-hematological disorders. Nevertheless, the insufficient number of UCB-HSPCs for graft represents a major challenge. HSPCs ex vivo expansion prior to transplantation is a valid strategy to overcome this limit. Several attempts to optimize the expansion conditions have been reported, including the use of mesenchymal stromal cells (MSCs) as feeder layer. Wharton's Jelly (WJ), the main component of umbilical cord (UC) matrix, is especially rich in MSCs, which are considered ideal candidates for feeder layer in co-culture systems. In fact, they can be easily harvested and grow robustly in culture, producing a confluent monolayer in a short time. Similarly to bone marrow-mesenchymal stromal cells (BM-MSCs), WJ-derived MSCs (WJ-MSCs) have been used to support hematopoiesis in vitro and in vivo. Here, we review the rationale for using MSCs, particularly WJ-MSCs, as a feeder layer for UCB-HSPCs ex vivo expansion. In addition, we report the main findings attesting the use of these MSCs as a support in hematopoiesis.

  4. Mesenchymal progenitor cells for the osteogenic lineage.

    PubMed

    Ono, Noriaki; Kronenberg, Henry M

    2015-09-01

    Mesenchymal progenitors of the osteogenic lineage provide the flexibility for bone to grow, maintain its function and homeostasis. Traditionally, colony-forming-unit fibroblasts (CFU-Fs) have been regarded as surrogates for mesenchymal progenitors; however, this definition cannot address the function of these progenitors in their native setting. Transgenic murine models including lineage-tracing technologies based on the cre-lox system have proven to be useful in delineating mesenchymal progenitors in their native environment. Although heterogeneity of cell populations of interest marked by a promoter-based approach complicates overall interpretation, an emerging complexity of mesenchymal progenitors has been revealed. Current literatures suggest two distinct types of bone progenitor cells; growth-associated mesenchymal progenitors contribute to explosive growth of bone in early life, whereas bone marrow mesenchymal progenitors contribute to the much slower remodeling process and response to injury that occurs mainly in adulthood. More detailed relationships of these progenitors need to be studied through further experimentation.

  5. The biochemistry of hematopoietic stem cell development.

    PubMed

    Kaimakis, P; Crisan, M; Dzierzak, E

    2013-02-01

    The cornerstone of the adult hematopoietic system and clinical treatments for blood-related disease is the cohort of hematopoietic stem cells (HSC) that is harbored in the adult bone marrow microenvironment. Interestingly, this cohort of HSCs is generated only during a short window of developmental time. In mammalian embryos, hematopoietic progenitor and HSC generation occurs within several extra- and intraembryonic microenvironments, most notably from 'hemogenic' endothelial cells lining the major vasculature. HSCs are made through a remarkable transdifferentiation of endothelial cells to a hematopoietic fate that is long-lived and self-renewable. Recent studies are beginning to provide an understanding of the biochemical signaling pathways and transcription factors/complexes that promote their generation. The focus of this review is on the biochemistry behind the generation of these potent long-lived self-renewing stem cells of the blood system. Both the intrinsic (master transcription factors) and extrinsic regulators (morphogens and growth factors) that affect the generation, maintenance and expansion of HSCs in the embryo will be discussed. The generation of HSCs is a stepwise process involving many developmental signaling pathways, morphogens and cytokines. Pivotal hematopoietic transcription factors are required for their generation. Interestingly, whereas these factors are necessary for HSC generation, their expression in adult bone marrow HSCs is oftentimes not required. Thus, the biochemistry and molecular regulation of HSC development in the embryo are overlapping, but differ significantly from the regulation of HSCs in the adult. HSC numbers for clinical use are limiting, and despite much research into the molecular basis of HSC regulation in the adult bone marrow, no panel of growth factors, interleukins and/or morphogens has been found to sufficiently increase the number of these important stem cells. An understanding of the biochemistry of HSC

  6. The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function.

    PubMed

    Gu, Yue; Jones, Amanda E; Yang, Wei; Liu, Shanrun; Dai, Qian; Liu, Yudong; Swindle, C Scott; Zhou, Dewang; Zhang, Zhuo; Ryan, Thomas M; Townes, Tim M; Klug, Christopher A; Chen, Dongquan; Wang, Hengbin

    2016-01-05

    Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.

  7. Tapak liman (Elephantopus scaber L) extract-induced CD4+ and CD8+ differentiation from hematopoietic stem cells and progenitor cell proliferation in mice (Mus musculus L)

    NASA Astrophysics Data System (ADS)

    Djati, Muhammad Sasmito; Habibu, Hindun; Jatiatmaja, Nabilah A.; Rifa'i, Muhaimin

    2017-11-01

    Tapak Liman (Elephantopus scaber L) is a traditional medicinal plant containing several active compounds that potentially affecting hematopoietic stem cells, such as epifrieelinol, lupeol, stigmasterol, triacontane-1-ol, dotriacontane-1-ol, lupeol acetate, deoxyelephan-topin, isodeoxyelephantopin, polyphenol luteolin-7, as well as various flavonoids and glucosides. The aim of this study was to elucidate the effect of leaf extract of Tapak Liman on hematopoietic stem cells in mice BALB/c, by observation of the relative number of cells expressing CD4/CD8, CD4/CD62L, and TER119/B220 in the spleen, and TER119/B220, TER119/VLA-4 and TER119/CD34 in bone marrow, after being administered leaf extract for 2 weeks. This experiment used 12 female mice, which were divided into three treatment groups, P1= 0.5 g.g bw-1.day-1, P2= 1.0 g.g bw-1.day-1 and P3=2.0 g.g bw-1.day-1 Tapak Liman leaf extract as well as a control. The relative numbers of cells expressing surface molecules were analyzed by flowcytometry and quantitative data were tested using one-way ANOVA. The results showed that the leaf extract of Tapak Liman has no significant effect on erythrocyte proliferation; on the other hand, it had a significant effect on both proliferation and differentiation of B lymphocytes (B220+) in bone marrow (p=0.044) and increased the expression of CD4+, CD8+ molecule in B cells (p=0.026) and erythroid cells in spleen and bone marrow, based on the estimation of cells that expressed TER119+VLA-4+, identified as important in the development pathway of erythrocytes. An increased cell percentage of TER11+VLA-4+ occurred for treatment P2, 12% higher than the control. The increased expression of TER119+VLA-4+ was assumed to be due to the iron content in Tapak Liman, which functioned to stimulate the progenitor hematopoietic cells to proliferate and differentiate into a precursor of erythroid cells (TER119+VLA-4+). There was an increasing number of cells expressing the surface molecules TER119

  8. USP10 Is an Essential Deubiquitinase for Hematopoiesis and Inhibits Apoptosis of Long-Term Hematopoietic Stem Cells.

    PubMed

    Higuchi, Masaya; Kawamura, Hiroki; Matsuki, Hideaki; Hara, Toshifumi; Takahashi, Masahiko; Saito, Suguru; Saito, Kousuke; Jiang, Shuying; Naito, Makoto; Kiyonari, Hiroshi; Fujii, Masahiro

    2016-12-13

    Self-renewal, replication, and differentiation of hematopoietic stem cells (HSCs) are regulated by cytokines produced by niche cells in fetal liver and bone marrow. HSCs must overcome stresses induced by cytokine deprivation during normal development. In this study, we found that ubiquitin-specific peptidase 10 (USP10) is a crucial deubiquitinase for mouse hematopoiesis. All USP10 knockout (KO) mice died within 1 year because of bone marrow failure with pancytopenia. Bone marrow failure in these USP10-KO mice was associated with remarkable reductions of long-term HSCs (LT-HSCs) in bone marrow and fetal liver. Such USP10-KO fetal liver exhibited enhanced apoptosis of hematopoietic stem/progenitor cells (HSPCs) including LT-HSCs but not of lineage-committed progenitor cells. Transplantation of USP10-competent bone marrow cells into USP10-KO mice reconstituted multilineage hematopoiesis. These results suggest that USP10 is an essential deubiquitinase in hematopoiesis and functions by inhibiting apoptosis of HSPCs including LT-HSCs. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  9. Mesenchymal Inflammation Drives Genotoxic Stress in Hematopoietic Stem Cells and Predicts Disease Evolution in Human Pre-leukemia.

    PubMed

    Zambetti, Noemi A; Ping, Zhen; Chen, Si; Kenswil, Keane J G; Mylona, Maria A; Sanders, Mathijs A; Hoogenboezem, Remco M; Bindels, Eric M J; Adisty, Maria N; Van Strien, Paulina M H; van der Leije, Cindy S; Westers, Theresia M; Cremers, Eline M P; Milanese, Chiara; Mastroberardino, Pier G; van Leeuwen, Johannes P T M; van der Eerden, Bram C J; Touw, Ivo P; Kuijpers, Taco W; Kanaar, Roland; van de Loosdrecht, Arjan A; Vogl, Thomas; Raaijmakers, Marc H G P

    2016-11-03

    Mesenchymal niche cells may drive tissue failure and malignant transformation in the hematopoietic system, but the underlying molecular mechanisms and relevance to human disease remain poorly defined. Here, we show that perturbation of mesenchymal cells in a mouse model of the pre-leukemic disorder Shwachman-Diamond syndrome (SDS) induces mitochondrial dysfunction, oxidative stress, and activation of DNA damage responses in hematopoietic stem and progenitor cells. Massive parallel RNA sequencing of highly purified mesenchymal cells in the SDS mouse model and a range of human pre-leukemic syndromes identified p53-S100A8/9-TLR inflammatory signaling as a common driving mechanism of genotoxic stress. Transcriptional activation of this signaling axis in the mesenchymal niche predicted leukemic evolution and progression-free survival in myelodysplastic syndrome (MDS), the principal leukemia predisposition syndrome. Collectively, our findings identify mesenchymal niche-induced genotoxic stress in heterotypic stem and progenitor cells through inflammatory signaling as a targetable determinant of disease outcome in human pre-leukemia. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. An integrated global regulatory network of hematopoietic precursor cell self-renewal and differentiation.

    PubMed

    You, Yanan; Cuevas-Diaz Duran, Raquel; Jiang, Lihua; Dong, Xiaomin; Zong, Shan; Snyder, Michael; Wu, Jia Qian

    2018-06-12

    Systematic study of the regulatory mechanisms of Hematopoietic Stem Cell and Progenitor Cell (HSPC) self-renewal is fundamentally important for understanding hematopoiesis and for manipulating HSPCs for therapeutic purposes. Previously, we have characterized gene expression and identified important transcription factors (TFs) regulating the switch between self-renewal and differentiation in a multipotent Hematopoietic Progenitor Cell (HPC) line, EML (Erythroid, Myeloid, and Lymphoid) cells. Herein, we report binding maps for additional TFs (SOX4 and STAT3) by using chromatin immunoprecipitation (ChIP)-Sequencing, to address the underlying mechanisms regulating self-renewal properties of lineage-CD34+ subpopulation (Lin-CD34+ EML cells). Furthermore, we applied the Assay for Transposase Accessible Chromatin (ATAC)-Sequencing to globally identify the open chromatin regions associated with TF binding in the self-renewing Lin-CD34+ EML cells. Mass spectrometry (MS) was also used to quantify protein relative expression levels. Finally, by integrating the protein-protein interaction database, we built an expanded transcriptional regulatory and interaction network. We found that MAPK (Mitogen-activated protein kinase) pathway and TGF-β/SMAD signaling pathway components were highly enriched among the binding targets of these TFs in Lin-CD34+ EML cells. The present study integrates regulatory information at multiple levels to paint a more comprehensive picture of the HSPC self-renewal mechanisms.

  11. Reduced hematopoietic reserves in DNA interstrand crosslink repair-deficient Ercc1−/− mice

    PubMed Central

    Prasher, Joanna M; Lalai, Astrid S; Heijmans-Antonissen, Claudia; Ploemacher, Robert E; Hoeijmakers, Jan H J; Touw, Ivo P; Niedernhofer, Laura J

    2005-01-01

    The ERCC1-XPF heterodimer is a structure-specific endonuclease involved in both nucleotide excision repair and interstrand crosslink repair. Mice carrying a genetic defect in Ercc1 display symptoms suggestive of a progressive, segmental progeria, indicating that disruption of one or both of these DNA damage repair pathways accelerates aging. In the hematopoietic system, there are defined age-associated changes for which the cause is unknown. To determine if DNA repair is critical to prolonged hematopoietic function, hematopoiesis in Ercc1−/− mice was compared to that in young and old wild-type mice. Ercc1−/− mice (3-week-old) exhibited multilineage cytopenia and fatty replacement of bone marrow, similar to old wild-type mice. In addition, the proliferative reserves of hematopoietic progenitors and stress erythropoiesis were significantly reduced in Ercc1−/− mice compared to age-matched controls. These features were not seen in nucleotide excision repair-deficient Xpa−/− mice, but are characteristic of Fanconi anemia, a human cancer syndrome caused by defects in interstrand crosslink repair. These data support the hypothesis that spontaneous interstrand crosslink damage contributes to the functional decline of the hematopoietic system associated with aging. PMID:15692571

  12. FGF-2 signal promotes proliferation of cerebellar progenitor cells and their oligodendrocytic differentiation at early postnatal stage

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Naruse, Masae; Shibasaki, Koji; Ishizaki, Yasuki, E-mail: yasukiishizaki@gunma-u.ac.jp

    The origins and developmental regulation of cerebellar oligodendrocytes are largely unknown, although some hypotheses of embryonic origins have been suggested. Neural stem cells exist in the white matter of postnatal cerebellum, but it is unclear whether these neural stem cells generate oligodendrocytes at postnatal stages. We previously showed that cerebellar progenitor cells, including neural stem cells, widely express CD44 at around postnatal day 3. In the present study, we showed that CD44-positive cells prepared from the postnatal day 3 cerebellum gave rise to neurospheres, while CD44-negative cells prepared from the same cerebellum did not. These neurospheres differentiated mainly into oligodendrocytesmore » and astrocytes, suggesting that CD44-positive neural stem/progenitor cells might generate oligodendrocytes in postnatal cerebellum. We cultured CD44-positive cells from the postnatal day 3 cerebellum in the presence of signaling molecules known as mitogens or inductive differentiation factors for oligodendrocyte progenitor cells. Of these, only FGF-2 promoted survival and proliferation of CD44-positive cells, and these cells differentiated into O4+ oligodendrocytes. Furthermore, we examined the effect of FGF-2 on cerebellar oligodendrocyte development ex vivo. FGF-2 enhanced proliferation of oligodendrocyte progenitor cells and increased the number of O4+ and CC1+ oligodendrocytes in slice cultures. These results suggest that CD44-positive cells might be a source of cerebellar oligodendrocytes and that FGF-2 plays important roles in their development at an early postnatal stage. - Highlights: • CD44 is expressed in cerebellar neural stem/progenitor cells at postnatal day 3 (P3). • FGF-2 promoted proliferation of CD44-positive progenitor cells from P3 cerebellum. • FGF-2 promoted oligodendrocytic differentiation of CD44-positive progenitor cells. • FGF-2 increased the number of oligodendrocytes in P3 cerebellar slice culture.« less

  13. NKL homeobox gene activities in hematopoietic stem cells, T-cell development and T-cell leukemia.

    PubMed

    Nagel, Stefan; Pommerenke, Claudia; Scherr, Michaela; Meyer, Corinna; Kaufmann, Maren; Battmer, Karin; MacLeod, Roderick A F; Drexler, Hans G

    2017-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) cells represent developmentally arrested T-cell progenitors, subsets of which aberrantly express homeobox genes of the NKL subclass, including TLX1, TLX3, NKX2-1, NKX2-5, NKX3-1 and MSX1. Here, we analyzed the transcriptional landscape of all 48 members of the NKL homeobox gene subclass in CD34+ hematopoietic stem and progenitor cells (HSPCs) and during lymphopoiesis, identifying activities of nine particular genes. Four of these were expressed in HSPCs (HHEX, HLX1, NKX2-3 and NKX3-1) and three in common lymphoid progenitors (HHEX, HLX1 and MSX1). Interestingly, our data indicated downregulation of NKL homeobox gene transcripts in late progenitors and mature T-cells, a phenomenon which might explain the oncogenic impact of this group of genes in T-ALL. Using MSX1-expressing T-ALL cell lines as models, we showed that HHEX activates while HLX1, NKX2-3 and NKX3-1 repress MSX1 transcription, demonstrating the mutual regulation and differential activities of these homeobox genes. Analysis of a public T-ALL expression profiling data set comprising 117 patient samples identified 20 aberrantly activated members of the NKL subclass, extending the number of known NKL homeobox oncogene candidates. While 7/20 genes were also active during hematopoiesis, the remaining 13 showed ectopic expression. Finally, comparative analyses of T-ALL patient and cell line profiling data of NKL-positive and NKL-negative samples indicated absence of shared target genes but instead highlighted deregulation of apoptosis as common oncogenic effect. Taken together, we present a comprehensive survey of NKL homeobox genes in early hematopoiesis, T-cell development and T-ALL, showing that these genes generate an NKL-code for the diverse stages of lymphoid development which might be fundamental for regular differentiation.

  14. NKL homeobox gene activities in hematopoietic stem cells, T-cell development and T-cell leukemia

    PubMed Central

    Pommerenke, Claudia; Scherr, Michaela; Meyer, Corinna; Kaufmann, Maren; Battmer, Karin; MacLeod, Roderick A. F.; Drexler, Hans G.

    2017-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) cells represent developmentally arrested T-cell progenitors, subsets of which aberrantly express homeobox genes of the NKL subclass, including TLX1, TLX3, NKX2-1, NKX2-5, NKX3-1 and MSX1. Here, we analyzed the transcriptional landscape of all 48 members of the NKL homeobox gene subclass in CD34+ hematopoietic stem and progenitor cells (HSPCs) and during lymphopoiesis, identifying activities of nine particular genes. Four of these were expressed in HSPCs (HHEX, HLX1, NKX2-3 and NKX3-1) and three in common lymphoid progenitors (HHEX, HLX1 and MSX1). Interestingly, our data indicated downregulation of NKL homeobox gene transcripts in late progenitors and mature T-cells, a phenomenon which might explain the oncogenic impact of this group of genes in T-ALL. Using MSX1-expressing T-ALL cell lines as models, we showed that HHEX activates while HLX1, NKX2-3 and NKX3-1 repress MSX1 transcription, demonstrating the mutual regulation and differential activities of these homeobox genes. Analysis of a public T-ALL expression profiling data set comprising 117 patient samples identified 20 aberrantly activated members of the NKL subclass, extending the number of known NKL homeobox oncogene candidates. While 7/20 genes were also active during hematopoiesis, the remaining 13 showed ectopic expression. Finally, comparative analyses of T-ALL patient and cell line profiling data of NKL-positive and NKL-negative samples indicated absence of shared target genes but instead highlighted deregulation of apoptosis as common oncogenic effect. Taken together, we present a comprehensive survey of NKL homeobox genes in early hematopoiesis, T-cell development and T-ALL, showing that these genes generate an NKL-code for the diverse stages of lymphoid development which might be fundamental for regular differentiation. PMID:28151996

  15. Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression

    PubMed Central

    Richter, Karin; Wirta, Valtteri; Dahl, Lina; Bruce, Sara; Lundeberg, Joakim; Carlsson, Leif; Williams, Cecilia

    2006-01-01

    Background Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types. Results Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development. Conclusion Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin. PMID:16600034

  16. Early-time spectra of supernovae and their precursor winds. The luminous blue variable/yellow hypergiant progenitor of SN 2013cu

    NASA Astrophysics Data System (ADS)

    Groh, Jose H.

    2014-12-01

    We present the first quantitative spectroscopic modeling of an early-time supernova (SN) that interacts with its progenitor wind. Using the radiative transfer code CMFGEN, we investigate the recently reported 15.5 h post-explosion spectrum of the type IIb SN 2013cu. We are able to directly measure the chemical abundances of a SN progenitor and find a relatively H-rich wind, with H and He abundances (by mass) of X = 0.46 ± 0.2 and Y = 0.52 ± 0.2, respectively. The wind is enhanced in N and depleted in C relative to solar values (mass fractions of 8.2 × 10-3 and 1.0 × 10-5, respectively). We obtain that a slow, dense wind or circumstellar medium surrounds the precursor at the pre-SN stage, with a wind terminal velocity vwind ≲ 100 km s-1 and mass-loss rate of Ṁ ≃ 3 × 10-3 (vwind/ 100 km s-1) M⊙ yr-1. These values are lower than previous analytical estimates, although Ṁ/υ∞ is consistent with previous work. We also compute a CMFGEN model to constrain the progenitor spectral type; the high Ṁ and low vwind imply that the star had an effective temperature of ≃ 8000 K immediately before the SN explosion. Our models suggest that the progenitor was either an unstable luminous blue variable or a yellow hypergiant undergoing an eruptive phase, and rule out a Wolf-Rayet star. We classify the post-explosion spectra at 15.5 h as XWN5(h) and advocate for the use of the prefix "X" (eXplosion) to avoid confusion between post-explosion, non-stellar spectra, and those of massive stars. We show that the XWN spectrum results from the ionization of the progenitor wind after the SN, and that the progenitor spectral type is significantly different from the early post-explosion spectral type owing to the huge differences in the ionization structure before and after the SN event. We find the following temporal evolution: LBV/YHG → XWN5(h) → SN IIb. Future early-time spectroscopy in the UV will further constrain the properties of SN precursors, such as their

  17. Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells.

    PubMed

    Li, LiQi; Jothi, Raja; Cui, Kairong; Lee, Jan Y; Cohen, Tsadok; Gorivodsky, Marat; Tzchori, Itai; Zhao, Yangu; Hayes, Sandra M; Bresnick, Emery H; Zhao, Keji; Westphal, Heiner; Love, Paul E

    2011-02-01

    The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non-DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex-binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs.

  18. Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells

    PubMed Central

    Li, LiQi; Jothi, Raja; Cui, Kairong; Lee, Jan Y; Cohen, Tsadok; Gorivodsky, Marat; Tzchori, Itai; Zhao, Yangu; Hayes, Sandra M; Bresnick, Emery H; Zhao, Keji; Westphal, Heiner; Love, Paul E

    2013-01-01

    The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non–DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex–binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs. PMID:21186366

  19. Quantitative analysis by next generation sequencing of hematopoietic stem and progenitor cells (LSK) and of splenic B cells transcriptomes from wild-type and Usp3-knockout mice.

    PubMed

    Lancini, Cesare; Gargiulo, Gaetano; van den Berk, Paul C M; Citterio, Elisabetta

    2016-03-01

    The data described here provide genome-wide expression profiles of murine primitive hematopoietic stem and progenitor cells (LSK) and of B cell populations, obtained by high throughput sequencing. Cells are derived from wild-type mice and from mice deficient for the ubiquitin-specific protease 3 (USP3; Usp3Δ/Δ). Modification of histone proteins by ubiquitin plays a crucial role in the cellular response to DNA damage (DDR) (Jackson and Durocher, 2013) [1]. USP3 is a histone H2A deubiquitinating enzyme (DUB) that regulates ubiquitin-dependent DDR in response to DNA double-strand breaks (Nicassio et al., 2007; Doil et al., 2008) [2], [3]. Deletion of USP3 in mice increases the incidence of spontaneous tumors and affects hematopoiesis [4]. In particular, Usp3-knockout mice show progressive loss of B and T cells and decreased functional potential of hematopoietic stem cells (HSCs) during aging. USP3-deficient cells, including HSCs, display enhanced histone ubiquitination, accumulate spontaneous DNA damage and are hypersensitive to ionizing radiation (Lancini et al., 2014) [4]. To address whether USP3 loss leads to deregulation of specific molecular pathways relevant to HSC homeostasis and/or B cell development, we have employed the RNA-sequencing technology and investigated transcriptional differences between wild-type and Usp3Δ/Δ LSK, naïve B cells or in vitro activated B cells. The data relate to the research article "Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells" (Lancini et al., 2014) [4]. The RNA-sequencing and analysis data sets have been deposited in NCBI׳s Gene Expression Omnibus (Edgar et al., 2002) [5] and are accessible through GEO Series accession number GSE58495 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58495). With this article, we present validation of the RNA-seq data set through quantitative real-time PCR and comparative analysis.

  20. Linking Doses with Clinical Scores of Hematopoietic Acute Radiation Syndrome.

    PubMed

    Hu, Shaowen

    2016-10-01

    In radiation accidents, determining the radiation dose the victim received is a key step for medical decision making and patient prognosis. To reconstruct and evaluate the absorbed dose, researchers have developed many physical devices and biological techniques during the last decades. However, using the physical parameter "absorbed dose" alone is not sufficient to predict the clinical development of the various organs injured in an individual patient. In operational situations for radiation accidents, medical responders need more urgently to classify the severity of the radiation injury based on the signs and symptoms of the patient. In this work, the author uses a unified hematopoietic model to describe dose-dependent dynamics of granulocytes, lymphocytes, and platelets, and the corresponding clinical grading of hematopoietic acute radiation syndrome. This approach not only visualizes the time course of the patient's probable outcome in the form of graphs but also indirectly gives information of the remaining stem and progenitor cells, which are responsible for the autologous recovery of the hematopoietic system. Because critical information on the patient's clinical evolution can be provided within a short time after exposure and only peripheral cell counts are required for the simulation, these modeling tools will be useful to assess radiation exposure and injury in human-involved radiation accident/incident scenarios.

  1. Preservation of Differentiation and Clonogenic Potential of Human Hematopoietic Stem and Progenitor Cells during Lyophilization and Ambient Storage

    PubMed Central

    Buchanan, Sandhya S.; Pyatt, David W.; Carpenter, John F.

    2010-01-01

    Progenitor cell therapies show great promise, but their potential for clinical applications requires improved storage and transportation. Desiccated cells stored at ambient temperature would provide economic and practical advantages over approaches employing cell freezing and subzero temperature storage. The objectives of this study were to assess a method for loading the stabilizing sugar, trehalose, into hematopoietic stem and progenitor cells (HPC) and to evaluate the effects of subsequent freeze-drying and storage at ambient temperature on differentiation and clonogenic potential. HPC were isolated from human umbilical cord blood and loaded with trehalose using an endogenous cell surface receptor, termed P2Z. Solution containing trehalose-loaded HPC was placed into vials, which were transferred to a tray freeze-dryer and removed during each step of the freeze-drying process to assess differentiation and clonogenic potential. Control groups for these experiments were freshly isolated HPC. Control cells formed 1450±230 CFU-GM, 430±140 BFU-E, and 50±40 CFU-GEMM per 50 µL. Compared to the values for the control cells, there was no statistical difference observed for cells removed at the end of the freezing step or at the end of primary drying. There was a gradual decrease in the number of CFU-GM and BFU-E for cells removed at different temperatures during secondary drying; however, there were no significant differences in the number of CFU-GEMM. To determine storage stability of lyophilized HPC, cells were stored for 4 weeks at 25°C in the dark. Cells reconstituted immediately after lyophilization produced 580±90 CFU-GM (∼40%, relative to unprocessed controls p<0.0001), 170±70 BFU-E (∼40%, p<0.0001), and 41±22 CFU-GEMM (∼82%, p = 0.4171), and cells reconstituted after 28 days at room temperature produced 513±170 CFU-GM (∼35%, relative to unprocessed controls, p<0.0001), 112±68 BFU-E (∼26%, p<0.0001), and 36±17 CFU-GEMM (∼82%, p = 0

  2. Declining lymphoid progenitor fitness promotes aging-associated leukemogenesis.

    PubMed

    Henry, Curtis J; Marusyk, Andriy; Zaberezhnyy, Vadym; Adane, Biniam; DeGregori, James

    2010-12-14

    Aging is associated with the functional decline of cells, tissues, and organs. At the same time, age is the single most important prognostic factor in the development of most human cancers, including chronic myelogenous and acute lymphoblastic leukemias initiated by Bcr-Abl oncogenic translocations. Prevailing paradigms attribute the association between aging and cancers to the accumulation of oncogenic mutations over time, because the accrual of oncogenic events is thought to be the rate-limiting step in initiation and progression of cancers. Conversely, aging-associated functional decline caused by both cell-autonomous and non-cell-autonomous mechanisms is likely to reduce the fitness of stem and progenitor cell populations. This reduction in fitness should be conducive for increased selection of oncogenic mutations that can at least partially alleviate fitness defects, thereby promoting the initiation of cancers. We tested this hypothesis using mouse hematopoietic models. Our studies indicate that the dramatic decline in the fitness of aged B-lymphopoiesis coincides with altered receptor-associated kinase signaling. We further show that Bcr-Abl provides a much greater competitive advantage to old B-lymphoid progenitors compared with young progenitors, coinciding with restored kinase signaling pathways, and that this enhanced competitive advantage translates into increased promotion of Bcr-Abl-driven leukemias. Moreover, impairing IL-7-mediated signaling is sufficient to promote selection for Bcr-Abl-expressing B progenitors. These studies support an unappreciated causative link between aging and cancer: increased selection of oncogenic mutations as a result of age-dependent alterations of the fitness landscape.

  3. The isolation and in vitro expansion of hepatic Sca-1 progenitor cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Clayton, Elizabeth, E-mail: Elizabeth.Clayton@ed.ac.uk; Forbes, Stuart J.

    2009-04-17

    The intra-hepatic population of liver progenitor cells expands during liver injury when hepatocyte proliferation is inhibited. These cells can be purified by density gradient centrifugation and cultured. Separated by size only this population contains small cells of hematopoietic, epithelial and endothelial lineages and is thought to contain liver stem cells. The identity of liver stem cells remains unknown although there is some evidence that tissue Sca1{sup +} CD45{sup -} cells display progenitor cell characteristics. We identified both intra-hepatic and gall bladder Sca1{sup +} cells following liver injury and expanded ex vivo Sca1 cells as part of heterogenous cell culture ormore » as a purified population. We found significant difference between the proliferation of Sca-1 cells when plated on laminin or collagen I while proliferation of heterogenous population was not affected by the extracellular matrix indicating the necessity for culture of Sca1{sup +} cells with laminin matrix or laminin producing cells in long term liver progenitor cell cultures.« less

  4. Parasitic Infections in Hematopoietic Stem Cell Transplantation

    PubMed Central

    Jarque, Isidro; Salavert, Miguel; Pemán, Javier

    2016-01-01

    Parasitic infections are rarely documented in hematopoietic stem cell transplant recipients. However they may be responsible for fatal complications that are only diagnosed at autopsy. Increased awareness of the possibility of parasitic diseases both in autologous and allogeneic stem cell transplant patients is relevant not only for implementing preventive measures but also for performing an early diagnosis and starting appropriate therapy for these unrecognized but fatal infectious complications in hematopoietic transplant recipients. In this review, we will focus on parasitic diseases occurring in this population especially those with major clinical relevance including toxoplasmosis, American trypanosomiasis, leishmaniasis, malaria, and strongyloidiasis, among others, highlighting the diagnosis and management in hematopoietic transplant recipients. PMID:27413527

  5. Bone marrow niche-inspired, multiphase expansion of megakaryocytic progenitors with high polyploidization potential.

    PubMed

    Panuganti, Swapna; Papoutsakis, Eleftherios T; Miller, William M

    2010-10-01

    Megakaryopoiesis encompasses hematopoietic stem and progenitor cell (HSPC) commitment to the megakaryocytic cell (Mk) lineage, expansion of Mk progenitors and mature Mks, polyploidization and platelet release. pH and pO2 increase from the endosteum to sinuses, and different cytokines are important for various stages of differentiation. We hypothesized that mimicking the changing conditions during Mk differentiation in the bone marrow would facilitate expansion of progenitors that could generate many high-ploidy Mks. CD34+ HSPCs were cultured at pH 7.2 and 5% O2 with stem cell factor (SCF), thrombopoietin (Tpo) and all combinations of Interleukin (IL)-3, IL-6, IL-11 and Flt-3 ligand to promote Mk progenitor expansion. Cells cultured with selected cytokines were shifted to pH 7.4 and 20% O2 to generate mature Mks, and treated with nicotinamide (NIC) to enhance polyploidization. Using Tpo + SCF + IL-3 + IL-11, we obtained 3.5 CD34+ CD41+ Mk progenitors per input HSPC, while increasing purity from 1% to 17%. Cytokine cocktails with IL-3 yielded more progenitors and mature Mks, although the purities were lower. Mk production was much greater at higher pH and pO2. Although fewer progenitors were present, shifting to 20% O2 /pH 7.4 at day 5 (versus days 7 or 9) yielded the greatest mature Mk production, 14 per input HSPC. NIC more than doubled the percentage of high-ploidy Mks to 40%. We obtained extensive Mk progenitor expansion, while ensuring that the progenitors could produce high-ploidy Mks. We anticipate that subsequent optimization of cytokines for mature Mk production and delayed NIC addition will greatly increase high-ploidy Mk production.

  6. Cellular innate immunity and restriction of viral infection: implications for lentiviral gene therapy in human hematopoietic cells.

    PubMed

    Kajaste-Rudnitski, Anna; Naldini, Luigi

    2015-04-01

    Hematopoietic gene therapy has tremendous potential to treat human disease. Nevertheless, for gene therapy to be efficacious, effective gene transfer into target cells must be reached without inducing detrimental effects on their biological properties. This remains a great challenge for the field as high vector doses and prolonged ex vivo culture conditions are still required to reach significant transduction levels of clinically relevant human hematopoietic stem and progenitor cells (HSPCs), while other potential target cells such as primary macrophages can hardly be transduced. The reasons behind poor permissiveness of primary human hematopoietic cells to gene transfer partly reside in the retroviral origin of lentiviral vectors (LVs). In particular, host antiviral factors referred to as restriction factors targeting the retroviral life cycle can hamper LV transduction efficiency. Furthermore, LVs may activate innate immune sensors not only in differentiated hematopoietic cells but also in HSPCs, with potential consequences on transduction efficiency as well as their biological properties. Therefore, better understanding of the vector-host interactions in the context of hematopoietic gene transfer is important for the development of safer and more efficient gene therapy strategies. In this review, we briefly summarize the current knowledge regarding innate immune recognition of lentiviruses in primary human hematopoietic cells as well as discuss its relevance for LV-based ex vivo gene therapy approaches.

  7. Generation of hematopoietic stem cells from human embryonic stem cells using a defined, stepwise, serum-free, and serum replacement-free monolayer culture method.

    PubMed

    Kim, So-Jung; Jung, Ji-Won; Ha, Hye-Yeong; Koo, Soo Kyung; Kim, Eung-Gook; Kim, Jung-Hyun

    2017-03-01

    Embryonic stem cells (ESCs) can be expanded infinitely in vitro and have the potential to differentiate into hematopoietic stem cells (HSCs); thus, they are considered a useful source of cells for HSC production. Although several technical in vitro methods for engineering HSCs from pluripotent stem cells have been developed, clinical application of HSCs engineered from pluripotent stem cells is restricted because of the possibility of xenogeneic contamination resulting from the use of murine materials. Human ESCs (CHA-hES15) were cultured on growth factor-reduced Matrigel-coated dishes in the mTeSR1 serum-free medium. When the cells were 70% confluent, we initiated HSC differentiation by three methods involving (1) knockout serum replacement (KSR), cytokines, TGFb1, EPO, and FLT3L; (2) KSR, cytokines, and bFGF; or (3) cytokines and bFGF. Among the three differentiation methods, the minimal number of cytokines without KSR resulted in the greatest production of HSCs. The optimized method resulted in a higher proportion of CD34 + CD43 + hematopoietic progenitor cells (HPCs) and CD34 + CD45 + HPCs compared to the other methods. In addition, the HSCs showed the potential to differentiate into multiple lineages of hematopoietic cells in vitro . In this study, we optimized a two-step, serum-free, animal protein-free, KSR-free, feeder-free, chemically defined monolayer culture method for generation of HSCs and hematopoietic stem and progenitor cells (HSPCs) from human ESCs.

  8. Hematopoietic stem cell injury induced by ionizing radiation.

    PubMed

    Shao, Lijian; Luo, Yi; Zhou, Daohong

    2014-03-20

    Exposure to ionizing radiation (IR) as the result of nuclear accidents or terrorist attacks is a significant threat and a major medical concern. Hematopoietic stem cell (HSC) injury is the primary cause of death after accidental or intentional exposure to a moderate or high dose of IR. Protecting HSCs from IR should be a primary goal in the development of novel medical countermeasures against radiation. Significant progress has been made in our understanding of the mechanisms by which IR causes HSC damage. The mechanisms include (i) induction of HSC apoptosis via the p53-Puma pathway; (ii) promotion of HSC differentiation via the activation of the G-CSF/Stat3/BATF-dependent differentiation checkpoint; (iii) induction of HSC senescence via the ROS-p38 pathway; and (iv) damage to the HSC niche. Induction of apoptosis in HSCs and hematopoietic progenitor cells is primarily responsible for IR-induced acute bone marrow (BM) injury. Long-term BM suppression caused by IR is mainly attributable to the induction of HSC senescence. However, the promotion of HSC differentiation and damage to the HSC niche can contribute to both the acute and long-term effects of IR on the hematopoietic system. In this review, we have summarized a number of recent findings that provide new insights into the mechanisms whereby IR damages HSCs. These findings will provide new opportunities for developing a mechanism-based strategy to prevent and/or mitigate IR-induced BM suppression. Antioxid.

  9. Ability of circulating human hematopoietic lineage negative cells to support hematopoiesis.

    PubMed

    Peris, Pilar; Roforth, Matthew M; Nicks, Kristy M; Fraser, Daniel; Fujita, Koji; Jilka, Robert L; Khosla, Sundeep; McGregor, Ulrike

    2015-01-01

    Hematopoietic stem cell (HSC) self-renewal is regulated by osteoblast and/or endothelial cells within the hematopoietic niche. However, the true identity of the supporting cells and the nature of the secreted factors remain uncertain. We developed a novel mouse model and analyzed whether circulating human peripheral hematopoietic lineage negative/AP+ (lin-/AP+) cells support hematopoiesis in vivo. Thus, immunocompromised (Rag) mice expressing thymidine kinase (Tk) under the control of the 3.6Col1α1 promoter (Tk-Rag) were treated with ganciclovir, resulting in osteoblast progenitor cell ablation and subsequent loss of hematopoiesis (evaluated by measuring mouse Ter119+ erythroid cells). Following hematopoietic cell depletion, human bone marrow-derived marrow stromal cells (MSCs) or lin-/AP+ cells were infused into Tk-Rag mice and compared with saline infusions. Ganciclovir significantly reduced (7.4-fold) Ter119+ cells in the bone marrow of Tk-Rag mice compared to saline injections. Infusion of either MSCs or lin-/AP+ cells into ganciclovir-treated mice resulted in a 3.3-fold and 2.7-fold increase (P < 0.01), respectively, in Ter119+ cells compared to mice receiving saline. Relative to lin-/AP- cells, lin-/AP+ cells expressed high levels of mesenchymal, endothelial, and hematopoiesis supporting genes. Thus, human peripheral blood lin-/AP+ cells represent a novel cell type capable of supporting hematopoiesis in a manner comparable to MSCs. © 2014 Wiley Periodicals, Inc.

  10. Mdm2 is required for survival of hematopoietic stem cells/progenitors via dampening of ROS-induced p53 activity

    USDA-ARS?s Scientific Manuscript database

    Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53(515C) (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2(-/-) mice. Mdm2(-/-) p53(515C/515C) mice, however, die by postnatal day 13 resulting from hematopoietic failure. Hematopoietic st...

  11. Regulation of long-term repopulating hematopoietic stem cells by EPCR/PAR1 signaling

    PubMed Central

    Gur-Cohen, Shiri; Kollet, Orit; Graf, Claudine; Esmon, Charles T.; Ruf, Wolfram; Lapidot, Tsvee

    2016-01-01

    The common developmental origin of endothelial and hematopoietic cells is manifested by coexpression of several cell surface receptors. Adult murine bone marrow (BM) long-term repopulating hematopoietic stem cells (LT-HSCs), endowed with the highest repopulation and self-renewal potential, express endothelial protein C receptor (EPCR), which is used as a marker to isolate them. EPCR/PAR1 signaling in endothelial cells has anticoagulant and anti-inflammatory roles, while thrombin/PAR1 signaling induces coagulation and inflammation. Recent studies define two new PAR1-mediated signaling cascades that regulate EPCR+ LT-HSC BM retention and egress. EPCR/PAR1 signaling facilitates LT-HSC BM repopulation, retention, survival, and chemotherapy resistance by restricting nitric oxide (NO) production, maintaining NOlow LT-HSC BM retention with increased VLA4 expression, affinity, and adhesion. Conversely, acute stress and clinical mobilization upregulate thrombin generation and activate different PAR1 signaling which overcomes BM EPCR+ LT-HSC retention, inducing their recruitment to the bloodstream. Thrombin/PAR1 signaling induces NO generation, TACE-mediated EPCR shedding, and upregulation of CXCR4 and PAR1, leading to CXCL12-mediated stem and progenitor cell mobilization. This review discusses new roles for factors traditionally viewed as coagulation related, which independently act in the BM to regulate PAR1 signaling in bone- and blood-forming progenitor cells, navigating their fate by controlling NO production. PMID:26928241

  12. Single-lineage transcriptome analysis reveals key regulatory pathways in primitive erythroid progenitors in the mouse embryo

    PubMed Central

    Isern, Joan; He, Zhiyong; Fraser, Stuart T.; Nowotschin, Sonja; Ferrer-Vaquer, Anna; Moore, Rebecca; Hadjantonakis, Anna-Katerina; Schulz, Vincent; Tuck, David; Gallagher, Patrick G.

    2011-01-01

    Primitive erythroid (EryP) progenitors are the first cell type specified from the mesoderm late in gastrulation. We used a transgenic reporter to image and purify the earliest blood progenitors and their descendants from developing mouse embryos. EryP progenitors exhibited remarkable proliferative capacity in the yolk sac immediately before the onset of circulation, when these cells comprise nearly half of all cells of the embryo. Global expression profiles generated at 24-hour intervals from embryonic day 7.5 through 2.5 revealed 2 abrupt changes in transcript diversity that coincided with the entry of EryPs into the circulation and with their late maturation and enucleation, respectively. These changes were paralleled by the expression of critical regulatory factors. Experiments designed to test predictions from these data demonstrated that the Wnt-signaling pathway is active in EryP progenitors, which display an aerobic glycolytic profile and the numbers of which are regulated by transforming growth factor-β1 and hypoxia. This is the first transcriptome assembled for a single hematopoietic lineage of the embryo over the course of its differentiation. PMID:21263157

  13. Bone marrow-on-a-chip replicates hematopoietic niche physiology in vitro.

    PubMed

    Torisawa, Yu-suke; Spina, Catherine S; Mammoto, Tadanori; Mammoto, Akiko; Weaver, James C; Tat, Tracy; Collins, James J; Ingber, Donald E

    2014-06-01

    Current in vitro hematopoiesis models fail to demonstrate the cellular diversity and complex functions of living bone marrow; hence, most translational studies relevant to the hematologic system are conducted in live animals. Here we describe a method for fabricating 'bone marrow-on-a-chip' that permits culture of living marrow with a functional hematopoietic niche in vitro by first engineering new bone in vivo, removing it whole and perfusing it with culture medium in a microfluidic device. The engineered bone marrow (eBM) retains hematopoietic stem and progenitor cells in normal in vivo-like proportions for at least 1 week in culture. eBM models organ-level marrow toxicity responses and protective effects of radiation countermeasure drugs, whereas conventional bone marrow culture methods do not. This biomimetic microdevice offers a new approach for analysis of drug responses and toxicities in bone marrow as well as for study of hematopoiesis and hematologic diseases in vitro.

  14. FGF/EGF signaling regulates the renewal of early nephron progenitors during embryonic development.

    PubMed

    Brown, Aaron C; Adams, Derek; de Caestecker, Mark; Yang, Xuehui; Friesel, Robert; Oxburgh, Leif

    2011-12-01

    Recent studies indicate that nephron progenitor cells of the embryonic kidney are arranged in a series of compartments of an increasing state of differentiation. The earliest progenitor compartment, distinguished by expression of CITED1, possesses greater capacity for renewal and differentiation than later compartments. Signaling events governing progression of nephron progenitor cells through stages of increasing differentiation are poorly understood, and their elucidation will provide key insights into normal and dysregulated nephrogenesis, as well as into regenerative processes that follow kidney injury. In this study, we found that the mouse CITED1(+) progenitor compartment is maintained in response to receptor tyrosine kinase (RTK) ligands that activate both FGF and EGF receptors. This RTK signaling function is dependent on RAS and PI3K signaling but not ERK. In vivo, RAS inactivation by expression of sprouty 1 (Spry1) in CITED1(+) nephron progenitors results in loss of characteristic molecular marker expression and in increased death of progenitor cells. Lineage tracing shows that surviving Spry1-expressing progenitor cells are impaired in their subsequent epithelial differentiation, infrequently contributing to epithelial structures. These findings demonstrate that the survival and developmental potential of cells in the earliest embryonic nephron progenitor cell compartment are dependent on FGF/EGF signaling through RAS.

  15. Hematopoietic Acute Radiation Syndrome (Bone marrow syndrome, Aplastic Anemia): Molecular Mechanisms of Radiation Toxicity.

    NASA Astrophysics Data System (ADS)

    Popov, Dmitri

    Key Words: Aplastic Anemia (AA), Pluripotential Stem Cells (PSC) Introduction: Aplastic Anemia (AA) is a disorder of the pluripotential stem cells involve a decrease in the number of cells of myeloid, erythroid and megakaryotic lineage [Segel et al. 2000 ]. The etiology of AA include idiopathic cases and secondary aplastic anemia after exposure to drugs, toxins, chemicals, viral infections, lympho-proliferative diseases, radiation, genetic causes, myelodisplastic syndromes and hypoplastic anemias, thymomas, lymphomas. [Brodskyet al. 2005.,Modan et al. 1975., Szklo et al. 1975]. Hematopoietic Acute Radiation Syndrome (or Bone marrow syndrome, or Radiation-Acquired Aplastic Anemia) is the acute toxic syndrome which usually occurs with a dose of irradiation between 0.7 and 10 Gy (70- 1000 rads), depending on the species irradiated. [Waselenko et al., 2004]. The etiology of bone morrow damage from high-level radiation exposure results depends on the radiosensitivity of certain bone marrow cell lines. [Waselenko et al. 2004] Aplastic anemia after radiation exposure is a clinical syndrome that results from a marked disorder of bone marrow blood cell production. [Waselenko et al. 2004] Radiation hematotoxicity is mediated via genotoxic and other specific toxic mechanisms, leading to aplasia, cell apoptosis or necrosis, initiation via genetic mechanisms of clonal disorders, in cases such as the acute radiation-acquired form of AA. AA results from radiation injury to pluripotential and multipotential stem cells in the bone marrow. The clinical signs displayed in reticulocytopenia, anemia, granulocytopenia, monocytopenia, and thrombocytopenia. The number of marrow CD34+ cells (multipotential hematopoietic progenitors) and their derivative colony-forming unit{granulocyte-macrophage (CFU-GM) and burst forming unit {erythroid (BFU{E) are reduced markedly in patients with AA. [Guinan 2011, Brodski et al. 2005, Beutler et al.,2000] Cells expressing CD34 (CD34+ cell) are normally

  16. Granulocyte-colony stimulating factor (G-CSF)-primed, delayed marrow harvests as a source of hematopoietic stem and progenitor cells for allogeneic transplantation.

    PubMed

    Phillips, G L; Davey, D D; Hale, G A; Marshall, K W; Munn, R K; Nath, R; Reece, D E; Van Zant, G

    1999-10-01

    We evaluated the ability of G-CSF to increase the number of hematopoietic stem cells obtained by "delayed" BM harvest for allogeneic transplantation. Five normal donors received G-CSF @ 10 mcg/kg/day x 5 followed by repeat PB and BM assays at day 6 and 16, and BM harvest at day 16. Stem cells were not increased in the BM at day 16. Five patients underwent BMT and engrafted at +10 to +19 days. While the tested strategy offers no intrinsic advantages, its potential cannot be evaluated fully without alternative timing and/or additional, "early acting" growth factors.

  17. Competence of failed endocrine progenitors to give rise to acinar but not ductal cells is restricted to early pancreas development

    PubMed Central

    Beucher, Anthony; Martín, Mercè; Spenle, Caroline; Poulet, Martine; Collin, Caitlin; Gradwohl, Gérard

    2011-01-01

    SUMMARY During mouse pancreas development, the transient expression of Neurogenin3 (Neurog3) in uncommitted pancreas progenitors is required to determine endocrine destiny. However it has been reported that Neurog3-expressing cells can eventually adopt acinar or ductal fates and that Neurog3 levels were important to secure the islet destiny. It is not known whether the competence of Neurog3-induced cells to give rise to non-endocrine lineages is an intrinsic property of these progenitors or depends on pancreas developmental stage. Using temporal genetic labeling approaches we examined the dynamic of endocrine progenitor differentiation and explored the plasticity of Neurog3-induced cells throughout development. We found that Neurog3+ progenitors develop into hormone-expressing cells in a fast process taking less then 10h. Furthermore, fate-mapping studies in heterozygote (Neurog3CreERT/+) and Neurog3-deficient (Neurog3CreERT/CreERT) embryos revealed that Neurog3-induced cells have different potential over time. At the early bud stage, failed endocrine progenitors can adopt acinar or ductal fate, whereas later in the branching pancreas they do not contribute to the acinar lineage but Neurog3-deficient cells eventually differentiate into duct cells. Thus these results provide evidence that the plasticity of Neurog3-induced cells becomes restricted during development. Furthermore these data suggest that during the secondary transition endocrine progenitor cells arise from single bipotent progenitor already committed to the duct/endocrine lineages and not from domain of cells having both potentialities. PMID:22056785

  18. Bone marrow niche-inspired, multi-phase expansion of megakaryocytic progenitors with high polyploidization potential

    PubMed Central

    Panuganti, Swapna; Papoutsakis, Eleftherios T.; Miller, William M.

    2010-01-01

    Background Megakaryopoiesis encompasses hematopoietic stem and progenitor cell (HSPC) commitment to the megakaryocytic cell (Mk) lineage, expansion of Mk progenitors and mature Mks, polyploidization, and platelet release. pH and pO2 increase from the endosteum to sinuses, and different cytokines are important for various stages of differentiation. We hypothesized that mimicking the changing conditions during Mk differentiation in the bone marrow would facilitate expansion of progenitors that could generate many high-ploidy Mks. Methods CD34+ HSPCs were cultured at pH 7.2 and 5% O2 with stem cell factor (SCF), thrombopoietin (Tpo), and all combinations of Interleukin (IL)-3, IL-6, IL-11, and Flt-3 ligand to promote Mk progenitor expansion. Cells cultured with selected cytokines were shifted to pH 7.4 and 20% O2 to generate mature Mks, and treated with nicotinamide to enhance polyploidization. Results Using Tpo+SCF+IL-3+IL-11, we obtained 3.5 CD34+CD41+ Mk progenitors per input HSPC, while increasing purity from 1% to 17%. Cytokine cocktails with IL-3 yielded more progenitors and mature Mks, although the purities were lower. Mk production was much greater at higher pH and pO2. Although fewer progenitors were present, shifting to 20% O2/pH 7.4 at day 5 (versus days 7 or 9) yielded the greatest mature Mk production, 14 per input HSPC. Nicotinamide more than doubled the percentage of high-ploidy Mks to 40%. Discussion We obtained extensive Mk progenitor expansion, while ensuring that the progenitors could produce high-ploidy Mks. We anticipate that subsequent optimization of cytokines for mature Mk production and delayed nicotinamide addition will greatly increase high-ploidy Mk production. PMID:20482285

  19. Adrenaline administration promotes the efficiency of granulocyte colony stimulating factor-mediated hematopoietic stem and progenitor cell mobilization in mice.

    PubMed

    Chen, Chong; Cao, Jiang; Song, Xuguang; Zeng, Lingyu; Li, Zhenyu; Li, Yong; Xu, Kailin

    2013-01-01

    A high dose of granulocyte colony stimulating factor (G-CSF) is widely used to mobilize hematopoietic stem and progenitor cells (HSPC), but G-CSF is relatively inefficient and may cause adverse effects. Recently, adrenaline has been found to play important roles in HSPC mobilization. In this study, we explored whether adrenaline combined with G-CSF could induce HSPC mobilization in a mouse model. Mice were treated with adrenaline and either a high or low dose of G-CSF alone or in combination. Peripheral blood HSPC counts were evaluated by flow cytometry. Levels of bone marrow SDF-1 were measured by ELISA, the transcription of CXCR4 and SDF-1 was measured by real-time RT-PCR, and CXCR4 protein was detected by Western blot. Our results showed that adrenaline alone fails to mobilize HSPCs into the peripheral blood; however, when G-CSF and adrenaline are combined, the WBC counts and percentages of HSPCs are significantly higher compared to those in mice that received G-CSF alone. The combined use of adrenaline and G-CSF not only accelerated HSPC mobilization, but also enabled the efficient mobilization of HSPCs into the peripheral blood at lower doses of G-CSF. Adrenaline/G-CSF treatment also extensively downregulated levels of SDF-1 and CXCR4 in mouse bone marrow. These results demonstrated that adrenaline combined with G-CSF can induce HSPC mobilization by down-regulating the CXCR4/SDF-1 axis, indicating that the use of adrenaline may enable the use of reduced dosages or durations of G-CSF treatment, minimizing G-CSF-associated complications.

  20. Transient CDK4/6 inhibition protects hematopoietic stem cells from chemotherapy-induced exhaustion.

    PubMed

    He, Shenghui; Roberts, Patrick J; Sorrentino, Jessica A; Bisi, John E; Storrie-White, Hannah; Tiessen, Renger G; Makhuli, Karenann M; Wargin, William A; Tadema, Henko; van Hoogdalem, Ewoud-Jan; Strum, Jay C; Malik, Rajesh; Sharpless, Norman E

    2017-04-26

    Conventional cytotoxic chemotherapy is highly effective in certain cancers but causes dose-limiting damage to normal proliferating cells, especially hematopoietic stem and progenitor cells (HSPCs). Serial exposure to cytotoxics causes a long-term hematopoietic compromise ("exhaustion"), which limits the use of chemotherapy and success of cancer therapy. We show that the coadministration of G1T28 (trilaciclib), which is a small-molecule inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), contemporaneously with cytotoxic chemotherapy protects murine hematopoietic stem cells (HSCs) from chemotherapy-induced exhaustion in a serial 5-fluorouracil treatment model. Consistent with a cell-intrinsic effect, we show directly preserved HSC function resulting in a more rapid recovery of peripheral blood counts, enhanced serial transplantation capacity, and reduced myeloid skewing. When administered to healthy human volunteers, G1T28 demonstrated excellent in vivo pharmacology and transiently inhibited bone marrow (BM) HSPC proliferation. These findings suggest that the combination of CDK4/6 inhibitors with cytotoxic chemotherapy should provide a means to attenuate therapy-induced BM exhaustion in patients with cancer. Copyright © 2017, American Association for the Advancement of Science.

  1. TIS21/(BTG2) negatively regulates estradiol-stimulated expansion of hematopoietic stem cells by derepressing Akt phosphorylation and inhibiting mTOR signal transduction.

    PubMed

    Kim, Bong Cho; Ryu, Min Sook; Oh, S Paul; Lim, In Kyoung

    2008-09-01

    It has been known that 12-O-tetradecanoyl phorbol-13-acetate-inducible sequence 21 (TIS21), ortholog of human B-cell translocation gene 2, regulates expansions of stage-specific thymocytes and hematopoietic progenitors. In the present study, lineage-negative (Lin(-))/stem cell antigen-1-positive (Sca-1+)/c-Kit+ (LSK) cell content was significantly elevated in bone marrow (BM) of TIS21-knockout (TIS21(-/-)) female mice, suggesting 17beta-estradiol (E(2))-regulated progenitor expansion. E(2) induced DNA synthesis and cell proliferation of mouse embryonic fibroblasts (MEFs) isolated from TIS21(-/-) mice, but not wild type (WT). In contrast to WT, E(2) failed to activate protein kinase B (Akt) in the TIS21(-/-) MEFs, independent of extracellular signal-regulated kinase 1/2 (Erk1/2) activation. Despite attenuation of Akt activation, mammalian target of rapamycin (mTOR) was constitutively activated in the TIS21(-/-) MEFs. Furthermore, mitogen-activated protein kinase 1/2 inhibitor or knockdown of Erk1 could restore activation of Akt and downregulate mTOR. Immunoprecipitation showed Akt preferentially bound to phosphorylated Erk1/2 (p-Erk1/2) in TIS21(-/-) cells, but reconstitution of TIS21 inhibited their interaction. E(2)-injected TIS21(-/-) male mice also increased LSK cells in BM. Taken together, expansion of hematopoietic progenitors in TIS21(-/-) female mice might be through inhibition of Akt activation, and constitutive activation of mTOR via preferential binding of TIS21 to E(2)-induced p-Erk1/2, compared with that of Akt. Our results suggest that TIS21 plays a pivotal role in maintaining the hematopoietic stem cell compartment and hematopoiesis.

  2. Many layers of embryonic hematopoiesis: new insights into B-cell ontogeny and the origin of hematopoietic stem cells.

    PubMed

    Hadland, Brandon; Yoshimoto, Momoko

    2018-04-01

    In adult hematopoiesis, the hematopoietic stem cell (HSC) sits at the top of a hierarchy of hematopoietic progenitors responsible for generating the diverse repertoire of blood and immune cells. During embryonic development, however, the initial waves of hematopoiesis provide the first functioning blood cells of the developing embryo, such as primitive erythrocytes arising in the yolk sac, independently of HSCs. In the field of developmental immunology, it has been recognized that some components of the immune system, such as B-1a lymphocytes, are uniquely produced during the embryonic and neonatal period, suggesting a "layered" development of immunity. Several recent studies have shed new light on the developmental origin of the layered immune system, suggesting complex and sometimes multiple contributions to unique populations of innate-like immune cells from both fetal HSCs and earlier HSC-independent progenitors. In this review, we will attempt to synthesize these studies to provide an integrated model of developmental hematopoiesis and layered immunity that may offer new insights into the origin of HSCs. Copyright © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  3. Reduction of Endoplasmic Reticulum Stress Improves Angiogenic Progenitor Cell function in a Mouse Model of Type 1 Diabetes.

    PubMed

    Bhatta, Maulasri; Chatpar, Krishna; Hu, Zihua; Wang, Joshua J; Zhang, Sarah X

    2018-04-27

    Persistent vascular injury and degeneration in diabetes are attributed in part to defective reparatory function of angiogenic cells. Our recent work implicates endoplasmic reticulum (ER) stress in high-glucose-induced bone marrow (BM) progenitor dysfunction. Herein, we investigated the in vivo role of ER stress in angiogenic abnormalities of streptozotocin-induced diabetic mice. Our data demonstrate that ER stress markers and inflammatory gene expression in BM mononuclear cells and hematopoietic progenitor cells increase dynamically with disease progression. Increased CHOP and cleaved caspase- 3 levels were observed in BM--derived early outgrowth cells (EOCs) after 3 months of diabetes. Inhibition of ER stress by ex vivo or in vivo chemical chaperone treatment significantly improved the generation and migration of diabetic EOCs while reducing apoptosis of these cells. Chemical chaperone treatment also increased the number of circulating angiogenic cells in peripheral blood, alleviated BM pathology, and enhanced retinal vascular repair following ischemia/reperfusion in diabetic mice. Mechanistically, knockdown of CHOP alleviated high-glucose-induced EOC dysfunction and mitigated apoptosis, suggesting a pivotal role of CHOP in mediating ER stress-associated angiogenic cell injury in diabetes. Together, our study suggests that targeting ER signaling may provide a promising and novel approach to enhancing angiogenic function in diabetes.

  4. Histone acetyltransferase activity of MOF is required for adult but not early fetal hematopoiesis in mice

    PubMed Central

    Valerio, Daria G.; Xu, Haiming; Eisold, Meghan E.; Woolthuis, Carolien M.; Pandita, Tej K.

    2017-01-01

    K(lysine) acetyltransferase 8 (KAT8, also known as MOF) mediates the acetylation of histone H4 at lysine 16 (H4K16ac) and is crucial for murine embryogenesis. Lysine acetyltransferases have been shown to regulate various stages of normal hematopoiesis. However, the function of MOF in hematopoietic stem cell (HSC) development has not yet been elucidated. We set out to study the role of MOF in general hematopoiesis by using a Vav1-cre–induced conditional murine Mof knockout system and found that MOF is critical for hematopoietic cell maintenance and HSC engraftment capacity in adult hematopoiesis. Rescue experiments with a MOF histone acetyltransferase domain mutant illustrated the requirement for MOF acetyltransferase activity in the clonogenic capacity of HSCs and progenitors. In stark contrast, fetal steady-state hematopoiesis at embryonic day (E) 14.5 was not affected by homozygous Mof deletion despite dramatic loss of global H4K16ac. Hematopoietic defects start manifesting in late gestation at E17.5. The discovery that MOF and its H4K16ac activity are required for adult but not early and midgestational hematopoiesis supports the notion that multiple chromatin regulators may be crucial for hematopoiesis at varying stages of development. MOF is therefore a developmental-stage–specific chromatin regulator found to be essential for adult but not early fetal hematopoiesis. PMID:27827827

  5. Reactive oxygen species-dependent Toll/NF-κB activation in the Drosophila hematopoietic niche confers resistance to wasp parasitism.

    PubMed

    Louradour, Isabelle; Sharma, Anurag; Morin-Poulard, Ismael; Letourneau, Manon; Vincent, Alain; Crozatier, Michèle; Vanzo, Nathalie

    2017-11-01

    Hematopoietic stem/progenitor cells in the adult mammalian bone marrow ensure blood cell renewal. Their cellular microenvironment, called 'niche', regulates hematopoiesis both under homeostatic and immune stress conditions. In the Drosophila hematopoietic organ, the lymph gland, the posterior signaling center (PSC) acts as a niche to regulate the hematopoietic response to immune stress such as wasp parasitism. This response relies on the differentiation of lamellocytes, a cryptic cell type, dedicated to pathogen encapsulation and killing. Here, we establish that Toll/NF-κB pathway activation in the PSC in response to wasp parasitism non-cell autonomously induces the lymph gland immune response. Our data further establish a regulatory network where co-activation of Toll/NF-κB and EGFR signaling by ROS levels in the PSC/niche controls lymph gland hematopoiesis under parasitism. Whether a similar regulatory network operates in mammals to control emergency hematopoiesis is an open question.

  6. Modulation of occluding junctions alters the hematopoietic niche to trigger immune activation

    PubMed Central

    Khadilkar, Rohan J; Vogl, Wayne; Goodwin, Katharine

    2017-01-01

    Stem cells are regulated by signals from their microenvironment, or niche. During Drosophila hematopoiesis, a niche regulates prohemocytes to control hemocyte production. Immune challenges activate cell-signalling to initiate the cellular and innate immune response. Specifically, certain immune challenges stimulate the niche to produce signals that induce prohemocyte differentiation. However, the mechanisms that promote prohemocyte differentiation subsequent to immune challenges are poorly understood. Here we show that bacterial infection induces the cellular immune response by modulating occluding-junctions at the hematopoietic niche. Occluding-junctions form a permeability barrier that regulates the accessibility of prohemocytes to niche derived signals. The immune response triggered by infection causes barrier breakdown, altering the prohemocyte microenvironment to induce immune cell production. Moreover, genetically induced barrier ablation provides protection against infection by activating the immune response. Our results reveal a novel role for occluding-junctions in regulating niche-hematopoietic progenitor signalling and link this mechanism to immune cell production following infection. PMID:28841136

  7. Prostaglandin E2 increases hematopoietic stem cell survival and accelerates hematopoietic recovery after radiation injury

    PubMed Central

    Porter, Rebecca L.; Georger, Mary; Bromberg, Olga; McGrath, Kathleen E.; Frisch, Benjamin J.; Becker, Michael W.; Calvi, Laura M.

    2013-01-01

    Hematopoietic stem and progenitor cells (HSPCs), which continuously maintain all mature blood cells, are regulated within the marrow microenvironment. We previously reported that pharmacologic treatment of naïve mice with prostaglandin E2 (PGE2) expands HSPCs. However, the cellular mechanisms mediating this expansion remain unknown. Here we demonstrate that PGE2 treatment in naïve mice inhibits apoptosis of HSPCs without changing their proliferation rate. In a murine model of sub-lethal total body irradiation (TBI), in which HSPCs are rapidly lost, treatment with a long-acting PGE2 analogue (dmPGE2) reversed the apoptotic program initiated by TBI. dmPGE2 treatment in vivo decreased the loss of functional HSPCs following radiation injury, as demonstrated both phenotypically and by their increased reconstitution capacity. The antiapoptotic effect of dmPGE2 on HSPCs did not impair their ability to differentiate in vivo, resulting instead in improved hematopoietic recovery after TBI. dmPGE2 also increased microenvironmental cyclooxygenase-2 expression and expanded the α-SMA+ subset of marrow macrophages, thus enhancing the bone marrow microenvironmental response to TBI. Therefore, in vivo treatment with PGE2 analogues may be particularly beneficial to HSPCs in the setting of injury by targeting them both directly and also through their niche. The current data provide rationale for in vivo manipulation of the HSPC pool as a strategy to improve recovery after myelosuppression. PMID:23169593

  8. Hematopoietic Stem Cell Injury Induced by Ionizing Radiation

    PubMed Central

    Shao, Lijian; Luo, Yi

    2014-01-01

    Abstract Significance: Exposure to ionizing radiation (IR) as the result of nuclear accidents or terrorist attacks is a significant threat and a major medical concern. Hematopoietic stem cell (HSC) injury is the primary cause of death after accidental or intentional exposure to a moderate or high dose of IR. Protecting HSCs from IR should be a primary goal in the development of novel medical countermeasures against radiation. Recent Advances: Significant progress has been made in our understanding of the mechanisms by which IR causes HSC damage. The mechanisms include (i) induction of HSC apoptosis via the p53-Puma pathway; (ii) promotion of HSC differentiation via the activation of the G-CSF/Stat3/BATF-dependent differentiation checkpoint; (iii) induction of HSC senescence via the ROS-p38 pathway; and (iv) damage to the HSC niche. Critical Issues: Induction of apoptosis in HSCs and hematopoietic progenitor cells is primarily responsible for IR-induced acute bone marrow (BM) injury. Long-term BM suppression caused by IR is mainly attributable to the induction of HSC senescence. However, the promotion of HSC differentiation and damage to the HSC niche can contribute to both the acute and long-term effects of IR on the hematopoietic system. Future Directions: In this review, we have summarized a number of recent findings that provide new insights into the mechanisms whereby IR damages HSCs. These findings will provide new opportunities for developing a mechanism-based strategy to prevent and/or mitigate IR-induced BM suppression. Antioxid. Redox Signal. 20, 1447–1462. PMID:24124731

  9. Memory T and memory B cells share a transcriptional program of self-renewal with long-term hematopoietic stem cells

    PubMed Central

    Luckey, Chance John; Bhattacharya, Deepta; Goldrath, Ananda W.; Weissman, Irving L.; Benoist, Christophe; Mathis, Diane

    2006-01-01

    The only cells of the hematopoietic system that undergo self-renewal for the lifetime of the organism are long-term hematopoietic stem cells and memory T and B cells. To determine whether there is a shared transcriptional program among these self-renewing populations, we first compared the gene-expression profiles of naïve, effector and memory CD8+ T cells with those of long-term hematopoietic stem cells, short-term hematopoietic stem cells, and lineage-committed progenitors. Transcripts augmented in memory CD8+ T cells relative to naïve and effector T cells were selectively enriched in long-term hematopoietic stem cells and were progressively lost in their short-term and lineage-committed counterparts. Furthermore, transcripts selectively decreased in memory CD8+ T cells were selectively down-regulated in long-term hematopoietic stem cells and progressively increased with differentiation. To confirm that this pattern was a general property of immunologic memory, we turned to independently generated gene expression profiles of memory, naïve, germinal center, and plasma B cells. Once again, memory-enriched and -depleted transcripts were also appropriately augmented and diminished in long-term hematopoietic stem cells, and their expression correlated with progressive loss of self-renewal function. Thus, there appears to be a common signature of both up- and down-regulated transcripts shared between memory T cells, memory B cells, and long-term hematopoietic stem cells. This signature was not consistently enriched in neural or embryonic stem cell populations and, therefore, appears to be restricted to the hematopoeitic system. These observations provide evidence that the shared phenotype of self-renewal in the hematopoietic system is linked at the molecular level. PMID:16492737

  10. Multiplex CRISPR/Cas9-Based Genome Editing in Human Hematopoietic Stem Cells Models Clonal Hematopoiesis and Myeloid Neoplasia.

    PubMed

    Tothova, Zuzana; Krill-Burger, John M; Popova, Katerina D; Landers, Catherine C; Sievers, Quinlan L; Yudovich, David; Belizaire, Roger; Aster, Jon C; Morgan, Elizabeth A; Tsherniak, Aviad; Ebert, Benjamin L

    2017-10-05

    Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord blood CD34 + human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes, including cohesin complex genes, observed in myeloid malignancies generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically defined models of human myeloid diseases, and they are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Inhibition of Aldehyde Dehydrogenase-Activity Expands Multipotent Myeloid Progenitor Cells with Vascular Regenerative Function.

    PubMed

    Cooper, Tyler T; Sherman, Stephen E; Kuljanin, Miljan; Bell, Gillian I; Lajoie, Gilles A; Hess, David A

    2018-05-01

    Blood-derived progenitor cell transplantation holds potential for the treatment of severe vascular diseases. Human umbilical cord blood (UCB)-derived hematopoietic progenitor cells purified using high aldehyde dehydrogenase (ALDH hi ) activity demonstrate pro-angiogenic functions following intramuscular (i.m.) transplantation into immunodeficient mice with hind-limb ischemia. Unfortunately, UCB ALDH hi cells are rare and prolonged ex vivo expansion leads to loss of high ALDH-activity and diminished vascular regenerative function. ALDH-activity generates retinoic acid, a potent driver of hematopoietic differentiation, creating a paradoxical challenge to expand UCB ALDH hi cells while limiting differentiation and retaining pro-angiogenic functions. We investigated whether inhibition of ALDH-activity during ex vivo expansion of UCB ALDH hi cells would prevent differentiation and expand progeny that retained pro-angiogenic functions after transplantation into non-obese diabetic/severe combined immunodeficient mice with femoral artery ligation-induced unilateral hind-limb ischemia. Human UCB ALDH hi cells were cultured under serum-free conditions for 9 days, with or without the reversible ALDH-inhibitor, diethylaminobenzaldehyde (DEAB). Although total cell numbers were increased >70-fold, the frequency of cells that retained ALDH hi /CD34+ phenotype was significantly diminished under basal conditions. In contrast, DEAB-inhibition increased total ALDH hi /CD34+ cell number by ≥10-fold, reduced differentiation marker (CD38) expression, and enhanced the retention of multipotent colony-forming cells in vitro. Proteomic analysis revealed that DEAB-treated cells upregulated anti-apoptotic protein expression and diminished production of proteins implicated with megakaryocyte differentiation. The i.m. transplantation of DEAB-treated cells into mice with hind-limb ischemia stimulated endothelial cell proliferation and augmented recovery of hind-limb perfusion. DEAB

  12. Iron overload may promote alteration of NK cells and hematopoietic stem/progenitor cells by JNK and P38 pathway in myelodysplastic syndromes.

    PubMed

    Hua, Yanni; Wang, Chaomeng; Jiang, Huijuan; Wang, Yihao; Liu, Chunyan; Li, Lijuan; Liu, Hui; Shao, Zonghong; Fu, Rong

    2017-08-01

    The objective of the study was to examine levels of intracellular iron, reactive oxygen species (ROS) and the expression of JNK and p38MAPK in NK cells and hematopoietic stem/progenitor cells (HSPCs) in MDS patients, and explore potential mechanisms by which iron overload (IOL) promotes MDS progression. Thirty-four cases of MDS and six cases of AML transformed from MDS (MDS/AML) were included. HSPCs and NK cells were isolated by magnetic absorption cell sorting. We used flow cytometry to detect the levels of ROS and intracellular JNK and P38 in NK cells and HSPCs. Total RNA and protein were extracted from NK cells and CD34 + cells to examine the expression of JNK and p38MAPK using RT-PCR and Western blotting. Intracellular iron concentration was detected. Data were analyzed by SPSS 21 statistical software. Intracellular iron concentration and ROS were increased in both NK cells and HSPCs in MDS patients with iron overload (P < 0.05). MDS patients with iron overload had higher JNK expression and lower p38 expression in NK cells, and higher p38 expression in HSPCs compared with non-iron overload group. IOL may cause alterations in NK cells and HSPCs through the JNK and p38 pathways, and play a role in the transformation to AML from MDS.

  13. Good manufacturing practice-compliant expansion of marrow-derived stem and progenitor cells for cell therapy.

    PubMed

    Gastens, Martin H; Goltry, Kristin; Prohaska, Wolfgang; Tschöpe, Diethelm; Stratmann, Bernd; Lammers, Dirk; Kirana, Stanley; Götting, Christian; Kleesiek, Knut

    2007-01-01

    Ex vivo expansion is being used to increase the number of stem and progenitor cells for autologous cell therapy. Initiation of pivotal clinical trials testing the efficacy of these cells for tissue repair has been hampered by the challenge of assuring safe and high-quality cell production. A strategy is described here for clinical-scale expansion of bone marrow (BM)-derived stem cells within a mixed cell population in a completely closed process from cell collection through postculture processing using sterile connectable devices. Human BM mononuclear cells (BMMNC) were isolated, cultured for 12 days, and washed postharvest using either standard open procedures in laminar flow hoods or using automated closed systems. Conditions for these studies were similar to long-term BM cultures in which hematopoietic and stromal components are cultured together. Expansion of marrow-derived stem and progenitor cells was then assessed. Cell yield, number of colony forming units (CFU), phenotype, stability, and multilineage differentiation capacity were compared from the single pass perfusion bioreactor and standard flask cultures. Purification of BMMNC using a closed Ficoll gradient process led to depletion of 98% erythrocytes and 87% granulocytes, compared to 100% and 70%, respectively, for manual processing. After closed system culture, mesenchymal progenitors, measured as CD105+CD166+CD14-CD45- and fibroblastic CFU, expanded 317- and 364-fold, respectively, while CD34+ hematopoietic progenitors were depleted 10-fold compared to starting BMMNC. Cultured cells exhibited multilineage differentiation by displaying adipogenic, osteogenic, and endothelial characteristics in vitro. No significant difference was observed between manual and bioreactor cultures. Automated culture and washing of the cell product resulted in 181 x 10(6) total cells that were viable and contained fibroblastic CFU for at least 24 h of storage. A combination of closed, automated technologies enabled

  14. Brief Reports: Nfix Promotes Survival of Immature Hematopoietic Cells via Regulation of c-Mpl.

    PubMed

    Hall, Trent; Walker, Megan; Ganuza, Miguel; Holmfeldt, Per; Bordas, Marie; Kang, Guolian; Bi, Wenjian; Palmer, Lance E; Finkelstein, David; McKinney-Freeman, Shannon

    2018-02-12

    Hematopoietic stem and progenitor cells (HSPCs) are necessary for life-long blood production and replenishment of the hematopoietic system during stress. We recently reported that nuclear factor I/X (Nfix) promotes HSPC survival post-transplant. Here, we report that ectopic expression of Nfix in primary mouse HSPCs extends their ex vivo culture from about 20 to 40 days. HSPCs overexpressing Nfix display hypersensitivity to supportive cytokines and reduced apoptosis when subjected to cytokine deprivation relative to controls. Ectopic Nfix resulted in elevated levels of c-Mpl transcripts and cell surface protein on primary murine HSPCs as well as increased phosphorylation of STAT5, which is known to be activated down-stream of c-MPL. Blocking c-MPL signaling by removal of thrombopoietin or addition of a c-MPL neutralizing antibody negated the antiapoptotic effect of Nfix overexpression on cultured HSPCs. Furthermore, NFIX was capable of binding to and transcriptionally activating a proximal c-Mpl promoter fragment. In sum, these data suggest that NFIX-mediated upregulation of c-Mpl transcription can protect primitive hematopoietic cells from stress ex vivo. Stem Cells 2018. © AlphaMed Press 2018.

  15. Neural Crossroads in the Hematopoietic Stem Cell Niche.

    PubMed

    Agarwala, Sobhika; Tamplin, Owen J

    2018-05-29

    The hematopoietic stem cell (HSC) niche supports steady-state hematopoiesis and responds to changing needs during stress and disease. The nervous system is an important regulator of the niche, and its influence is established early in development when stem cells are specified. Most research has focused on direct innervation of the niche, however recent findings show there are different modes of neural control, including globally by the central nervous system (CNS) and hormone release, locally by neural crest-derived mesenchymal stem cells, and intrinsically by hematopoietic cells that express neural receptors and neurotransmitters. Dysregulation between neural and hematopoietic systems can contribute to disease, however new therapeutic opportunities may be found among neuroregulator drugs repurposed to support hematopoiesis. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. Cytokine-free directed differentiation of human pluripotent stem cells efficiently produces hemogenic endothelium with lymphoid potential.

    PubMed

    Galat, Yekaterina; Dambaeva, Svetlana; Elcheva, Irina; Khanolkar, Aaruni; Beaman, Kenneth; Iannaccone, Philip M; Galat, Vasiliy

    2017-03-17

    The robust generation of human hematopoietic progenitor cells from induced or embryonic pluripotent stem cells would be beneficial for multiple areas of research, including mechanistic studies of hematopoiesis, the development of cellular therapies for autoimmune diseases, induced transplant tolerance, anticancer immunotherapies, disease modeling, and drug/toxicity screening. Over the past years, significant progress has been made in identifying effective protocols for hematopoietic differentiation from pluripotent stem cells and understanding stages of mesodermal, endothelial, and hematopoietic specification. Thus, it has been shown that variations in cytokine and inhibitory molecule treatments in the first few days of hematopoietic differentiation define primitive versus definitive potential of produced hematopoietic progenitor cells. The majority of current feeder-free, defined systems for hematopoietic induction from pluripotent stem cells include prolonged incubations with various cytokines that make the differentiation process complex and time consuming. We established that the application of Wnt agonist CHIR99021 efficiently promotes differentiation of human pluripotent stem cells in the absence of any hematopoietic cytokines to the stage of hemogenic endothelium capable of definitive hematopoiesis. The hemogenic endothelium differentiation was accomplished in an adherent, serum-free culture system by applying CHIR99021. Hemogenic endothelium progenitor cells were isolated on day 5 of differentiation and evaluated for their endothelial, myeloid, and lymphoid potential. Monolayer induction based on GSK3 inhibition, described here, yielded a large number of CD31 + CD34 + hemogenic endothelium cells. When isolated and propagated in adherent conditions, these progenitors gave rise to mature endothelium. When further cocultured with OP9 mouse stromal cells, these progenitors gave rise to various cells of myeloid lineages as well as natural killer lymphoid, T

  17. Hematopoietic stem cells with controllable tEpoR transgenes have a competitive advantage in bone marrow transplantation.

    PubMed

    Kirby, S; Walton, W; Smithies, O

    2000-06-15

    In a previous study, it was found that a truncated erythropoietin receptor transgene (tEpoR tg) enables multilineage hematopoietic progenitor amplification after treatment with erythropoietin (epo) in vitro and in vivo. This study used competitive bone marrow (BM) repopulation to show that tEpoR tg facilitates transplantation by hematopoietic stem cells (HSC). Individual multilineage colonies, committed myeloid progenitor colonies, and lymphoid colonies (pre-B colony-forming units) were grown from the marrow of animals 6 months after they received a 50/50 mixture of transgene and wild-type BM cells. In epo-treated recipients, the transgene-bearing cells significantly outcompeted the wild-type cells (84%-100% versus 16%-0%, respectively). In recipients treated with phosphate-buffered saline, the repopulation was minimally different from the donor mixture (49%-64% transgene versus 51%-36% wild-type). The epo-induced repopulation advantage is maintained in secondary transplants. In addition, neither accelerated HSC depletion nor uncontrollable proliferation occurred during epo-stimulated serial transplants of transgene-containing BM. Thus, the tEpoR tg functions in a benign fashion in HSC and allows for a significant and controllable repopulation advantage in vivo without excessive HSC depletion relative to wild-type BM. (Blood. 2000;95:3710-3715)

  18. Competence of failed endocrine progenitors to give rise to acinar but not ductal cells is restricted to early pancreas development.

    PubMed

    Beucher, Anthony; Martín, Mercè; Spenle, Caroline; Poulet, Martine; Collin, Caitlin; Gradwohl, Gérard

    2012-01-15

    During mouse pancreas development, the transient expression of Neurogenin3 (Neurog3) in uncommitted pancreas progenitors is required to determine endocrine destiny. However it has been reported that Neurog3-expressing cells can eventually adopt acinar or ductal fates and that Neurog3 levels were important to secure the islet destiny. It is not known whether the competence of Neurog3-induced cells to give rise to non-endocrine lineages is an intrinsic property of these progenitors or depends on pancreas developmental stage. Using temporal genetic labeling approaches we examined the dynamic of endocrine progenitor differentiation and explored the plasticity of Neurog3-induced cells throughout development. We found that Neurog3(+) progenitors develop into hormone-expressing cells in a fast process taking less then 10h. Furthermore, fate-mapping studies in heterozygote (Neurog3(CreERT/+)) and Neurog3-deficient (Neurog3(CreERT/CreERT)) embryos revealed that Neurog3-induced cells have different potential over time. At the early bud stage, failed endocrine progenitors can adopt acinar or ductal fate, whereas later in the branching pancreas they do not contribute to the acinar lineage but Neurog3-deficient cells eventually differentiate into duct cells. Thus these results provide evidence that the plasticity of Neurog3-induced cells becomes restricted during development. Furthermore these data suggest that during the secondary transition, endocrine progenitor cells arise from bipotent precursors already committed to the duct/endocrine lineages and not from domain of cells having distinct potentialities. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Murine thymic lymphoma is associated with a species-specific hematopoietic progenitor cell subpopulation

    NASA Technical Reports Server (NTRS)

    Irons, R. D.; Colagiovanni, D. B.; Stillman, W. S.; Clarkson, T. W. (Principal Investigator)

    1996-01-01

    Many strains of laboratory mouse are uniquely susceptible to the development of T cell lymphoma/leukemia, either spontaneously or as a result of chemical or radiation exposure. In contrast, T cell leukemias or lymphomas which are relatively uncommon in human populations, are not easily induced by radiation, and are not generally associated with chemotherapy or chemical exposure. Evidence is presented to suggest that differences in the susceptibility to the development of these malignancies is related to subtle but important variations in the regulation of hematopoietic stem cell differentiation between these two species.

  20. [Stem and progenitor cells in biostructure of blood vessel walls].

    PubMed

    Korta, Krzysztof; Kupczyk, Piotr; Skóra, Jan; Pupka, Artur; Zejler, Paweł; Hołysz, Marcin; Gajda, Mariusz; Nowakowska, Beata; Barć, Piotr; Dorobisz, Andrzej T; Dawiskiba, Tomasz; Szyber, Piotr; Bar, Julia

    2013-09-18

    Development of vascular and hematopoietic systems during organogenesis occurs at the same time. During vasculogenesis, a small part of cells does not undergo complete differentiation but stays on this level, "anchored" in tissue structures described as stem cell niches. The presence of blood vessels within tissue stem cell niches is typical and led to identification of niches and ensures that they are functioning. The three-layer biostructure of vessel walls for artery and vein, tunica: intima, media and adventitia, for a long time was defined as a mechanical barrier between vessel light and the local tissue environment. Recent findings from vascular biology studies indicate that vessel walls are dynamic biostructures, which are equipped with stem and progenitor cells, described as vascular wall-resident stem cells/progenitor cells (VW-SC/PC). Distinct zones for vessel wall harbor heterogeneous subpopulations of VW-SC/PC, which are described as "subendothelial or vasculogenic zones". Recent evidence from in vitro and in vivo studies show that prenatal activity of stem and progenitor cells is not only limited to organogenesis but also exists in postnatal life, where it is responsible for vessel wall homeostasis, remodeling and regeneration. It is believed that VW-SC/PC could be engaged in progression of vascular disorders and development of neointima. We would like to summarize current knowledge about mesenchymal and progenitor stem cell phenotype with special attention to distribution and biological properties of VW-SC/PC in biostructures of intima, media and adventitia niches. It is postulated that in the near future, niches for VW-SC/PC could be a good source of stem and progenitor cells, especially in the context of vessel tissue bioengineering as a new alternative to traditional revascularization therapies.

  1. Busulfan-conditioned bone marrow transplantation results in high-level allogeneic chimerism in mice made tolerant by in utero hematopoietic cell transplantation.

    PubMed

    Ashizuka, Shuichi; Peranteau, William H; Hayashi, Satoshi; Flake, Alan W

    2006-03-01

    In utero hematopoietic cell transplantation (IUHCT) is a non-ablative approach that achieves mixed allogeneic chimerism and donor-specific tolerance. However, clinical application of IUHCT has been limited by minimal engraftment. We have previously demonstrated in the murine model that low-level allogeneic chimerism achieved by IUHCT can be enhanced to near-complete donor chimerism by postnatal minimally myeloablative total body irradiation (TBI) followed by same-donor bone marrow transplantation. Because of concerns of toxicity related to even low-dose TBI in early life, we wondered if a potentially less toxic strategy utilizing a single myelosuppressive agent, Busulfan (BU), would provide similar enhancement of engraftment. In this study, mixed chimerism was created by IUHCT in a fully allogeneic strain combination. After birth, chimeric mice were conditioned with BU followed by transplantation of bone marrow cells congenic to the prenatal donor. We demonstrate that: 1) low-level chimerism after IUHCT can be converted to high-level chimerism by this protocol; 2) enhancement of chimerism is BU dose-dependent; and 3) BU reduces the proliferative potential of hematopoietic progenitor cells thus conferring a competitive advantage to the non-BU-treated postnatal donor cells. This study confirms the potential of IUHCT for facilitation of minimally toxic postnatal regimens to achieve therapeutic levels of allogeneic engraftment.

  2. Coordinated transcriptional regulation of bone homeostasis by Ebf1 and Zfp521 in both mesenchymal and hematopoietic lineages

    PubMed Central

    Kiviranta, Riku; Yamana, Kei; Saito, Hiroaki; Ho, Daniel K.; Laine, Julius; Tarkkonen, Kati; Nieminen-Pihala, Vappu; Hesse, Eric; Correa, Diego; Määttä, Jorma; Tessarollo, Lino; Rosen, Evan D.; Horne, William C.; Jenkins, Nancy A.; Copeland, Neal G.; Warming, Soren

    2013-01-01

    Bone homeostasis is maintained by the coupled actions of hematopoietic bone-resorbing osteoclasts (OCs) and mesenchymal bone-forming osteoblasts (OBs). Here we identify early B cell factor 1 (Ebf1) and the transcriptional coregulator Zfp521 as components of the machinery that regulates bone homeostasis through coordinated effects in both lineages. Deletion of Zfp521 in OBs led to impaired bone formation and increased OB-dependent osteoclastogenesis (OC-genesis), and deletion in hematopoietic cells revealed a strong cell-autonomous role for Zfp521 in OC progenitors. In adult mice, the effects of Zfp521 were largely caused by repression of Ebf1, and the bone phenotype of Zfp521+/− mice was rescued in Zfp521+/−:Ebf1+/− mice. Zfp521 interacted with Ebf1 and repressed its transcriptional activity. Accordingly, deletion of Zfp521 led to increased Ebf1 activity in OBs and OCs. In vivo, Ebf1 overexpression in OBs resulted in suppressed bone formation, similar to the phenotype seen after OB-targeted deletion of Zfp521. Conversely, Ebf1 deletion led to cell-autonomous defects in both OB-dependent and cell-intrinsic OC-genesis, a phenotype opposite to that of the Zfp521 knockout. Thus, we have identified the interplay between Zfp521 and Ebf1 as a novel rheostat for bone homeostasis. PMID:23569325

  3. Dietary restriction improves repopulation but impairs lymphoid differentiation capacity of hematopoietic stem cells in early aging

    PubMed Central

    Tang, Duozhuang; Tao, Si; Chen, Zhiyang; Koliesnik, Ievgen Oleksandrovich; Calmes, Philip Gerald; Hoerr, Verena; Han, Bing; Gebert, Nadja; Zörnig, Martin; Löffler, Bettina

    2016-01-01

    Dietary restriction (DR) improves health, delays tissue aging, and elongates survival in flies and worms. However, studies on laboratory mice and nonhuman primates revealed ambiguous effects of DR on lifespan despite improvements in health parameters. In this study, we analyzed consequences of adult-onset DR (24 h to 1 yr) on hematopoietic stem cell (HSC) function. DR ameliorated HSC aging phenotypes, such as the increase in number of HSCs and the skewing toward myeloid-biased HSCs during aging. Furthermore, DR increased HSC quiescence and improved the maintenance of the repopulation capacity of HSCs during aging. In contrast to these beneficial effects, DR strongly impaired HSC differentiation into lymphoid lineages and particularly inhibited the proliferation of lymphoid progenitors, resulting in decreased production of peripheral B lymphocytes and impaired immune function. The study shows that DR-dependent suppression of growth factors and interleukins mediates these divergent effects caused by DR. Supplementation of insulin-like growth factor 1 partially reverted the DR-induced quiescence of HSCs, whereas IL-6/IL-7 substitutions rescued the impairment of B lymphopoiesis exposed to DR. Together, these findings delineate positive and negative effects of long-term DR on HSC functionality involving distinct stress and growth signaling pathways. PMID:26951333

  4. Uncaria tomentosa stimulates the proliferation of myeloid progenitor cells.

    PubMed

    Farias, Iria; do Carmo Araújo, Maria; Zimmermann, Estevan Sonego; Dalmora, Sergio Luiz; Benedetti, Aloisio Luiz; Alvarez-Silva, Marcio; Asbahr, Ana Carolina Cavazzin; Bertol, Gustavo; Farias, Júlia; Schetinger, Maria Rosa Chitolina

    2011-09-01

    The Asháninkas, indigenous people of Peru, use cat's claw (Uncaria tomentosa) to restore health. Uncaria tomentosa has antioxidant activity and works as an agent to repair DNA damage. It causes different effects on cell proliferation depending on the cell type involved; specifically, it can stimulate the proliferation of myeloid progenitors and cause apoptosis of neoplastic cells. Neutropenia is the most common collateral effect of chemotherapy. For patients undergoing cancer treatment, the administration of a drug that stimulates the proliferation of healthy hematopoietic tissue cells is very desirable. It is important to assess the acute effects of Uncaria tomentosa on granulocyte-macrophage colony-forming cells (CFU-GM) and in the recovery of neutrophils after chemotherapy-induced neutropenia, by establishing the correlation with filgrastim (rhG-CSF) treatment to evaluate its possible use in clinical oncology. The in vivo assay was performed in ifosfamide-treated mice receiving oral doses of 5 and 15 mg of Uncaria tomentosa and intraperitoneal doses of 3 and 9 μg of filgrastim, respectively, for four days. Colony-forming cell (CFC) assays were performed with human hematopoietic stem/precursor cells (hHSPCs) obtained from umbilical cord blood (UCB). Bioassays showed that treatment with Uncaria tomentosa significantly increased the neutrophil count, and a potency of 85.2% was calculated in relation to filgrastim at the corresponding doses tested. An in vitro CFC assay showed an increase in CFU-GM size and mixed colonies (CFU-GEMM) size at the final concentrations of 100 and 200 μg extract/mL. At the tested doses, Uncaria tomentosa had a positive effect on myeloid progenitor number and is promising for use with chemotherapy to minimize the adverse effects of this treatment. These results support the belief of the Asháninkas, who have classified Uncaria tomentosa as a 'powerful plant'. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  5. Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells.

    PubMed

    Macaulay, Iain C; Svensson, Valentine; Labalette, Charlotte; Ferreira, Lauren; Hamey, Fiona; Voet, Thierry; Teichmann, Sarah A; Cvejic, Ana

    2016-02-02

    The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Exercise training improves in vivo endothelial repair capacity of early endothelial progenitor cells in subjects with metabolic syndrome.

    PubMed

    Sonnenschein, Kristina; Horváth, Tibor; Mueller, Maja; Markowski, Andrea; Siegmund, Tina; Jacob, Christian; Drexler, Helmut; Landmesser, Ulf

    2011-06-01

    Endothelial dysfunction and injury are considered to contribute considerably to the development and progression of atherosclerosis. It has been suggested that intense exercise training can increase the number and angiogenic properties of early endothelial progenitor cells (EPCs). However, whether exercise training stimulates the capacity of early EPCs to promote repair of endothelial damage and potential underlying mechanisms remain to be determined. The present study was designed to evaluate the effects of moderate exercise training on in vivo endothelial repair capacity of early EPCs, and their nitric oxide and superoxide production as characterized by electron spin resonance spectroscopy analysis in subjects with metabolic syndrome. Twenty-four subjects with metabolic syndrome were randomized to an 8 weeks exercise training or a control group. Superoxide production and nitric oxide (NO) availability of early EPCs were characterized by using electron spin resonance (ESR) spectroscopy analysis. In vivo endothelial repair capacity of EPCs was examined by transplantation into nude mice with defined carotid endothelial injury. Endothelium-dependent, flow-mediated vasodilation was analysed using high-resolution ultrasound. Importantly, exercise training resulted in a substantially improved in vivo endothelial repair capacity of early EPCs (24.0 vs 12.7%; p < 0.05) and improved endothelium-dependent vasodilation. Nitric oxide production of EPCs was substantially increased after exercise training, but not in the control group. Moreover, exercise training reduced superoxide production of EPCs, which was not observed in the control group. The present study suggests for the first time that moderate exercise training increases nitric oxide production of early endothelial progenitor cells and reduces their superoxide production. Importantly, this is associated with a marked beneficial effect on the in vivo endothelial repair capacity of early EPCs in subjects with

  7. Generating autologous hematopoietic cells from human-induced pluripotent stem cells through ectopic expression of transcription factors.

    PubMed

    Hwang, Yongsung; Broxmeyer, Hal E; Lee, Man Ryul

    2017-07-01

    Hematopoietic cell transplantation (HCT) is a successful treatment modality for patients with malignant and nonmalignant disorders, usually when no other treatment option is available. The cells supporting long-term reconstitution after HCT are the hematopoietic stem cells (HSCs), which can be limited in numbers. Moreover, finding an appropriate human leukocyte antigen-matched donor can be problematic. If HSCs can be stably produced in large numbers from autologous or allogeneic cell sources, it would benefit HCT. Induced pluripotent stem cells (iPSCs) established from patients' own somatic cells can be differentiated into hematopoietic cells in vitro. This review will highlight recent methods for regulating human (h) iPSC production of HSCs and more mature blood cells. Advancements in transcription factor-mediated regulation of the developmental stages of in-vivo hematopoietic lineage commitment have begun to provide an understanding of the molecular mechanism of hematopoiesis. Such studies involve not only directed differentiation in which transcription factors, specifically expressed in hematopoietic lineage-specific cells, are overexpressed in iPSCs, but also direct conversion in which transcription factors are introduced into patient-derived somatic cells which are dedifferentiated to hematopoietic cells. As iPSCs derived from patients suffering from genetically mutated diseases would express the same mutated genetic information, CRISPR-Cas9 gene editing has been utilized to differentiate genetically corrected iPSCs into normal hematopoietic cells. IPSCs provide a model for molecular understanding of disease, and also may function as a cell population for therapy. Efficient differentiation of patient-specific iPSCs into HSCs and progenitor cells is a potential means to overcome limitations of such cells for HCT, as well as for providing in-vitro drug screening templates as tissue-on-a-chip models.

  8. Alpha-tocopherol succinate- and AMD3100-mobilized progenitors mitigate radiation combined injury in mice.

    PubMed

    Singh, Vijay K; Wise, Stephen Y; Fatanmi, Oluseyi O; Beattie, Lindsay A; Ducey, Elizabeth J; Seed, Thomas M

    2014-01-01

    The purpose of this study was to elucidate the role of alpha-tocopherol succinate (TS)- and AMD3100-mobilized progenitors in mitigating combined injury associated with acute radiation exposure in combination with secondary physical wounding. CD2F1 mice were exposed to high doses of cobalt-60 gamma-radiation and then transfused intravenously with 5 million peripheral blood mononuclear cells (PBMCs) from TS- and AMD3100-injected mice after irradiation. Within 1 h after irradiation, mice were exposed to secondary wounding. Mice were observed for 30 d after irradiation and cytokine analysis was conducted by multiplex Luminex assay at various time-points after irradiation and wounding. Our results initially demonstrated that transfusion of TS-mobilized progenitors from normal mice enhanced survival of acutely irradiated mice exposed 24 h prior to transfusion to supralethal doses (11.5-12.5 Gy) of (60)Co gamma-radiation. Subsequently, comparable transfusions of TS-mobilized progenitors were shown to significantly mitigate severe combined injuries in acutely irradiated mice. TS administered 24 h before irradiation was able to protect mice against combined injury as well. Cytokine results demonstrated that wounding modulates irradiation-induced cytokines. This study further supports the conclusion that the infusion of TS-mobilized progenitor-containing PBMCs acts as a bridging therapy in radiation-combined-injury mice. We suggest that this novel bridging therapeutic approach involving the infusion of TS-mobilized hematopoietic progenitors following acute radiation exposure or combined injury might be applicable to humans.

  9. Improved hematopoietic differentiation efficiency of gene-corrected beta-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system.

    PubMed

    Song, Bing; Fan, Yong; He, Wenyin; Zhu, Detu; Niu, Xiaohua; Wang, Ding; Ou, Zhanhui; Luo, Min; Sun, Xiaofang

    2015-05-01

    The generation of beta-thalassemia (β-Thal) patient-specific induced pluripotent stem cells (iPSCs), subsequent homologous recombination-based gene correction of disease-causing mutations/deletions in the β-globin gene (HBB), and their derived hematopoietic stem cell (HSC) transplantation offers an ideal therapeutic solution for treating this disease. However, the hematopoietic differentiation efficiency of gene-corrected β-Thal iPSCs has not been well evaluated in the previous studies. In this study, we used the latest gene-editing tool, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), to correct β-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and full pluripotency as human embryonic stem cells (hESCs) showed no off-targeting effects. Then, we evaluated the differentiation efficiency of the gene-corrected β-Thal iPSCs. We found that during hematopoietic differentiation, gene-corrected β-Thal iPSCs showed an increased embryoid body ratio and various hematopoietic progenitor cell percentages. More importantly, the gene-corrected β-Thal iPSC lines restored HBB expression and reduced reactive oxygen species production compared with the uncorrected group. Our study suggested that hematopoietic differentiation efficiency of β-Thal iPSCs was greatly improved once corrected by the CRISPR/Cas9 system, and the information gained from our study would greatly promote the clinical application of β-Thal iPSC-derived HSCs in transplantation.

  10. Heparan sulfates and the decrease of N-glycans promote early adipogenic differentiation rather than myogenesis of murine myogenic progenitor cells.

    PubMed

    Grassot, Vincent; Bouchatal, Amel; Da Silva, Anne; Chantepie, Sandrine; Papy-Garcia, Dulce; Maftah, Abderrahman; Gallet, Paul-François; Petit, Jean-Michel

    In vitro, extracted muscle satellite cells, called myogenic progenitor cells, can differentiate either in myotubes or preadipocytes, depending on environmental factors and the medium. Transcriptomic analyses on glycosylation genes during satellite cells differentiation into myotubes showed that 31 genes present a significant variation of expression at the early stages of murine myogenic progenitor cells (MPC) differentiation. In the present study, we analyzed the expression of 383 glycosylation related genes during murine MPC differentiation into preadipocytes and compared the data to those previously obtained during their differentiation into myotubes. Fifty-six glycosylation related genes are specifically modified in their expression during early adipogenesis. The variations correspond mainly to: a decrease of N-glycans, and of alpha (2,3) and (2,6) linked sialic acids, and to a high level of heparan sulfates. A high amount of TGF-β1 in extracellular media during early adipogenesis was also observed. It seems that the increases of heparan sulfates and TGF-β1 favor pre-adipogenic differentition of MPC and possibly prevent their myogenic differentiation. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  11. Heterogeneity of Clonal Expansion and Maturation-Linked Mutation Acquisition in Hematopoietic Progenitors in Human Acute Myeloid Leukemia

    PubMed Central

    Walter, Roland B.; Laszlo, George S.; Lionberger, Jack M.; Pollard, Jessica A.; Harrington, Kimberly H.; Gudgeon, Chelsea J.; Othus, Megan; Rafii, Shahin; Meshinchi, Soheil; Appelbaum, Frederick R.; Bernstein, Irwin D.

    2014-01-01

    Recent technological advances led to an appreciation of the genetic complexity of human acute myeloid leukemia (AML) but underlying progenitor cells remain poorly understood because their rarity precludes direct study. We developed a co-culture method integrating hypoxia, aryl hydrocarbon receptor inhibition, and micro-environmental support via human endothelial cells to isolate these cells. X-chromosome inactivation studies of the least mature precursors derived following prolonged culture of CD34+/CD33− cells revealed polyclonal growth in highly curable AMLs, suggesting mutations necessary for clonal expansion were acquired in more mature progenitors. Consistently, in core-binding factor (CBF) leukemias with known complementing mutations, immature precursors derived following prolonged culture of CD34+/CD33− cells harbored neither mutation or the CBF mutation alone, whereas more mature precursors often carried both mutations. These results were in contrast to those with leukemias with poor prognosis that showed clonal dominance in the least mature precursors. These data indicate heterogeneity among progenitors in human AML that may have prognostic and therapeutic implications. PMID:24721792

  12. Promoting effects of serotonin on hematopoiesis: ex vivo expansion of cord blood CD34+ stem/progenitor cells, proliferation of bone marrow stromal cells, and antiapoptosis.

    PubMed

    Yang, Mo; Li, Karen; Ng, Pak Cheung; Chuen, Carmen Ka Yee; Lau, Tze Kin; Cheng, Yuan Shan; Liu, Yuan Sheng; Li, Chi Kong; Yuen, Patrick Man Pan; James, Anthony Edward; Lee, Shuk Man; Fok, Tai Fai

    2007-07-01

    Serotonin is a monoamine neurotransmitter that has multiple extraneuronal functions. We previously reported that serotonin exerted mitogenic stimulation on megakaryocytopoiesis mediated by 5-hydroxytryptamine (5-HT)2 receptors. In this study, we investigated effects of serotonin on ex vivo expansion of human cord blood CD34+ cells, bone marrow (BM) stromal cell colony-forming unit-fibroblast (CFU-F) formation, and antiapoptosis of megakaryoblastic M-07e cells. Our results showed that serotonin at 200 nM significantly enhanced the expansion of CD34+ cells to early stem/progenitors (CD34+ cells, colony-forming unit-mixed [CFU-GEMM]) and multilineage committed progenitors (burst-forming unit/colony-forming unit-erythroid [BFU/CFU-E], colony-forming unit-granulocyte macrophage, colony-forming unit-megakaryocyte, CD61+ CD41+ cells). Serotonin also increased nonobese diabetic/severe combined immunodeficient repopulating cells in the expansion culture in terms of human CD45+, CD33+, CD14+ cells, BFU/CFU-E, and CFU-GEMM engraftment in BM of animals 6 weeks post-transplantation. Serotonin alone or in addition to fibroblast growth factor, platelet-derived growth factor, or vascular endothelial growth factor stimulated BM CFU-F formation. In M-07e cells, serotonin exerted antiapoptotic effects (annexin V, caspase-3, and propidium iodide staining) and reduced mitochondria membrane potential damage. The addition of ketanserin, a competitive antagonist of 5-HT2 receptor, nullified the antiapoptotic effects of serotonin. Our data suggest the involvement of serotonin in promoting hematopoietic stem cells and the BM microenvironment. Serotonin could be developed for clinical ex vivo expansion of hematopoietic stem cells for transplantation. Disclosure of potential conflicts of interest is found at the end of this article.

  13. Developmental regulation of myeloerythroid progenitor function by the Lin28b–let-7–Hmga2 axis

    PubMed Central

    Rowe, R. Grant; Wang, Leo D.; Coma, Silvia; Pearson, Daniel S.; Nguyen, Phi T.; Wagers, Amy J.

    2016-01-01

    For appropriate development, tissue and organ system morphogenesis and maturation must occur in synchrony with the overall developmental requirements of the host. Mistiming of such developmental events often results in disease. The hematopoietic system matures from the fetal state, characterized by robust erythrocytic output that supports prenatal growth in the hypoxic intrauterine environment, to the postnatal state wherein granulocytes predominate to provide innate immunity. Regulation of the developmental timing of these myeloerythroid states is not well understood. In this study, we find that expression of the heterochronic factor Lin28b decreases in common myeloid progenitors during hematopoietic maturation to adulthood in mice. This decrease in Lin28b coincides with accumulation of mature let-7 microRNAs, whose biogenesis is regulated by Lin28 proteins. We find that inhibition of let-7 in the adult hematopoietic system recapitulates fetal erythroid-dominant hematopoiesis. Conversely, deletion of Lin28b or ectopic activation of let-7 microRNAs in the fetal state induces a shift toward adult-like myeloid-dominant output. Furthermore, we identify Hmga2 as an effector of this genetic switch. These studies provide the first detailed analysis of the roles of endogenous Lin28b and let-7 in the timing of hematopoietic states during development. PMID:27401346

  14. Histone acetyltransferase activity of MOF is required for adult but not early fetal hematopoiesis in mice.

    PubMed

    Valerio, Daria G; Xu, Haiming; Eisold, Meghan E; Woolthuis, Carolien M; Pandita, Tej K; Armstrong, Scott A

    2017-01-05

    K(lysine) acetyltransferase 8 (KAT8, also known as MOF) mediates the acetylation of histone H4 at lysine 16 (H4K16ac) and is crucial for murine embryogenesis. Lysine acetyltransferases have been shown to regulate various stages of normal hematopoiesis. However, the function of MOF in hematopoietic stem cell (HSC) development has not yet been elucidated. We set out to study the role of MOF in general hematopoiesis by using a Vav1-cre-induced conditional murine Mof knockout system and found that MOF is critical for hematopoietic cell maintenance and HSC engraftment capacity in adult hematopoiesis. Rescue experiments with a MOF histone acetyltransferase domain mutant illustrated the requirement for MOF acetyltransferase activity in the clonogenic capacity of HSCs and progenitors. In stark contrast, fetal steady-state hematopoiesis at embryonic day (E) 14.5 was not affected by homozygous Mof deletion despite dramatic loss of global H4K16ac. Hematopoietic defects start manifesting in late gestation at E17.5. The discovery that MOF and its H4K16ac activity are required for adult but not early and midgestational hematopoiesis supports the notion that multiple chromatin regulators may be crucial for hematopoiesis at varying stages of development. MOF is therefore a developmental-stage-specific chromatin regulator found to be essential for adult but not early fetal hematopoiesis. © 2017 by The American Society of Hematology.

  15. Identification of Multipotent Stem/Progenitor Cells in Murine Sclera

    PubMed Central

    Tsai, Chia-Ling; Wu, Pei-Chang; Fini, M. Elizabeth; Shi, Songtao

    2011-01-01

    Purpose. The sclera forms the fibrous outer coat of the eyeball and acts as a supportive framework. The purpose of this study was to examine whether the sclera contains mesenchymal stem/progenitor cells. Method. Scleral tissue from C57BL6/J mice was separated from the retina and choroid and subsequently enzyme digested to release single cells. Proliferation capacity, self-renewal capacity, and ability for multipotent differentiation were analyzed by BrdU labeling, flow cytometry, reverse transcriptase–polymerase chain reaction, immunocytochemistry, and in vivo transplantation. Results. The scleral stem/progenitor cells (SSPCs) possessed clonogenic and high doubling capacities. These cells were positive for the mesenchymal markers Sca-1, CD90.2, CD44, CD105, and CD73 and negative for the hematopoietic markers CD45, CD11b, Flk1, CD34, and CD117. In addition to expressing stem cell genes ABCG2, Six2, Notch1, and Pax6, SSPCs were able to differentiate to adipogenic, chondrogenic, and neurogenic lineages. Conclusions. This study indicates that the sclera contains multipotent mesenchymal stem cells. Further study of SSPCs may help elucidate the cellular and molecular mechanism of scleral diseases such as scleritis and myopia. PMID:21788434

  16. Combined mitigation of the gastrointestinal and hematopoietic acute radiation syndromes by an LPA2 receptor-specific nonlipid agonist.

    PubMed

    Patil, Renukadevi; Szabó, Erzsébet; Fells, James I; Balogh, Andrea; Lim, Keng G; Fujiwara, Yuko; Norman, Derek D; Lee, Sue-Chin; Balazs, Louisa; Thomas, Fridtjof; Patil, Shivaputra; Emmons-Thompson, Karin; Boler, Alyssa; Strobos, Jur; McCool, Shannon W; Yates, C Ryan; Stabenow, Jennifer; Byrne, Gerrald I; Miller, Duane D; Tigyi, Gábor J

    2015-02-19

    Pharmacological mitigation of injuries caused by high-dose ionizing radiation is an unsolved medical problem. A specific nonlipid agonist of the type 2 G protein coupled receptor for lysophosphatidic acid (LPA2) 2-[4-(1,3-dioxo-1H,3H-benzoisoquinolin-2-yl)butylsulfamoyl]benzoic acid (DBIBB) when administered with a postirradiation delay of up to 72 hr reduced mortality of C57BL/6 mice but not LPA2 knockout mice. DBIBB mitigated the gastrointestinal radiation syndrome, increased intestinal crypt survival and enterocyte proliferation, and reduced apoptosis. DBIBB enhanced DNA repair by augmenting the resolution of γ-H2AX foci, increased clonogenic survival of irradiated IEC-6 cells, attenuated the radiation-induced death of human CD34(+) hematopoietic progenitors and enhanced the survival of the granulocyte/macrophage lineage. DBIBB also increased the survival of mice suffering from the hematopoietic acute radiation syndrome after total-body irradiation. DBIBB represents a drug candidate capable of mitigating acute radiation syndrome caused by high-dose γ-radiation to the hematopoietic and gastrointestinal system. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Distinct Roles for Matrix Metalloproteinases 2 and 9 in Embryonic Hematopoietic Stem Cell Emergence, Migration, and Niche Colonization.

    PubMed

    Theodore, Lindsay N; Hagedorn, Elliott J; Cortes, Mauricio; Natsuhara, Kelsey; Liu, Sarah Y; Perlin, Julie R; Yang, Song; Daily, Madeleine L; Zon, Leonard I; North, Trista E

    2017-05-09

    Hematopoietic stem/progenitor cells (HSPCs) are formed during ontogeny from hemogenic endothelium in the ventral wall of the dorsal aorta (VDA). Critically, the cellular mechanism(s) allowing HSPC egress and migration to secondary niches are incompletely understood. Matrix metalloproteinases (MMPs) are inflammation-responsive proteins that regulate extracellular matrix (ECM) remodeling, cellular interactions, and signaling. Here, inhibition of vascular-associated Mmp2 function caused accumulation of fibronectin-rich ECM, retention of runx1/cmyb + HSPCs in the VDA, and delayed caudal hematopoietic tissue (CHT) colonization; these defects were absent in fibronectin mutants, indicating that Mmp2 facilitates endothelial-to-hematopoietic transition via ECM remodeling. In contrast, Mmp9 was dispensable for HSPC budding, being instead required for proper colonization of secondary niches. Significantly, these migration defects were mimicked by overexpression and blocked by knockdown of C-X-C motif chemokine-12 (cxcl12), suggesting that Mmp9 controls CHT homeostasis through chemokine regulation. Our findings indicate Mmp2 and Mmp9 play distinct but complementary roles in developmental HSPC production and migration. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Early molecular events during retinoic acid induced differentiation of neuromesodermal progenitors

    PubMed Central

    Cunningham, Thomas J.; Colas, Alexandre

    2016-01-01

    ABSTRACT Bipotent neuromesodermal progenitors (NMPs) residing in the caudal epiblast drive coordinated body axis extension by generating both posterior neuroectoderm and presomitic mesoderm. Retinoic acid (RA) is required for body axis extension, however the early molecular response to RA signaling is poorly defined, as is its relationship to NMP biology. As endogenous RA is first seen near the time when NMPs appear, we used WNT/FGF agonists to differentiate embryonic stem cells to NMPs which were then treated with a short 2-h pulse of 25 nM RA or 1 µM RA followed by RNA-seq transcriptome analysis. Differential expression analysis of this dataset indicated that treatment with 25 nM RA, but not 1 µM RA, provided physiologically relevant findings. The 25 nM RA dataset yielded a cohort of previously known caudal RA target genes including Fgf8 (repressed) and Sox2 (activated), plus novel early RA signaling targets with nearby conserved RA response elements. Importantly, validation of top-ranked genes in vivo using RA-deficient Raldh2−/− embryos identified novel examples of RA activation (Nkx1-2, Zfp503, Zfp703, Gbx2, Fgf15, Nt5e) or RA repression (Id1) of genes expressed in the NMP niche or progeny. These findings provide evidence for early instructive and permissive roles of RA in controlling differentiation of NMPs to neural and mesodermal lineages. PMID:27793834

  19. Epigenetic Loss of MLH1 Expression in Normal Human Hematopoietic Stem Cell Clones is Defined by the Promoter CpG Methylation Pattern Observed by High-Throughput Methylation Specific Sequencing

    PubMed Central

    Kenyon, Jonathan; Nickel-Meester, Gabrielle; Qing, Yulan; Santos-Guasch, Gabriela; Drake, Ellen; PingfuFu; Sun, Shuying; Bai, Xiaodong; Wald, David; Arts, Eric; Gerson, Stanton L.

    2016-01-01

    Normal human hematopoietic stem and progenitor cells (HPC) lose expression of MLH1, an important mismatch repair (MMR) pathway gene, with age. Loss of MMR leads to replication dependent mutational events and microsatellite instability observed in secondary acute myelogenous leukemia and other hematologic malignancies. Epigenetic CpG methylation upstream of the MLH1 promoter is a contributing factor to acquired loss of MLH1 expression in tumors of the epithelia and proximal mucosa. Using single molecule high-throughput bisulfite sequencing we have characterized the CpG methylation landscape from −938 to −337 bp upstream of the MLH1 transcriptional start site (position +0), from 30 hematopoietic colony forming cell clones (CFC) either expressing or not expressing MLH1. We identify a correlation between MLH1 promoter methylation and loss of MLH1 expression. Additionally, using the CpG site methylation frequencies obtained in this study we were able to generate a classification algorithm capable of sorting the expressing and non-expressing CFC. Thus, as has been previously described for many tumor cell types, we report for the first time a correlation between the loss of MLH1 expression and increased MLH1 promoter methylation in CFC derived from CD34+ selected hematopoietic stem and progenitor cells. PMID:27570841

  20. Epigenetic Loss of MLH1 Expression in Normal Human Hematopoietic Stem Cell Clones is Defined by the Promoter CpG Methylation Pattern Observed by High-Throughput Methylation Specific Sequencing.

    PubMed

    Kenyon, Jonathan; Nickel-Meester, Gabrielle; Qing, Yulan; Santos-Guasch, Gabriela; Drake, Ellen; PingfuFu; Sun, Shuying; Bai, Xiaodong; Wald, David; Arts, Eric; Gerson, Stanton L

    Normal human hematopoietic stem and progenitor cells (HPC) lose expression of MLH1 , an important mismatch repair (MMR) pathway gene, with age. Loss of MMR leads to replication dependent mutational events and microsatellite instability observed in secondary acute myelogenous leukemia and other hematologic malignancies. Epigenetic CpG methylation upstream of the MLH1 promoter is a contributing factor to acquired loss of MLH1 expression in tumors of the epithelia and proximal mucosa. Using single molecule high-throughput bisulfite sequencing we have characterized the CpG methylation landscape from -938 to -337 bp upstream of the MLH1 transcriptional start site (position +0), from 30 hematopoietic colony forming cell clones (CFC) either expressing or not expressing MLH1 . We identify a correlation between MLH1 promoter methylation and loss of MLH1 expression. Additionally, using the CpG site methylation frequencies obtained in this study we were able to generate a classification algorithm capable of sorting the expressing and non-expressing CFC. Thus, as has been previously described for many tumor cell types, we report for the first time a correlation between the loss of MLH1 expression and increased MLH1 promoter methylation in CFC derived from CD34 + selected hematopoietic stem and progenitor cells.

  1. Bone marrow hematopoietic stem cells behavior with or without growth factors in trauma hemorrhagic shock

    PubMed Central

    Kumar, Manoj; Bhoi, Sanjeev; Mohanty, Sujata; Kamal, Vineet Kumar; Rao, D. N.; Mishra, Pravas; Galwankar, Sagar

    2016-01-01

    Background: Hemorrhagic shock (HS) is the major leading cause of death after trauma. Up to 50% of early deaths are due to massive hemorrhage. Excessive release of pro-inflammatory cytokine and hypercatecholamine induces hematopoietic progenitor cells (HPCs) apoptosis, leading to multiorgan failure and death. However, still, result remains elusive for hematopoietic stem cells (HSCs) behavior in trauma HS (T/HS). Objectives: Therefore, our aim was to evaluate the in vitro HSCs behavior with or without recombinant human erythropoietin (rhEPO), recombinant human granulocyte macrophage-colony-stimulating factor (rhGM-CSF), recombinant human interleukin-3 (rhIL-3) alone, and combination with rhEPO + rhGM-CSF + rhIL-3 (EG3) in T/HS patients. Methodology: Bone marrow (BM) aspirates (n = 14) were collected from T/HS patients, those survived on day 3. BM cells were cultured for HPCs: Colony-forming unit-erythroid (CFU-E), burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte, monocyte/macrophage colonies growth. HPCs were counted with or without rhEPO, rhGM-CSF, rhIL-3 alone, and combination with EG3 in T/HS patients. Results: BM HSCs growth significantly suppressed in T/HS when compared with control group (P < 0.05). In addition, CFU-E and BFU-E colony growth were increased with additional growth factor (AGF) (rhEPO, rhGM-CSF, and rhIL-3) as compared to baseline (without AGF) (P < 0.05). Conclusion: Suppressed HPCs may be reactivated by addition of erythropoietin, GM-CSF, IL-3 alone and with combination in T/HS. PMID:27722113

  2. Decursin inhibits vasculogenesis in early tumor progression by suppression of endothelial progenitor cell differentiation and function.

    PubMed

    Jung, Seok Yun; Choi, Jin Hwa; Kwon, Sang-Mo; Masuda, Haruchika; Asahara, Takayuki; Lee, You-Mie

    2012-05-01

    Endothelial progenitor cells (EPCs) contribute to the tumor vasculature during tumor progression. Decursin isolated from the herb Angelica gigas is known to possess potent anti-inflammatory activities. Recently, we reported that decursin is a novel candidate for an angiogenesis inhibitor [Jung et al., 2009]. In this study, we investigated whether decursin regulates EPC differentiation and function to inhibit tumor vasculogenesis. We isolated AC133+ cells from human cord blood and decursin significantly decreased the number of EPC colony forming units of human cord blood-derived AC133+ cells that produce functional EPC progenies. Decursin dose-dependently decreased the cell number of EPC committing cells as demonstrated by EPC expansion studies. Decursin inhibited EPC differentiation from progenitor cells into spindle-shaped EPC colonies. Additionally, decursin inhibited proliferation and migration of early EPCs isolated from mouse bone marrow. Furthermore, decursin suppressed expression of angiopoietin-2, angiopoietin receptor Tie-2, Flk-1 (vascular endothelial growth factor receptor-2), and endothelial nitric oxide synthase in mouse BM derived EPCs in a dose-dependent manner. Decursin suppressed tube formation ability of EPCs in collaboration with HUVEC. Decursin (4 mg/kg) inhibited tumor-induced mobilization of circulating EPCs (CD34 + /VEGFR-2+ cells) from bone marrow and early incorporation of Dil-Ac-LDL-labeled or green fluorescent protein (GFP)+ EPCs into neovessels of xenograft Lewis lung carcinoma tumors in wild-type- or bone-marrow-transplanted mice. Accordingly, decursin attenuated EPC-derived endothelial cells in neovessels of Lewis lung carcinoma tumor masses grown in mice. Together, decursin likely affects EPC differentiation and function, thereby inhibiting tumor vasculogenesis in early tumorigenesis. Copyright © 2012 Wiley Periodicals, Inc.

  3. Mouse Bone Marrow VSELs Exhibit Differentiation into Three Embryonic Germ Lineages and Germ & Hematopoietic Cells in Culture.

    PubMed

    Shaikh, Ambreen; Anand, Sandhya; Kapoor, Sona; Ganguly, Ranita; Bhartiya, Deepa

    2017-04-01

    Very small embryonic-like stem cells (VSELs) have been reported in various adult tissues, express pluripotent and primordial germ cells (PGCs) specific markers, are mobilized under stress/disease conditions, give rise to tissue committed progenitors and thus help regenerate and maintain homeostasis. The aim of the present study was to evaluate in vitro differentiation potential of VSELs using a quantitative approach. VSELs were collected from mouse bone marrow after 4 days of 5-fluorouracil (5-FU, 150 mg/Kg) treatment, further enriched by size based filtration and cultured on a feeder support in the presence of specific differentiation media. Cultured VSELs were found to differentiate into all three embryonic germ cell lineages, germ and hematopoietic cells after 14 days in culture. This was confirmed by studying Nestin, PDX-1, NKX2.5, DAZL, CD45 and other markers expression by various approaches. Very small, CD45 negative cells collected and enriched from GFP positive 5-FU treated mice bone marrow transitioned into CD45 positive cells in vitro thus demonstrating that VSELs can give rise to hematopoietic stem cells (HSCs). We envision that VSELs may be responsible for plasticity and ability of bone marrow cells to give rise to non-hematopoietic tissue progenitors of all 3 germ layers. Moreover the ability of VSELs to differentiate into germ cells as well as all the three lineages provides further evidence to support their pluripotent state and confirms developmental link between bone marrow VSELs and PGCs. The property of quiescence, no risk of teratoma formation and autologus source, make pluripotent VSELs a potential candidate to facilitate endogenous regeneration compared to cell replacement strategy envisioned using embryonic and induced pluripotent stem cells.

  4. Functional interleukin-33 receptors are expressed in early progenitor stages of allergy-related granulocytes.

    PubMed

    Tsuzuki, Hirofumi; Arinobu, Yojiro; Miyawaki, Kohta; Takaki, Ayako; Ota, Shun-Ichiro; Ota, Yuri; Mitoma, Hiroki; Akahoshi, Mitsuteru; Mori, Yasuo; Iwasaki, Hiromi; Niiro, Hiroaki; Tsukamoto, Hiroshi; Akashi, Koichi

    2017-01-01

    Interleukin-33 (IL-33) induces T helper type 2 (Th2) cytokine production and eosinophilia independently of acquired immunity, leading to innate immunity-mediated allergic inflammation. Allergy-related innate myeloid cells such as eosinophils, basophils and mast cells express the IL-33 receptor (IL-33R), but it is still unknown how IL-33 regulates allergic inflammation involving these cells and their progenitors. Here, we revealed that the functional IL-33R was expressed on eosinophil progenitors (EoPs), basophil progenitors (BaPs) and mast cell progenitors (MCPs). In the presence of IL-33, these progenitors did not expand, but produced a high amount of Th2 and pro-inflammatory cytokines such as IL-9, IL-13, IL-1β and IL-6. The amount of cytokines produced by these progenitors was greater than that by mature cells. In vivo, IL-33 stimulated the expansion of EoPs, but it was dependent upon the elevated serum IL-5 that is presumably derived from type 2 innate lymphoid cells that express functional IL-33R. These data collectively suggest that EoPs, BaPs and MCPs are not only the sources of allergy-related granulocytes, but can also be sources of allergy-related cytokines in IL-33-induced inflammation. Because such progenitors can differentiate into mature granulocytes at the site of inflammation, they are potential therapeutic targets in IL-33-related allergic diseases. © 2016 John Wiley & Sons Ltd.

  5. Symmetry of initial cell divisions among primitive hematopoietic progenitors is independent of ontogenic age and regulatory molecules.

    PubMed

    Huang, S; Law, P; Francis, K; Palsson, B O; Ho, A D

    1999-10-15

    We have developed a time-lapse camera system to follow the replication history and the fate of hematopoietic stem cells (HSC) at a single-cell level. Combined with single-cell culture, we correlated the early replication behavior with colony development after 14 days. The membrane dye PKH26 was used to monitor cell division. In addition to multiple, synchronous, and symmetric divisions, single-sorted CD34(+)/CD38(-) cells derived from fetal liver (FLV) also gave rise to a daughter cell that remained quiescent for up to 8 days, whereas the other daughter cell proliferated exponentially. Upon separation and replating as single cells onto medium containing a cytokine cocktail, 60.6% +/- 9.8% of the initially quiescent cells (PKH26 bright) gave rise again to colonies and 15.8% +/- 7.8% to blast colonies that could be replated. We have then determined the effects of various regulatory molecules on symmetry of initial cell divisions. After single-cell sorting, the CD34(+)/CD38(-) cells derived from FLV were exposed to flt3-ligand, thrombopoietin, stem cell factor (SCF), or medium containing a cytokine cocktail (with SCF, interleukin-3, interleukin-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin). Whereas mitotic rate, colony efficiency, and asymmetric divisions could be altered using various regulatory molecules, the asymmetric division index, defined as the number of asymmetric divisions versus the number of dividing cells, was not altered significantly. This observation suggests that, although lineage commitment and cell proliferation can be skewed by extrinsic signaling, symmetry of early divisions is probably under the control of intrinsic factors.

  6. The Chromatin Remodeler BPTF Activates a Stemness Gene-Expression Program Essential for the Maintenance of Adult Hematopoietic Stem Cells.

    PubMed

    Xu, Bowen; Cai, Ling; Butler, Jason M; Chen, Dongliang; Lu, Xiongdong; Allison, David F; Lu, Rui; Rafii, Shahin; Parker, Joel S; Zheng, Deyou; Wang, Gang Greg

    2018-03-13

    Self-renewal and differentiation of adult stem cells are tightly regulated partly through configuration of chromatin structure by chromatin remodelers. Using knockout mice, we here demonstrate that bromodomain PHD finger transcription factor (BPTF), a component of the nucleosome remodeling factor (NURF) chromatin-remodeling complex, is essential for maintaining the population size of hematopoietic stem/progenitor cells (HSPCs), including long-term hematopoietic stem cells (HSCs). Bptf-deficient HSCs are defective in reconstituted hematopoiesis, and hematopoietic-specific knockout of Bptf caused profound defects including bone marrow failure and anemia. Genome-wide transcriptome profiling revealed that BPTF loss caused downregulation of HSC-specific gene-expression programs, which contain several master transcription factors (Meis1, Pbx1, Mn1, and Lmo2) required for HSC maintenance and self-renewal. Furthermore, we show that BPTF potentiates the chromatin accessibility of key HSC "stemness" genes. These results demonstrate an essential requirement of the chromatin remodeler BPTF and NURF for activation of "stemness" gene-expression programs and proper function of adult HSCs. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. Infection of hematopoietic stem cells by Leishmania infantum increases erythropoiesis and alters the phenotypic and functional profiles of progeny.

    PubMed

    Carvalho-Gontijo, Raquel; Moreira, Diana Raquel; Resende, Mariana; Costa-Silva, Matheus Fernandes; Peruhype-Magalhães, Vanessa; Ribeiro, Cláudia Maria Franco; Ribeiro, Daniel Dias; Silvestre, Ricardo; Cordeiro-da-Silva, Anabela; Martins-Filho, Olindo Assis; Teixeira-Carvalho, Andréa

    2018-04-01

    Immunosuppression is a well-established risk factor for Visceral Leishmaniasis. Post-immunosuppression leishmaniasis is characterized by an increase of parasite burden, hematopoietic disorders and unusual clinical manifestations. Although there are many reports on bone marrow findings in VL, less is known about the relationship between parasite dynamics in this organ and the function of either hematopoietic stem cells and progenitor cells themselves. In the present study, we tackle these issues using a new approach of infecting human stem cells derived from bone marrow with L. infantum. Using this strategy, we show that human hematopoietic stem cells (hHSC) are able to phagocytize L. infantum promastigotes and release modulatory and pro-inflammatory cytokines, mainly TNF-α. Our results demonstrated that L. infantum infection in vitro enhances hematopoiesis, favoring the development of erythrocitic lineage through a mechanism yet unknown. Moreover, we found that L. infantum infection alters the phenotypic profile of the hematopoietic progeny; modifying the surface markers expression of differentiated cells. Thus, our study represents a rare opportunity to monitor the in vitro differentiation of human stem cells experimentally infected by L. infantum to better understand the consequences of the infection on phenotypic and functional profile of the cell progeny. Copyright © 2017. Published by Elsevier Inc.

  8. The Progenitor Dependence of Core-collapse Supernovae from Three-dimensional Simulations with Progenitor Models of 12–40 M ⊙

    NASA Astrophysics Data System (ADS)

    Ott, Christian D.; Roberts, Luke F.; da Silva Schneider, André; Fedrow, Joseph M.; Haas, Roland; Schnetter, Erik

    2018-03-01

    We present a first study of the progenitor star dependence of the three-dimensional (3D) neutrino mechanism of core-collapse supernovae. We employ full 3D general-relativistic multi-group neutrino radiation-hydrodynamics and simulate the postbounce evolutions of progenitors with zero-age main sequence masses of 12, 15, 20, 27, and 40 M ⊙. All progenitors, with the exception of the 12 M ⊙ star, experience shock runaway by the end of their simulations. In most cases, a strongly asymmetric explosion will result. We find three qualitatively distinct evolutions that suggest a complex dependence of explosion dynamics on progenitor density structure, neutrino heating, and 3D flow. (1) Progenitors with massive cores, shallow density profiles, and high post-core-bounce accretion rates experience very strong neutrino heating and neutrino-driven turbulent convection, leading to early shock runaway. Accretion continues at a high rate, likely leading to black hole formation. (2) Intermediate progenitors experience neutrino-driven, turbulence-aided explosions triggered by the arrival of density discontinuities at the shock. These occur typically at the silicon/silicon–oxygen shell boundary. (3) Progenitors with small cores and density profiles without strong discontinuities experience shock recession and develop the 3D standing-accretion shock instability (SASI). Shock runaway ensues late, once declining accretion rate, SASI, and neutrino-driven convection create favorable conditions. These differences in explosion times and dynamics result in a non-monotonic relationship between progenitor and compact remnant mass.

  9. Cryopreservation of cord blood CD34+ cells before or after thrombopoietin expansion differentially affects early platelet recovery in NOD SCID mice.

    PubMed

    van Hensbergen, Yvette; van der Garde, Mark; Brand, Anneke; Slot, Manon C; de Graaf-Dijkstra, Alice; Watt, Suzanne; Zwaginga, Jaap Jan

    2015-07-01

    Expansion of human cord blood (CB) CD34+ cells with thrombopoietin (TPO) can accelerate delayed platelet (PLT) recovery after transplantation into immunodeficient mice. Clinical implementation, however, will depend on practical and effective protocols. The best timing of TPO expansion in relation to cryopreservation in this respect is unknown. In this study, we evaluated whether the order of cryopreservation and TPO expansion affected the expansion rate and numbers of clonogenic hematopoietic progenitor cells in vitro or PLT and longer-term hematopoietic repopulation in NOD SCID mice in vivo. Our results demonstrate higher expansion rates and the generation of higher numbers of multilineage and megakaryocytic progenitors (granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming units and megakaryocyte colony-forming units) in vitro when freshly isolated CB CD34+ cells are first cultured with TPO and then cryopreserved and thawed as compared to TPO expansion after CD34+ cell cryopreservation. In contrast, the cells produced with the latter strategy showed higher expression of CD62L and a superior stromal cell-derived factor-1α-mediated migration. This might play a role in an also observed superior early PLT recovery after transplantation of these cells into NOD SCID mice. The hematopoietic engraftment in the marrow 6 weeks after transplantation was not different between the two strategies. Although TPO expansion before cryopreservation would yield higher nucleated cell and clonogenic myeloid and megakaryocyte cell numbers and enable earlier availability, CB TPO expansion after cryopreservation is likely to be clinically more effective, despite the lower number of cells obtained after expansion. Moreover, the latter strategy is logistically more feasible. © 2015 AABB.

  10. Ten years of iPSC: clinical potential and advances in vitro hematopoietic differentiation.

    PubMed

    Paes, Bárbara Cristina Martins Fernandes; Moço, Pablo Diego; Pereira, Cristiano Gonçalves; Porto, Geciane Silveira; de Sousa Russo, Elisa Maria; Reis, Luiza Cunha Junqueira; Covas, Dimas Tadeu; Picanço-Castro, Virginia

    2017-06-01

    Ten years have passed since the first publication announcing the generation of induced pluripotent stem cells (iPSCs). Issues related to ethics, immune rejection, and cell availability seemed to be solved following this breakthrough. The development of iPSC technology allows advances in in vitro cell differentiation for cell therapy purpose and other clinical applications. This review provides a perspective on the iPSC potential for cell therapies, particularly for hematological applications. We discuss the advances in in vitro hematopoietic differentiation, the possibilities to employ iPSC in hematology studies, and their potential clinical application in hematologic diseases. The generation of red blood cells and functional T cells and the genome editing technology applied to mutation correction are also covered. We highlight some of the requirements and obstacles to be overcome before translating these cells from research to the clinic, for instance, iPSC variability, genotoxicity, the differentiation process, and engraftment. Also, we evaluate the patent landscape and compile the clinical trials in the field of pluripotent stem cells. Currently, we know much more about iPSC than in 2006, but there are still challenges that must be solved. A greater understanding of molecular mechanisms underlying the generation of hematopoietic stem cells is necessary to produce suitable and transplantable hematopoietic stem progenitor cells from iPSC.

  11. Radiation-induced hematopoietic myelosuppression and genotoxicity get significantly countered by active principles of Podophyllum hexandrum: A study in strain 'A' mice.

    PubMed

    Verma, Savita; Gupta, Manju Lata

    2015-01-01

    To investigate the protective role of a novel formulation, prepared by a combination of three active principles isolated from Podophyllum hexandrum (G-002M), against radiation- mediated hematopoietic suppression and cytogenetic aberrations in lethally irradiated mice. G-002M, a combination of podophyllotoxin, podophyllotoxin-β-D glucoside and rutin, was administered intramuscularly in mice (- 1 h) to radiation (9 Gy) exposure. The animals were autopsied at different time intervals for further studies. Loss of bone marrow progenitor cells, altered myeloid/erythroid ratio, serum erythropoietin and pancytopenia in irradiated mice was found significantly (p < 0.001) ameliorated in G-002M pre-administered mice within 30 d. Bcl-2 (B-cell lymphoma 2) and BAX (Bcl-2-associated X) protein expression was also positively (p < 0.001) countered in these mice. Chromosomal aberrations in 30 d were found remarkably (p < 0.001) reduced in marrow of G-002M pretreated mice. Accelerated antioxidants, reduced DNA damage, stimulated lymphocyte proliferation and minimal cellular atrophy in spleen were some of the other key features observed in G-002M administered mice. Reduction in hematopoietic aplasia and chromosomal aberrations, besides, early recovery in bone marrow and spleen of G-002M pretreated mice, could be attributed to its free radical scavenging, DNA protecting and apoptotic proteins modulating ability against radiation.

  12. Marker-free detection of progenitor cell differentiation by analysis of Brownian motion in micro-wells.

    PubMed

    Sekhavati, Farzad; Endele, Max; Rappl, Susanne; Marel, Anna-Kristina; Schroeder, Timm; Rädler, Joachim O

    2015-02-01

    The kinetics of stem and progenitor cell differentiation at the single-cell level provides essential clues to the complexity of the underlying decision-making circuits. In many hematopoietic progenitor cells, differentiation is accompanied by the expression of lineage-specific markers and by a transition from a non-adherent to an adherent state. Here, using the granulocyte-macrophage progenitor (GMP) as a model, we introduce a label-free approach that allows one to follow the course of this transition in hundreds of single cells in parallel. We trap single cells in patterned arrays of micro-wells and use phase-contrast time-lapse movies to distinguish non-adherent from adherent cells by an analysis of Brownian motion. This approach allowed us to observe the kinetics of induced differentiation of primary bone-marrow-derived GMPs into macrophages. The time lapse started 2 hours after addition of the cytokine M-CSF, and nearly 80% of the population had accomplished the transition within the first 20 h. The analysis of Brownian motion proved to be a sensitive and robust tool for monitoring the transition, and thus provides a high-throughput method for the study of cell differentiation at the single-cell level.

  13. Distinguishing autocrine and paracrine signals in hematopoietic stem cell culture using a biofunctional microcavity platform

    NASA Astrophysics Data System (ADS)

    Müller, Eike; Wang, Weijia; Qiao, Wenlian; Bornhäuser, Martin; Zandstra, Peter W.; Werner, Carsten; Pompe, Tilo

    2016-08-01

    Homeostasis of hematopoietic stem cells (HSC) in the mammalian bone marrow stem cell niche is regulated by signals of the local microenvironment. Besides juxtacrine, endocrine and metabolic cues, paracrine and autocrine signals are involved in controlling quiescence, proliferation and differentiation of HSC with strong implications on expansion and differentiation ex vivo as well as in vivo transplantation. Towards this aim, a cell culture analysis on a polymer microcavity carrier platform was combined with a partial least square analysis of a mechanistic model of cell proliferation. We could demonstrate the discrimination of specific autocrine and paracrine signals from soluble factors as stimulating and inhibitory effectors in hematopoietic stem and progenitor cell culture. From that we hypothesize autocrine signals to be predominantly involved in maintaining the quiescent state of HSC in single-cell niches and advocate our analysis platform as an unprecedented option for untangling convoluted signaling mechanisms in complex cell systems being it of juxtacrine, paracrine or autocrine origin.

  14. Paracrine effects and heterogeneity of marrow-derived stem/progenitor cells: relevance for the treatment of respiratory diseases.

    PubMed

    Conese, Massimo; Carbone, Annalucia; Castellani, Stefano; Di Gioia, Sante

    2013-01-01

    Stem cell-based treatment may represent a hope for the treatment of acute lung injury and pulmonary fibrosis, and other chronic lung diseases, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease (COPD). It is well established in preclinical models that bone marrow-derived stem and progenitor cells exert beneficial effects on inflammation, immune responses and repairing of damage in virtually all lung-borne diseases. While it was initially thought that the positive outcome was due to a direct engraftment of these cells into the lung as endothelial and epithelial cells, paracrine factors are now considered the main mechanism through which stem and progenitor cells exert their therapeutic effect. This knowledge has led to the clinical use of marrow cells in pulmonary hypertension with endothelial progenitor cells (EPCs) and in COPD with mesenchymal stromal (stem) cells (MSCs). Bone marrow-derived stem cells, including hematopoietic stem/progenitor cells, MSCs, EPCs and fibrocytes, encompass a wide array of cell subsets with different capacities of engraftment and injured tissue-regenerating potential. The characterization/isolation of the stem cell subpopulations represents a major challenge to improve the efficacy of transplantation protocols used in regenerative medicine and applied to lung disorders. Copyright © 2013 S. Karger AG, Basel.

  15. Bag1 is essential for differentiation and survival of hematopoietic and neuronal cells.

    PubMed

    Götz, Rudolf; Wiese, Stefan; Takayama, Shinichi; Camarero, Guadalupe C; Rossoll, Wilfried; Schweizer, Ulrich; Troppmair, Jakob; Jablonka, Sibylle; Holtmann, Bettina; Reed, John C; Rapp, Ulf R; Sendtner, Michael

    2005-09-01

    Bag1 is a cochaperone for the heat-shock protein Hsp70 that interacts with C-Raf, B-Raf, Akt, Bcl-2, steroid hormone receptors and other proteins. Here we use targeted gene disruption in mice to show that Bag1 has an essential role in the survival of differentiating neurons and hematopoietic cells. Cells of the fetal liver and developing nervous system in Bag1-/- mice underwent massive apoptosis. Lack of Bag1 did not disturb the primary function of Akt or Raf, as phosphorylation of the forkhead transcription factor FKHR and activation of extracellular signal-regulated kinase (Erk)-1/2 were not affected. However, the defect was associated with the disturbance of a tripartite complex formed by Akt, B-Raf and Bag1, in addition to the absence of Bad phosphorylation at Ser136. We also observed reduced expression of members of the inhibitor of apoptosis (IAP) family. Our data show that Bag1 is a physiological mediator of extracellular survival signals linked to the cellular mechanisms that prevent apoptosis in hematopoietic and neuronal progenitor cells.

  16. Rapid expansion of preexisting nonleukemic hematopoietic clones frequently follows induction therapy for de novo AML.

    PubMed

    Wong, Terrence N; Miller, Christopher A; Klco, Jeffery M; Petti, Allegra; Demeter, Ryan; Helton, Nichole M; Li, Tiandao; Fulton, Robert S; Heath, Sharon E; Mardis, Elaine R; Westervelt, Peter; DiPersio, John F; Walter, Matthew J; Welch, John S; Graubert, Timothy A; Wilson, Richard K; Ley, Timothy J; Link, Daniel C

    2016-02-18

    There is interest in using leukemia-gene panels and next-generation sequencing to assess acute myelogenous leukemia (AML) response to induction chemotherapy. Studies have shown that patients with AML in morphologic remission may continue to have clonal hematopoiesis with populations closely related to the founding AML clone and that this confers an increased risk of relapse. However, it remains unknown how induction chemotherapy influences the clonal evolution of a patient's nonleukemic hematopoietic population. Here, we report that 5 of 15 patients with genetic clearance of their founding AML clone after induction chemotherapy had a concomitant expansion of a hematopoietic population unrelated to the initial AML. These populations frequently harbored somatic mutations in genes recurrently mutated in AML or myelodysplastic syndromes and were detectable at very low frequencies at the time of AML diagnosis. These results suggest that nonleukemic hematopoietic stem and progenitor cells, harboring specific aging-acquired mutations, may have a competitive fitness advantage after induction chemotherapy, expand, and persist long after the completion of chemotherapy. Although the clinical importance of these "rising" clones remains to be determined, it will be important to distinguish them from leukemia-related populations when assessing for molecular responses to induction chemotherapy. © 2016 by The American Society of Hematology.

  17. Purified hematopoietic stem cell engraftment of rare niches corrects severe lymphoid deficiencies without host conditioning

    PubMed Central

    Bhattacharya, Deepta; Rossi, Derrick J.; Bryder, David; Weissman, Irving L.

    2006-01-01

    In the absence of irradiation or other cytoreductive conditioning, endogenous hematopoietic stem cells (HSCs) are thought to fill the unique niches within the bone marrow that allow maintenance of full hematopoietic potential and thus prevent productive engraftment of transplanted donor HSCs. By transplantation of purified exogenous HSCs into unconditioned congenic histocompatible strains of mice, we show that ∼0.1–1.0% of these HSC niches are available for engraftment at any given point and find no evidence that endogenous HSCs can be displaced from the niches they occupy. We demonstrate that productive engraftment of HSCs within these empty niches is inhibited by host CD4+ T cells that recognize very subtle minor histocompatibility differences. Strikingly, transplantation of purified HSCs into a panel of severe combined immunodeficient (SCID) mice leads to a rapid and complete rescue of lymphoid deficiencies through engraftment of these very rare niches and expansion of donor lymphoid progenitors. We further demonstrate that transient antibody-mediated depletion of CD4+ T cells allows short-term HSC engraftment and regeneration of B cells in a mouse model of B(-) non-SCID. These experiments provide a general mechanism by which transplanted HSCs can correct hematopoietic deficiencies without any host conditioning or with only highly specific and transient lymphoablation. PMID:16380511

  18. Flow-cytometric separation and enrichment of hepatic progenitor cells in the developing mouse liver.

    PubMed

    Suzuki, A; Zheng, Y; Kondo, R; Kusakabe, M; Takada, Y; Fukao, K; Nakauchi, H; Taniguchi, H

    2000-12-01

    Stem cells responsible for tissue maintenance and repair are found in a number of organs. However, hepatic stem cells assumed to play a key role in liver development and regeneration remain to be well characterized. To address this issue, we set up a culture system in which primitive hepatic progenitor cells formed colonies. By combining this culture system with fluorescence-activated cell sorting (FACS), cells forming colonies containing distinct hepatocytes and cholangiocytes were identified in the fetal mouse liver. These cells express both CD49f and CD29 (alpha6 and beta1 integrin subunits), but do not mark for hematopoietic antigens such as CD45, TER119, and c-Kit. When transplanted into the spleen, these cells migrated to the recipient liver and differentiated into liver parenchymal cells. Our data demonstrate that hepatic progenitor cells are enriched by FACS and suggest approaches to supplanting organ allografting and improving artificial-organ hepatic support.

  19. Neurotrophins promote revascularization by local recruitment of TrkB+ endothelial cells and systemic mobilization of hematopoietic progenitors

    PubMed Central

    Kermani, Pouneh; Rafii, Dahlia; Jin, David K.; Whitlock, Paul; Schaffer, Wendy; Chiang, Anne; Vincent, Loic; Friedrich, Matthias; Shido, Koji; Hackett, Neil R.; Crystal, Ronald G.; Rafii, Shahin; Hempstead, Barbara L.

    2005-01-01

    The neurotrophin brain-derived neurotrophic factor (BDNF) is required for the maintenance of cardiac vessel wall stability during embryonic development through direct angiogenic actions on endothelial cells expressing the tropomysin receptor kinase B (TrkB). However, the role of BDNF and a related neurotrophin ligand, neurotrophin-4 (NT-4), in the regulation of revascularization of the adult tissues is unknown. To study the potential angiogenic capacity of BDNF in mediating the neovascularization of ischemic and non-ischemic adult mouse tissues, we utilized a hindlimb ischemia and a subcutaneous Matrigel model. Recruitment of endothelial cells and promotion of channel formation within the Matrigel plug by BDNF and NT-4 was comparable to that induced by VEGF-A. The introduction of BDNF into non-ischemic ears or ischemic limbs induced neoangiogenesis, with a 2-fold increase in the capillary density. Remarkably, treatment with BDNF progressively increased blood flow in the ischemic limb over 21 days, similar to treatment with VEGF-A. The mechanism by which BDNF enhances capillary formation is mediated in part through local activation of the TrkB receptor and also by recruitment of Sca-1+CD11b+ pro-angiogenic hematopoietic cells. BDNF induces a potent direct chemokinetic action on subsets of marrow-derived Sca-1+ hematopoietic cells co-expressing TrkB. These studies suggest that local regional delivery of BDNF may provide a novel mechanism for inducing neoangiogenesis through both direct actions on local TrkB-expressing endothelial cells in skeletal muscle and recruitment of specific subsets of TrkB+ bone marrow–derived hematopoietic cells to provide peri-endothelial support for the newly formed vessels. PMID:15765148

  20. Impact of modeled microgravity on migration, differentiation, and cell cycle control of primitive human hematopoietic progenitor cells.

    PubMed

    Plett, P Artur; Abonour, Rafat; Frankovitz, Stacy M; Orschell, Christie M

    2004-08-01

    Migration, proliferation, and differentiation of bone marrow (BM) hematopoietic stem cells (HSC) are important factors in maintaining hematopoietic homeostasis. Homeostatic control of erythrocytes and lymphocytes is perturbed in humans exposed to microgravity (micro-g), resulting in space flight-induced anemia and immunosuppression. We sought to determine whether any of these anomalies can be explained by micro-g-induced changes in migration, proliferation, and differentiation of human BM CD34+ cells, and whether such changes can begin to explain any of the shifts in hematopoietic homeostasis observed in astronauts. BM CD34+ cells were cultured in modeled micro-g (mmicro-g) using NASA's rotating wall vessels (RWV), or in control cultures at earth gravity for 2 to 18 days. Cells were harvested at different times and CD34+ cells assessed for migration potential, cell-cycle kinetics and regulatory proteins, and maturation status. Culture of BM CD34+ cells in RWV for 2 to 3 days resulted in a significant reduction of stromal cell-derived factor 1 (SDF-1alpha)-directed migration, which correlated with decreased expression of F-actin. Modeled micro-g induced alterations in cell-cycle kinetics that were characterized by prolonged S phase and reduced cyclin A expression. Differentiation of primitive CD34+ cells cultured for 14 to 18 days in RWV favored myeloid cell development at the expense of erythroid development, which was significantly reduced compared to controls. These results illustrate that mmicro-g significantly inhibits the migration potential, cell-cycle progression, and differentiation patterns of primitive BM CD34+ cells, which may contribute to some of the hematologic abnormalities observed in humans during space flight.

  1. Supportive Care of Hematopoietic Cell Transplant Patients

    PubMed Central

    Jim, Heather S. L.; Syrjala, Karen L.; Rizzo, Doug

    2012-01-01

    Hematopoietic cell transplant survivors face a number of challenges including low energy and stamina, “chemo-brain” and emotional distress, and late effects that can compromise functioning or lead to early mortality. This session will review the most recent interventions and recommendations to avoid or mitigate these complications. PMID:22226095

  2. Invited review: mesenchymal progenitor cells in intramuscular connective tissue development.

    PubMed

    Miao, Z G; Zhang, L P; Fu, X; Yang, Q Y; Zhu, M J; Dodson, M V; Du, M

    2016-01-01

    The abundance and cross-linking of intramuscular connective tissue contributes to the background toughness of meat, and is thus undesirable. Connective tissue is mainly synthesized by intramuscular fibroblasts. Myocytes, adipocytes and fibroblasts are derived from a common pool of progenitor cells during the early embryonic development. It appears that multipotent mesenchymal stem cells first diverge into either myogenic or non-myogenic lineages; non-myogenic mesenchymal progenitors then develop into the stromal-vascular fraction of skeletal muscle wherein adipocytes, fibroblasts and derived mesenchymal progenitors reside. Because non-myogenic mesenchymal progenitors mainly undergo adipogenic or fibrogenic differentiation during muscle development, strengthening progenitor proliferation enhances the potential for both intramuscular adipogenesis and fibrogenesis, leading to the elevation of both marbling and connective tissue content in the resulting meat product. Furthermore, given the bipotent developmental potential of progenitor cells, enhancing their conversion to adipogenesis reduces fibrogenesis, which likely results in the overall improvement of marbling (more intramuscular adipocytes) and tenderness (less connective tissue) of meat. Fibrogenesis is mainly regulated by the transforming growth factor (TGF) β signaling pathway and its regulatory cascade. In addition, extracellular matrix, a part of the intramuscular connective tissue, provides a niche environment for regulating myogenic differentiation of satellite cells and muscle growth. Despite rapid progress, many questions remain in the role of extracellular matrix on muscle development, and factors determining the early differentiation of myogenic, adipogenic and fibrogenic cells, which warrant further studies.

  3. The miR-23a~27a~24-2 microRNA cluster buffers transcription and signaling pathways during hematopoiesis

    PubMed Central

    Kurkewich, Jeffrey L.; Klopfenstein, Nathan; Wood, Christian; Boucher, Austin

    2017-01-01

    MicroRNA cluster mirn23a has previously been shown to promote myeloid development at the expense of lymphoid development in overexpression and knockout mouse models. This polarization is observed early in hematopoietic development, with an increase in common lymphoid progenitors (CLPs) and a decrease in all myeloid progenitor subsets in adult bone marrow. The pool size of multipotential progenitors (MPPs) is unchanged; however, in this report we observe by flow cytometry that polarized subsets of MPPs are changed in the absence of mirn23a. Additionally, in vitro culture of MPPs and sorted MPP transplants showed that these cells have decreased myeloid and increased lymphoid potential in vitro and in vivo. We investigated the mechanism by which mirn23a regulates hematopoietic differentiation and observed that mirn23a promotes myeloid development of hematopoietic progenitors through regulation of hematopoietic transcription factors and signaling pathways. Early transcription factors that direct the commitment of MPPs to CLPs (Ikzf1, Runx1, Satb1, Bach1 and Bach2) are increased in the absence of mirn23a miRNAs as well as factors that commit the CLP to the B cell lineage (FoxO1, Ebf1, and Pax5). Mirn23a appears to buffer transcription factor levels so that they do not stochastically reach a threshold level to direct differentiation. Intriguingly, mirn23a also inversely regulates the PI3 kinase (PI3K)/Akt and BMP/Smad signaling pathways. Pharmacological inhibitor studies, coupled with dominant active/dominant negative biochemical experiments, show that both signaling pathways are critical to mirn23a’s regulation of hematopoietic differentiation. Lastly, consistent with mirn23a being a physiological inhibitor of B cell development, we observed that the essential B cell transcription factor EBF1 represses expression of mirn23a. In summary, our data demonstrates that mirn23a regulates a complex array of transcription and signaling pathways to modulate adult hematopoiesis

  4. Endothelial cells and hematopoiesis: a light microscopic study of fetal, normal, and pathologic human bone marrow in plastic-embedded sections.

    PubMed

    Islam, A; Glomski, C; Henderson, E S

    1992-07-01

    The origin and morphological identity of hematopoietic progenitor cells, as well as their precursor, the pleuripotential hematopoietic stem cell (HSC), has not been established. Our studies of 2 microns sectioned undecalcified plastic-embedded bone marrow (BM) from healthy human fetuses; normal adults; patients with acute myeloblastic leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic granulocytic leukemia (CGL) in various stages (chronic, accelerated, acute blastic phase, and after autografting); and patients recovering from therapy-induced marrow hypoplasia suggest that proliferative hematopoietic zones exist near the endosteum (endosteal marrow) and the vascular endothelium (capillary and sinus-lining endothelium) and a maturational zone distal to these regions. In some of these areas, morphologically recognizable hematopoietic cells were seen and interpreted as emerging and maturing in a sequential progression, suggesting an origin from the endosteal or endothelial progenitors. In other loci, early hematopoietic cells were seen in close contact with the endosteal or vascular endothelial (VE) cells. This latter relationship suggested that these areas of cellular contact were important and represented sites of cell to cell interaction that may be associated with the liberation of growth factors by endosteal and endothelial cells and their action on hematopoietic progenitor cells. Following treatment-induced hypoplasia, the endosteal and VE cells were seen to modulate, transform, and migrate into the surrounding empty and edematous marrow space as fibroblasts. Later, as hemopoietic regeneration began, clusters of regenerating hematopoietic cells were seen adjacent to bone trabecule (BT) and near the vascular endothelium. We postulate that endosteal and VE cells are the equivalent of embryonal-stage, undifferentiated mesenchyme and, under the appropriate regulatory influence, are capable of modulation and transformation (differentiation) into stromal

  5. Direct Toll-like receptor-mediated stimulation of hematopoietic stem and progenitor cells occurs in vivo and promotes differentiation toward macrophages.

    PubMed

    Megías, Javier; Yáñez, Alberto; Moriano, Silvia; O'Connor, José-Enrique; Gozalbo, Daniel; Gil, María-Luisa

    2012-07-01

    As Toll-like receptors (TLRs) are expressed by hematopoietic stem and progenitor cells (HSPCs), they may play a role in hematopoiesis in response to pathogens during infection. We show here that TLR2, TLR4, and TLR9 agonists (tripalmitoyl-S-glyceryl-L-Cys-Ser-(Lys)4 [Pam3CSK4], lipopolysaccharide [LPS], and CpG oligodeoxynucleotide [ODN]) induce the in vitro differentiation of purified murine lineage negative cells (Lin(-) ) as well as HSPCs (identified as Lin(-) c-Kit(+) Sca-1(+) IL-7Rα(-) [LKS] cells) toward macrophages (Mph), through a myeloid differentiation factor 88 (MyD88)-dependent pathway. In order to investigate the possible direct interaction of soluble microorganism-associated molecular patterns and TLRs on HSPCs in vivo, we designed a new experimental approach: purified Lin(-) and LKS cells from bone marrow of B6Ly5.1 mice (CD45.1 alloantigen) were transplanted into TLR2(-/-) , TLR4(-/-) , or MyD88(-/-) mice (CD45.2 alloantigen), which were then injected with soluble TLR ligands (Pam3CSK4, LPS, or ODN, respectively). As recipient mouse cells do not recognize the TLR ligands injected, interference by soluble mediators secreted by recipient cells is negligible. Transplanted cells were detected in the spleen and bone marrow of recipient mice, and in response to soluble TLR ligands, cells differentiated preferentially to Mph. These results show, for the first time, that HSPCs may be directly stimulated by TLR agonists in vivo, and that the engagement of these receptors induces differentiation toward Mph. Therefore, HSPCs may sense pathogen or pathogen-derived products directly during infection, inducing a rapid generation of cells of the innate immune system. Copyright © 2012 AlphaMed Press.

  6. Impaired Endothelial Progenitor Cell Mobilization and Dysfunctional Bone Marrow Stroma in Diabetes Mellitus

    PubMed Central

    Rafii, Shahin; Jaspers, Janneke E.; White, Ian A.; Hooper, Andrea T.; Doevendans, Pieter A.; Verhaar, Marianne C.

    2013-01-01

    Background Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired –at least partly– due to dysfunction of the bone marrow stromal compartment. Methods Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1+Flk-1+ EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34+ hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell–endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed. Results In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro. Conclusion EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients. PMID:23555959

  7. Impaired endothelial progenitor cell mobilization and dysfunctional bone marrow stroma in diabetes mellitus.

    PubMed

    Westerweel, Peter E; Teraa, Martin; Rafii, Shahin; Jaspers, Janneke E; White, Ian A; Hooper, Andrea T; Doevendans, Pieter A; Verhaar, Marianne C

    2013-01-01

    Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment. Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+)Flk-1(+) EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+) hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed. In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro. EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.

  8. Mobilization of hematopoietic stem cells with highest self-renewal by G-CSF precedes clonogenic cell mobilization peak.

    PubMed

    Winkler, Ingrid G; Wiercinska, Eliza; Barbier, Valerie; Nowlan, Bianca; Bonig, Halvard; Levesque, Jean-Pierre

    2016-04-01

    Harvest of granulocyte colony-stimulating factor (G-CSF)-mobilized hematopoietic stem cells (HSCs) begins at day 5 of G-CSF administration, when most donors have achieved maximal mobilization. This is based on surrogate markers for HSC mobilization, such as CD34(+) cells and colony-forming activity in blood. However, CD34(+) cells or colony-forming units in culture (CFU-C) are heterogeneous cell populations with hugely divergent long-term repopulation potential on transplantation. HSC behavior is influenced by the vascular bed in the vicinity of which they reside. We hypothesized that G-CSF may mobilize sequentially cells proximal and more distal to bone marrow venous sinuses where HSCs enter the blood. We addressed this question with functional serial transplantation assays using blood and bone marrow after specific time points of G-CSF treatment in mice. We found that in mice, blood collected after only 48 hours of G-CSF administration was as enriched in serially reconstituting HSCs as blood collected at 5 days of G-CSF treatment. Similarly, mobilized Lin(-)CD34(+) cells were relatively enriched in more primitive Lin(-)CD34(+)CD38(-) cells at day 2 of G-CSF treatment compared with later points in half of human donors tested (n = 6). This suggests that in both humans and mice, hematopoietic progenitor and stem cells do not mobilize uniformly according to their maturation stage, with most potent HSCs mobilizing as early as day 2 of G-CSF. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  9. The scaffold protein Nde1 safeguards the brain genome during S phase of early neural progenitor differentiation

    PubMed Central

    Houlihan, Shauna L; Feng, Yuanyi

    2014-01-01

    Successfully completing the S phase of each cell cycle ensures genome integrity. Impediment of DNA replication can lead to DNA damage and genomic disorders. In this study, we show a novel function for NDE1, whose mutations cause brain developmental disorders, in safeguarding the genome through S phase during early steps of neural progenitor fate restrictive differentiation. Nde1 mutant neural progenitors showed catastrophic DNA double strand breaks concurrent with the DNA replication. This evoked DNA damage responses, led to the activation of p53-dependent apoptosis, and resulted in the reduction of neurons in cortical layer II/III. We discovered a nuclear pool of Nde1, identified the interaction of Nde1 with cohesin and its associated chromatin remodeler, and showed that stalled DNA replication in Nde1 mutants specifically occurred in mid-late S phase at heterochromatin domains. These findings suggest that NDE1-mediated heterochromatin replication is indispensible for neuronal differentiation, and that the loss of NDE1 function may lead to genomic neurological disorders. DOI: http://dx.doi.org/10.7554/eLife.03297.001 PMID:25245017

  10. Identification, characterization and isolation of a common progenitor for osteoclasts, macrophages and dendritic cells from murine bone marrow and periphery

    PubMed Central

    Jacome-Galarza, Christian E.; Lee, Sun-Kyeong; Lorenzo, Joseph A.; LeonardoAguila, Hector

    2012-01-01

    Osteoclasts are specialized bone resorbing cells that derive from monocyte precursors. We have identified three populations of cells with high osteoclastogenic potential in murine bone marrow, which expressed the phenotype: B220−CD3−CD11b−/low CD115+ and either CD117hi, CD117intermediate or CD117low. We have evaluated these populations for their ability to also generate macrophages and dendritic cells. At a single cell level, the population expressing higher CD117 levels was able to generate bone-resorbing osteoclasts, phagocytic macrophages and antigen-presenting dendritic cells in vitro with efficiencies of over 90 percent, indicating that there exists a common developmental pathway for these cell types. Cells with osteoclastogenic potential also exist in blood and peripheral hematopoietic organs. Their functional meaning and/or their relationship with bone marrow progenitors is not well established. Hence, we characterized murine peripheral cell populations for their ability to form osteoclasts, macrophages and dendritic cells in vitro. The spleen and peripheral blood monocyte progenitors share phenotypic markers with bone marrow progenitors, but differ in their expression of CD11b, which was low in bone marrow but high in periphery. We propose that circulating monocyte progenitors are derived from a common bone marrow osteoclasts/macrophage/dendritic cell progenitor (OcMDC), which we have now characterized at a clonal level. However, the lineage relationship between the bone marrow and peripheral monocyte progenitors has yet to be defined. PMID:23165930

  11. Teleost growth factor independence (gfi) genes differentially regulate successive waves of hematopoiesis.

    PubMed

    Cooney, Jeffrey D; Hildick-Smith, Gordon J; Shafizadeh, Ebrahim; McBride, Paul F; Carroll, Kelli J; Anderson, Heidi; Shaw, George C; Tamplin, Owen J; Branco, Diana S; Dalton, Arthur J; Shah, Dhvanit I; Wong, Clara; Gallagher, Patrick G; Zon, Leonard I; North, Trista E; Paw, Barry H

    2013-01-15

    Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Effects of long-term cryopreservation on peripheral blood progenitor cells.

    PubMed

    Vosganian, Gregory S; Waalen, Jill; Kim, Kevin; Jhatakia, Sejal; Schram, Ethan; Lee, Tracey; Riddell, Dan; Mason, James R

    2012-11-01

    The long-term stability of cryopreserved peripheral blood progenitor cells is an important issue for patients experiencing disease relapse. However, there is no consensus on how to evaluate the long-term effects of cryopreservation. We describe the effect of cryopreservation on viability and progenitor colony activity from 87 individual samples processed at the Scripps Green Hospital Stem Cell Processing Center (La Jolla, CA, USA). We randomly selected 87 peripheral blood hematopoietic stem cell (PBHSC) samples from 60 patients and evaluated the effect of cryopreservation on sample viability and red and white cell colony activity after < 24 h and 7, 10 and 15 years of cryopreservation. Viability was assayed via trypan blue dye exclusion and activity was measured following 14 days of culture. An age at collection older than 50 years may result in suboptimal activity and viability following long-term cryopreservation, while gender and disease status had no effect. Cryopreservation did not significantly affect white or red cell activity following 10 years of cryopreservation. However, for samples stored longer than 10 years, viability and activity significantly decreased. We noted a positive association between higher pre-cryopreservation %CD34 count and colony activity. Cryopreservation of peripheral blood progenitor cells for up to 10 years results in no loss of clonogenic capacity, as determined by culture activity, although longer durations of storage may affect activity. Until validated methods are developed, cryopreserved grafts should be evaluated based on pre-freeze CD34(+) cell counts as assayed by flow cytometry, and post-thaw sample evaluation should be reserved for patients identified as poor mobilizers.

  13. Neural differentiation of novel multipotent progenitor cells from cryopreserved human umbilical cord blood

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Myoung Woo; Moon, Young Joon; Yang, Mal Sook

    2007-06-29

    Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells, with practical and ethical advantages. To date, the presence of other stem cells in UCB remains to be established. We investigated whether other stem cells are present in cryopreserved UCB. Seeded mononuclear cells formed adherent colonized cells in optimized culture conditions. Over a 4- to 6-week culture period, colonized cells gradually developed into adherent mono-layer cells, which exhibited homogeneous fibroblast-like morphology and immunophenotypes, and were highly proliferative. Isolated cells were designated 'multipotent progenitor cells (MPCs)'. Under appropriate conditions for 2 weeks, MPCs differentiated into neural tissue-specific cell types,more » including neuron, astrocyte, and oligodendrocyte. Differentiated cells presented their respective markers, specifically, NF-L and NSE for neurons, GFAP for astrocytes, and myelin/oligodendrocyte for oligodendrocytes. In this study, we successfully isolated MPCs from cryopreserved UCB, which differentiated into the neural tissue-specific cell types. These findings suggest that cryopreserved human UCB is a useful alternative source of neural progenitor cells, such as MPCs, for experimental and therapeutic applications.« less

  14. Hematopoietic Stem Cells in Neonates: Any Differences between Very Preterm and Term Neonates?

    PubMed Central

    Wisgrill, Lukas; Schüller, Simone; Bammer, Markus; Berger, Angelika; Pollak, Arnold; Radke, Teja Falk; Kögler, Gesine; Spittler, Andreas; Helmer, Hanns; Husslein, Peter; Gortner, Ludwig

    2014-01-01

    Background In the last decades, human full-term cord blood was extensively investigated as a potential source of hematopoietic stem and progenitor cells (HSPCs). Despite the growing interest of regenerative therapies in preterm neonates, only little is known about the biological function of HSPCs from early preterm neonates under different perinatal conditions. Therefore, we investigated the concentration, the clonogenic capacity and the influence of obstetric/perinatal complications and maternal history on HSPC subsets in preterm and term cord blood. Methods CD34+ HSPC subsets in UCB of 30 preterm and 30 term infants were evaluated by flow cytometry. Clonogenic assays suitable for detection of the proliferative potential of HSPCs were conducted. Furthermore, we analyzed the clonogenic potential of isolated HSPCs according to the stem cell marker CD133 and aldehyde dehydrogenase (ALDH) activity. Results Preterm cord blood contained a significantly higher concentration of circulating CD34+ HSPCs, especially primitive progenitors, than term cord blood. The clonogenic capacity of HSPCs was enhanced in preterm cord blood. Using univariate analysis, the number and clonogenic potential of circulating UCB HSPCs was influenced by gestational age, birth weight and maternal age. Multivariate analysis showed that main factors that significantly influenced the HSPC count were maternal age, gestational age and white blood cell count. Further, only gestational age significantly influenced the clonogenic potential of UCB HSPCs. Finally, isolated CD34+/CD133+, CD34+/CD133– and ALDHhigh HSPC obtained from preterm cord blood showed a significantly higher clonogenic potential compared to term cord blood. Conclusion We demonstrate that preterm cord blood exhibits a higher HSPC concentration and increased clonogenic capacity compared to term neonates. These data may imply an emerging use of HSPCs in autologous stem cell therapy in preterm neonates. PMID:25181353

  15. Do all β-blockers attenuate the excess hematopoietic progenitor cell mobilization from the bone marrow following trauma/hemorrhagic shock?

    PubMed

    Pasupuleti, Latha V; Cook, Kristin M; Sifri, Ziad C; Alzate, Walter D; Livingston, David H; Mohr, Alicia M

    2014-04-01

    Severe injury results in increased mobilization of hematopoietic progenitor cells (HPC) from the bone marrow (BM) to sites of injury, which may contribute to persistent BM dysfunction after trauma. Norepinephrine is a known inducer of HPC mobilization, and nonselective β-blockade with propranolol has been shown to decrease mobilization after trauma and hemorrhagic shock (HS). This study will determine the role of selective β-adrenergic receptor blockade in HPC mobilization in a combined model of lung contusion (LC) and HS. Male Sprague-Dawley rats were subjected to LC, followed by 45 minutes of HS. Animals were then randomized to receive atenolol (LCHS + β1B), butoxamine (LCHS + β2B), or SR59230A (LCHS + β3B) immediately after resuscitation and daily for 6 days. Control groups were composed of naive animals. BM cellularity, %HPCs in peripheral blood, and plasma granulocyte-colony stimulating factor levels were assessed at 3 hours and 7 days. Systemic plasma-mediated effects were evaluated in vitro by assessment of BM HPC growth. Injured lung tissue was graded histologically by a blinded reader. The use of β2B or β3B following LCHS restored BM cellularity and significantly decreased HPC mobilization. In contrast, β1B had no effect on HPC mobilization. Only β3B significantly reduced plasma G-CSF levels. When evaluating the plasma systemic effects, both β2B and β3B significantly improved BM HPC growth as compared with LCHS alone. The use of β2 and β3 blockade did not affect lung injury scores. Both β2 and β3 blockade can prevent excess HPC mobilization and BM dysfunction when given after trauma and HS, and the effects seem to be mediated systemically, without adverse effects on subsequent healing. Only treatment with β3 blockade reduced plasma G-CSF levels, suggesting different mechanisms for adrenergic-induced G-CSF release and mobilization of HPCs. This study adds to the evidence that therapeutic strategies that reduce the exaggerated sympathetic

  16. Mycobacterium tuberculosis-Infected Hematopoietic Stem and Progenitor Cells Unable to Express Inducible Nitric Oxide Synthase Propagate Tuberculosis in Mice.

    PubMed

    Reece, Stephen T; Vogelzang, Alexis; Tornack, Julia; Bauer, Wolfgang; Zedler, Ulrike; Schommer-Leitner, Sandra; Stingl, Georg; Melchers, Fritz; Kaufmann, Stefan H E

    2018-04-23

    Persistence of Mycobacterium tuberculosis within human bone marrow stem cells has been identified as a potential bacterial niche during latent tuberculosis. Using a murine model of tuberculosis, we show here that bone marrow stem and progenitor cells containing M. tuberculosis propagated tuberculosis when transferred to naive mice, given that both transferred cells and recipient mice were unable to express inducible nitric oxide synthase, which mediates killing of intracellular bacteria via nitric oxide. Our findings suggest that bone marrow stem and progenitor cells containing M. tuberculosis propagate hallmarks of disease if nitric oxide-mediated killing of bacteria is defective.

  17. Mycobacterium tuberculosis-Infected Hematopoietic Stem and Progenitor Cells Unable to Express Inducible Nitric Oxide Synthase Propagate Tuberculosis in Mice

    PubMed Central

    Reece, Stephen T; Vogelzang, Alexis; Tornack, Julia; Bauer, Wolfgang; Zedler, Ulrike; Schommer-Leitner, Sandra; Stingl, Georg; Melchers, Fritz; Kaufmann, Stefan H E

    2018-01-01

    Abstract Persistence of Mycobacterium tuberculosis within human bone marrow stem cells has been identified as a potential bacterial niche during latent tuberculosis. Using a murine model of tuberculosis, we show here that bone marrow stem and progenitor cells containing M. tuberculosis propagated tuberculosis when transferred to naive mice, given that both transferred cells and recipient mice were unable to express inducible nitric oxide synthase, which mediates killing of intracellular bacteria via nitric oxide. Our findings suggest that bone marrow stem and progenitor cells containing M. tuberculosis propagate hallmarks of disease if nitric oxide-mediated killing of bacteria is defective. PMID:29471332

  18. Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia.

    PubMed

    Mettananda, Sachith; Fisher, Chris A; Hay, Deborah; Badat, Mohsin; Quek, Lynn; Clark, Kevin; Hublitz, Philip; Downes, Damien; Kerry, Jon; Gosden, Matthew; Telenius, Jelena; Sloane-Stanley, Jackie A; Faustino, Paula; Coelho, Andreia; Doondeea, Jessica; Usukhbayar, Batchimeg; Sopp, Paul; Sharpe, Jacqueline A; Hughes, Jim R; Vyas, Paresh; Gibbons, Richard J; Higgs, Douglas R

    2017-09-04

    β-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and β-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with β-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity. Here we demonstrate the use of CRISPR/Cas9 genome editing of primary human hematopoietic stem/progenitor (CD34+) cells to emulate a natural mutation, which deletes the MCS-R2 α-globin enhancer and causes α-thalassemia. When edited CD34+ cells are differentiated into erythroid cells, we observe the expected reduction in α-globin expression and a correction of the pathologic globin chain imbalance in cells from patients with β-thalassemia. Xenograft assays show that a proportion of the edited CD34+ cells are long-term repopulating hematopoietic stem cells, demonstrating the potential of this approach for translation into a therapy for β-thalassemia.β-thalassemia is characterised by the presence of an excess of α-globin chains, which contribute to erythrocyte pathology. Here the authors use CRISP/Cas9 to reduce α-globin expression in hematopoietic precursors, and show effectiveness in xenograft assays in mice.

  19. Cloning and expression profile of FLT3 gene during progenitor cell-dependent liver regeneration.

    PubMed

    Aydin, Iraz T; Tokcaer, Zeynep; Dalgic, Aydin; Konu, Ozlen; Akcali, Kamil C

    2007-12-01

    The liver has a unique capacity to regenerate upon exposure to viral infections, toxic reactions and cancer formation. Liver regeneration is a complex phenomenon in which several factors participate during its onset. Cellular proliferation is an important component of this process and the factors that regulate this proliferation have a vital role. FLT3, a well-known hematopoietic stem cell and hepatic lineage surface marker, is involved in proliferative events of hematopoietic stem cells. However, its contribution to liver regeneration is not known. Therefore, the aim of this study was to clone and examine the role of FLT3 during liver regeneration in rats. Partial cDNA of rat homolog of FLT3 gene was cloned from thymus and the tissue specific expression of this gene at mRNA and protein levels was examined by RT-PCR and Western blot. After treating with 2-AAF and performing hepatectomy in rats to induce progenitor-dependent liver regeneration, the mRNA and protein expression profile of FLT3 was investigated by real-time PCR and Western blot during liver regeneration. In addition, cellular localization of FLT3 protein was determined by immunohistochemistry. The results indicated that rat FLT3 cDNA has high homology with mouse and human FLT3 cDNA. It was also found that FLT3 is expressed in most of the rat tissues and during liver regeneration. In addition, its intracellular localization is altered during the late stages of liver regeneration. The FLT3 receptor is activated at the late stages of liver regeneration and participates in the proliferation response that is observed during progenitor-dependent liver regeneration.

  20. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9

    PubMed Central

    Mandal, Pankaj K.; Ferreira, Leonardo M. R.; Collins, Ryan; Meissner, Torsten B.; Boutwell, Christian L.; Friesen, Max; Vrbanac, Vladimir; Garrison, Brian S.; Stortchevoi, Alexei; Bryder, David; Musunuru, Kiran; Brand, Harrison; Tager, Andrew M.; Allen, Todd M.; Talkowski, Michael E.; Rossi, Derrick J.; Cowan, Chad A.

    2014-01-01

    SUMMARY Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9 mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multi-lineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy. PMID:25517468

  1. [Alteration of Microparticle Levels in Early Complications During Hematopoietic Stem Cell Transplantation].

    PubMed

    Zhou, Li-Li; Han, Yue; Zhu, Qian; Zhao, Shi-Xiang; Wang, Qian; Zhu, Ming-Qing; Dai, Lan; Shen, Wen-Hong; Wu, De-Pei

    2015-12-01

    To investigate the alteration of microparticles (MP) in the recipients following hematopoietic stem cell transplantation (HSCT) and its significance, and to search the early diagnostic indicators of thrombotic complications after transplantation. According to the occurrence of transplantation-associated complications, 94 allo-HSCT patients were divided into 4 groups: thrombotic group (VOD n = 7, TMA n = 2), acute graft-versus-host disease (aGVHD) group (n = 27), infection group (n = 41) and non-complication group (n = 17). Alterations of serum concentration of tissue factor positive microparticles (TF(+) MP) and endothelial microparticles (EMP) were analyzed by flow cytometry during the process of conditioning treatment and the early stage after transplantation. The relation of these 2 kinds of MP with complications was analysed. (1) The levels of TF(+) MP and EMP of patients undogoing allo-HSCT before conditioning treatment were obviously higher than those in normal controls, and showed some elevation during different times, but there was no significant statistical difference. Although the levels of TF(+) MP and EMP at the end of conditioning treatment were some higher than those before conditioning treatment, but there was no statistical difference between them. (2)The levels of TF(+) MP and EMP in thrombotic group were obviously higher than those in aGVHD group and infection group (P < 0.05). (3)The levels of TF(+) MP and EMP in thrombotic group at different times were significant differences from those in other groups (P < 0.05), and the levels of TF(+) MP and EMP were no significant difference from those in non-complication group. The increase of the TF(+) MP and EMP levels may be associated with occurrence of thrombosis after transplantation, indicating occurrence of the thrombotic complications, like hepatic vein occulusive disease (HVOD). The dynamically monitoring levels of TF(+) MP and EMP contributes to early discovery of thrombotic complications.

  2. CCND1-CDK4-mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo.

    PubMed

    Mende, Nicole; Kuchen, Erika E; Lesche, Mathias; Grinenko, Tatyana; Kokkaliaris, Konstantinos D; Hanenberg, Helmut; Lindemann, Dirk; Dahl, Andreas; Platz, Alexander; Höfer, Thomas; Calegari, Federico; Waskow, Claudia

    2015-07-27

    Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1-CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1-CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1-CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis. © 2015 Mende et al.

  3. Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

    PubMed Central

    Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

    1997-01-01

    We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate

  4. Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

    PubMed

    Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

    1997-03-01

    We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate

  5. Mobilization of peripheral blood progenitor cells by chemotherapy and granulocyte-macrophage colony-stimulating factor for hematologic support after high-dose intensification for breast cancer.

    PubMed

    Elias, A D; Ayash, L; Anderson, K C; Hunt, M; Wheeler, C; Schwartz, G; Tepler, I; Mazanet, R; Lynch, C; Pap, S

    1992-06-01

    High-dose therapy with autologous marrow support results in durable complete remissions in selected patients with relapsed lymphoma and leukemia who cannot be cured with conventional dose therapy. However, substantial morbidity and mortality result from the 3- to 6-week period of marrow aplasia until the reinfused marrow recovers adequate hematopoietic function. Hematopoietic growth factors, particularly used after chemotherapy, can increase the number of peripheral blood progenitor cells (PBPCs) present in systemic circulation. The reinfusion of PBPCs with marrow has recently been reported to reduce the time to recovery of adequate marrow function. This study was designed to determine whether granulocyte-macrophage colony-stimulating factor (GM-CSF)-mobilized PBPCs alone (without marrow) would result in rapid and reliable hematopoietic reconstitution. Sixteen patients with metastatic breast cancer were treated with four cycles of doxorubicin, 5-fluorouracil, and methotrexate (AFM induction). Patients responding after the first two cycles were administered GM-CSF after the third and fourth cycles to recruit PBPCs for collection by two leukapheresis per cycle. These PBPCs were reinfused as the sole source of hematopoietic support after high doses of cyclophosphamide, thiotepa, and carboplatin. No marrow or hematopoietic cytokines were used after progenitor cell reinfusion. Granulocytes greater than or equal to 500/microL was observed on a median of day 14 (range, 8 to 57). Transfusion independence of platelets greater than or equal to 20,000/microL occurred on a median day of 12 (range, 8 to 134). However, three patients required the use of a reserve marrow for slow platelet engraftment. In retrospect, these patients were characterized by poor baseline bone marrow cellularity and poor platelet recovery after AFM induction therapy. When compared with 29 historical control patients who had received the same high-dose intensification chemotherapy using autologous

  6. Prenatal stress delays inhibitory neuron progenitor migration in the developing neocortex

    PubMed Central

    Stevens, Hanna E.; Su, Tina; Yanagawa, Yuchio; Vaccarino, Flora M.

    2012-01-01

    Summary Prenatal stress has been widely demonstrated to have links with behavioral problems in clinical populations and animal models, however, few investigations have examined the immediate developmental events that are affected by prenatal stress. Here, we utilize GAD67GFP transgenic mice in which GABAergic progenitors express green fluorescent protein (GFP) to examine the impact of prenatal stress on the development of these precursors to inhibitory neurons. Pregnant female mice were exposed to restraint stress three times daily from embryonic day 12 (E12) onwards. Their offspring demonstrated changes in the distribution of GFP-positive (GFP+) GABAergic progenitors in the telencephalon as early as E13 and persisting until postnatal day 0. Changes in distribution reflected alterations in tangential migration and radial integration of GFP+ cells into the developing cortical plate. Fate mapping of GAD67GFP+progenitors with bromodeoxyuridine injected at E13 demonstrated a significant increase of these cells at P0 in anterior white matter. An overall decrease in GAD67GFP+ progenitors at P0 in medial frontal cortex could not be attributed to a reduction in cell proliferation. Significant changes in dlx2, nkx2.1 and their downstream target erbb4, transcription factors which regulate interneuron migration, were found within the prenatally-stressed developing forebrain, while no differences were seen in mash1, a determinant of interneuron fate, bdnf, a maturation factor for GABAergic cells or fgf2, an early growth/differentiation factor. These results demonstrate that early disruption in GABAergic progenitor migration caused by prenatal stress may be responsible for neuronal defects in disorders with GABAergic abnormalities like schizophrenia. PMID:22910687

  7. Regulation and Function of TIFAB in Myelodysplastic Syndrome

    DTIC Science & Technology

    2014-08-01

    knockdown of TIFAB in human CD34+ hematopoietic stem /progenitor cells results in increased survival and proliferation, TIFAB inhibits TRAF6 protein...regulator of human hematopoietic cells . Our key observations show that knockdown of TIFAB in human CD34+ hematopoietic stem /progenitor cells results in...reveals an essential role of mTOR in hematopoietic stem cell engraftment and hematopoiesis. (2013). Haematologica,!98(9):!135381358.! PMID:!23716557

  8. Leptin promotes the mobilization of vascular progenitor cells and neovascularization by NOX2-mediated activation of MMP9.

    PubMed

    Schroeter, Marco R; Stein, Susanne; Heida, Nana-Maria; Leifheit-Nestler, Maren; Cheng, I-Fen; Gogiraju, Rajinikanth; Christiansen, Hans; Maier, Lars S; Shah, Ajay M; Hasenfuss, Gerd; Konstantinides, Stavros; Schäfer, Katrin

    2012-01-01

    Bone marrow (BM) progenitors participate in new vessel formation and endothelial repair. The leptin receptor (ObR) is expressed on hematopoietic cells; however, the effects of leptin on BM progenitor cells and their angiogenic potential are unknown. In the present study, we show that the short-term administration of leptin (over five consecutive days) into wild-type mice increased the number of circulating, BM-derived sca-1(+), flk-1(+) vascular progenitors, 95 ± 1.7% of which also expressed ObR. Ex vivo stimulation of BM cells with leptin enhanced the expression of NADPH oxidase isoform 2 (NOX2), and the leptin-induced increase in reactive oxygen species production, matrix metalloproteinase-9 (MMP9) expression and circulating soluble KitL levels was absent in mice lacking NOX2. Furthermore, intraperitoneal injections of leptin improved perfusion and increased the number of BM-derived, CD31-positive endothelial cells in ischaemic hindlimbs after femoral artery ligation. The effects of leptin on the mobilization of sca-1(+), flk-1(+) cells and neovascularization were abolished in mice transplanted with BM from ObR-deficient and in NOX2(-/-) mice. Our findings suggest that the angiogenic effects of leptin involve sca-1(+), flk-1(+) vascular progenitor cells mobilized from the BM in response to ObR-mediated activation of NOX2, increased MMP9 expression, and sKitL release.

  9. Multipotent progenitor cells are present in human peripheral blood.

    PubMed

    Cesselli, Daniela; Beltrami, Antonio Paolo; Rigo, Silvia; Bergamin, Natascha; D'Aurizio, Federica; Verardo, Roberto; Piazza, Silvano; Klaric, Enio; Fanin, Renato; Toffoletto, Barbara; Marzinotto, Stefania; Mariuzzi, Laura; Finato, Nicoletta; Pandolfi, Maura; Leri, Annarosa; Schneider, Claudio; Beltrami, Carlo Alberto; Anversa, Piero

    2009-05-22

    To determine whether the peripheral blood in humans contains a population of multipotent progenitor cells (MPCs), products of leukapheresis were obtained from healthy donor volunteers following the administration of granulocyte colony-stimulating factor. Small clusters of adherent proliferating cells were collected, and these cells continued to divide up to 40 population doublings without reaching replicative senescence and growth arrest. MPCs were positive for the transcription factors Nanog, Oct3/4, Sox2, c-Myc, and Klf4 and expressed several antigens characteristic of mesenchymal stem cells. However, they were negative for markers of hematopoietic stem/progenitor cells and bone marrow cell lineages. MPCs had a cloning efficiency of approximately 3%, and following their expansion, retained a highly immature phenotype. Under permissive culture conditions, MPCs differentiated into neurons, glial cells, hepatocytes, cardiomyocytes, endothelial cells, and osteoblasts. Moreover, the gene expression profile of MPCs partially overlapped with that of neural and embryonic stem cells, further demonstrating their primitive, uncommitted phenotype. Following subcutaneous transplantation in nonimmunosuppressed mice, MPCs migrated to distant organs and integrated structurally and functionally within the new tissue, acquiring the identity of resident parenchymal cells. In conclusion, undifferentiated cells with properties of embryonic stem cells can be isolated and expanded from human peripheral blood after granulocyte colony-stimulating factor administration. This cell pool may constitute a unique source of autologous cells with critical clinical import.

  10. The non-canonical Wnt receptor Ryk regulates hematopoietic stem cell repopulation in part by controlling proliferation and apoptosis.

    PubMed

    Famili, Farbod; Perez, Laura Garcia; Naber, Brigitta Ae; Noordermeer, Jasprina N; Fradkin, Lee G; Staal, Frank Jt

    2016-11-24

    The development of blood and immune cells requires strict control by various signaling pathways in order to regulate self-renewal, differentiation and apoptosis in stem and progenitor cells. Recent evidence indicates critical roles for the canonical and non-canonical Wnt pathways in hematopoiesis. The non-canonical Wnt pathway is important for establishment of cell polarity and cell migration and regulates apoptosis in the thymus. We here investigate the role of the non-canonical Wnt receptor Ryk in hematopoiesis and lymphoid development. We show that there are dynamic changes in Ryk expression during development and in different hematopoietic tissues. Functionally, Ryk regulates NK cell development in a temporal fashion. Moreover, Ryk-deficient mice show diminished, but not absent self-renewal of hematopoietic stem cells (HSC), via effects on mildly increased proliferation and apoptosis. Thus, Ryk deficiency in HSCs from fetal liver reduces their quiescence, leading to proliferation-induced apoptosis and decreased self-renewal.

  11. Lin28 sustains early renal progenitors and induces Wilms tumor

    PubMed Central

    Urbach, Achia; Yermalovich, Alena; Zhang, Jin; Spina, Catherine S.; Zhu, Hao; Perez-Atayde, Antonio R.; Shukrun, Rachel; Charlton, Jocelyn; Sebire, Neil; Mifsud, William; Dekel, Benjamin; Pritchard-Jones, Kathy; Daley, George Q.

    2014-01-01

    Wilms Tumor, the most common pediatric kidney cancer, evolves from the failure of terminal differentiation of the embryonic kidney. Here we show that overexpression of the heterochronic regulator Lin28 during kidney development in mice markedly expands nephrogenic progenitors by blocking their final wave of differentiation, ultimately resulting in a pathology highly reminiscent of Wilms tumor. Using lineage-specific promoters to target Lin28 to specific cell types, we observed Wilms tumor only when Lin28 is aberrantly expressed in multiple derivatives of the intermediate mesoderm, implicating the cell of origin as a multipotential renal progenitor. We show that withdrawal of Lin28 expression reverts tumorigenesis and markedly expands the numbers of glomerulus-like structures and that tumor formation is suppressed by enforced expression of Let-7 microRNA. Finally, we demonstrate overexpression of the LIN28B paralog in a significant percentage of human Wilms tumor. Our data thus implicate the Lin28/Let-7 pathway in kidney development and tumorigenesis. PMID:24732380

  12. Negative feedback on the effects of stem cell factor on hematopoiesis is partly mediated through neutral endopeptidase activity on substance P: a combined functional and proteomic study.

    PubMed

    Joshi, D D; Dang, A; Yadav, P; Qian, J; Bandari, P S; Chen, K; Donnelly, R; Castro, T; Gascon, P; Haider, A; Rameshwar, P

    2001-11-01

    Hematopoietic regulation is a complex but dynamic process regulated by intercellular and intracellular interactions within the bone marrow (BM) microenvironment. Through neurokinin-1 (NK-1) and NK-2 receptors, peptides (eg, substance P [SP]) encoded by the preprotachykinin-I gene mediate distinct hematopoietic effects. Cytokines, associated with hematopoietic stimulation, and SP regulate the expression of each other in BM mesenchymal and immune cells. Neutral endopeptidase (NEP) uses SP as a substrate to produce SP(1-4), which inhibits the proliferation of matured myeloid progenitor. This study determines whether the degradation of SP to SP(1-4) by endogenous NEP in BM stroma could be a feedback on hematopoietic stimulation by stem cell factor (SCF). SP(1-4) induced the production of transforming growth factor (TGF)-beta and tumor necrosis factor-alpha in BM stroma. TGF-beta production accounted for part of the inhibitory effects by SP(1-4) on the proliferation of early (granulocyte-macrophage colony-forming units) and late (long-term culture-initiating cells) hematopoietic progenitors. Enzyme-linked immunosorbent assays and/or protein-chip arrays indicated a timeline change of SP to SP(1-4) in BM stroma stimulated with SCF, which correlated with increase in NEP messenger RNA. Since SP and its fragment, SP(1-4), interact with the same receptor to mediate opposing hematopoietic effects, 2 interactive studies were done to understand the dual responses of NK-1: (1) a 3-dimensional molecular model of NK-1 and SP and (2) screening of a random dodecapeptide library for SP(1-4) interacting sites. The effects of SP(1-4) on hematopoietic progenitors and the timeline change of SP to SP(1-4), together with the 3-dimensional model, provide a partial explanation for the feedback on the stimulatory effects of SCF and SP on hematopoiesis.

  13. Pancreatic β-cell regeneration: Facultative or dedicated progenitors?

    PubMed

    Afelik, Solomon; Rovira, Meritxell

    2017-04-15

    The adult pancreas is only capable of limited regeneration. Unlike highly regenerative tissues such as the skin, intestinal crypts and hematopoietic system, no dedicated adult stem cells or stem cell niche have so far been identified within the adult pancreas. New β cells have been shown to form in the adult pancreas, in response to high physiological demand or experimental β-cell ablation, mostly by replication of existing β cells. The possibility that new β cells are formed from other sources is currently a point of major controversy. Under particular injury conditions, fully differentiated pancreatic duct and acinar cells have been shown to dedifferentiate into a progenitor-like state, however the extent, to which ductal, acinar or other endocrine cells contribute to restoring pancreatic β-cell mass remains to be resolved. In this review we focus on regenerative events in the pancreas with emphasis on the restoration of β-cell mass. We present an overview of regenerative responses noted within the different pancreatic lineages, following injury. We also highlight the intrinsic plasticity of the adult pancreas that allows for inter-conversion of fully differentiated pancreatic lineages through manipulation of few genes or growth factors. Taken together, evidence from a number of studies suggest that differentiated pancreatic lineages could act as facultative progenitor cells, but the extent to which these contribute to β-cell regeneration in vivo is still a matter of contention. Copyright © 2016. Published by Elsevier B.V.

  14. Huntingtin Interacting Protein 1 mutations lead to abnormal hematopoiesis, spinal defects and cataracts.

    PubMed

    Oravecz-Wilson, Katherine I; Kiel, Mark J; Li, Lina; Rao, Dinesh S; Saint-Dic, Djenann; Kumar, Priti D; Provot, Melissa M; Hankenson, Kurt D; Reddy, Venkat N; Lieberman, Andrew P; Morrison, Sean J; Ross, Theodora S

    2004-04-15

    Huntingtin Interacting Protein 1 (HIP1) binds clathrin and AP2, is overexpressed in multiple human tumors, and transforms fibroblasts. The function of HIP1 is unknown although it is thought to play a fundamental role in clathrin trafficking. Gene-targeted Hip1-/- mice develop premature testicular degeneration and severe spinal deformities. Yet, although HIP1 is expressed in many tissues including the spleen and bone marrow and was part of a leukemogenic translocation, its role in hematopoiesis has not been examined. In this study we report that three different mutations of murine Hip1 lead to hematopoietic abnormalities reflected by diminished early progenitor frequencies and resistance to 5-FU-induced bone marrow toxicity. Two of the Hip1 mutant lines also display the previously described spinal defects. These observations indicate that, in addition to being required for the survival/proliferation of cancer cells and germline progenitors, HIP1 is also required for the survival/proliferation of diverse types of somatic cells, including hematopoietic progenitors.

  15. Targeting of the BLT2 in chronic myeloid leukemia inhibits leukemia stem/progenitor cell function

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xiao, Meifang; Ai, Hongmei; Li, Tao

    Imatinib, a tyrosine kinase inhibitor (TKI) has significantly improved clinical outcome for chronic myeloid leukemia (CML) patients. However, patients develop resistance when the disease progresses to the blast phase (BP) and the mechanisms are not well understood. Here we show that BCR-ABL activates BLT2 in hematopoietic stem/progenitor cells to promote leukemogenesis and this involves the p53 signaling pathway. Compared to normal bone marrow (NBM), the mRNA and protein levels of BLT2 are significantly increased in BP-CML CD34{sup +} stem/progenitor cells. This is correlated with increasing BCR-ABL expression. In contrast, knockdown of BCR-ABL or inhibition of its tyrosine kinase activity decreasesmore » Blt2 protein level. BLT2 inhibition induces apoptosis, inhibits proliferation, colony formation and self-renewal capacity of CD34{sup +} cells from TKI-resistant BP-CML patients. Importantly, the inhibitory effects of BCR-ABL TKI on CML stem/progenitor cells are further enhanced upon combination with BLT2 inhibition. We further show that BLT2 activation selectively suppresses p53 but not Wnt or BMP-mediated luciferase activity and transcription. Our results demonstrate that BLT2 is a novel pathway activated by BCR-ABL and critically involved in the resistance of BP-CML CD34{sup +} stem/progenitors to TKIs treatment. Our findings suggest that BLT2 and p53 can serve as therapeutic targets for CML treatment. - Highlights: • BCR-ABL regulates BLT2 expression to promote leukemogenesis. • BLT2 is essential to maintain CML cell function. • Activation of BLT2 suppresses p53 signaling pathway in CML cells. • Inhibition of BLT2 and BCR-ABL synergize in eliminating CML CD34{sup +} stem/progenitors.« less

  16. Nitrogen-containing bisphosphonate induces a newly discovered hematopoietic structure in the omentum of an anemic mouse model by stimulating G-CSF production.

    PubMed

    Otsuka, Hirotada; Yagi, Hideki; Endo, Yasuo; Soeta, Satoshi; Nonaka, Naoko; Nakamura, Masanori

    2017-02-01

    We previously reported that the injection of nitrogen-containing bisphosphonate (NBP) induced the site of erythropoiesis to shift from the bone marrow (BM) to the spleen. Our previous study established a severely anemic mouse model that was treated with a combination of NBP with phenylhydrazine (PHZ), which induced newly discovered hematopoietic organs in the omentum. No reports have shown that new hematopoietic organs form under any condition. We characterized the structures and factors related to the formation of these new organs. Splenectomized mice were treated with NBP to inhibit erythropoiesis in the BM and then injected with PHZ to induce hemolytic anemia. The mice showed severe anemia and wine-colored structures appeared in the omentum. Some hematopoietic cells, including megakaryocytes, and well-developed sinuses were observed in these structures. Numerous TER119-positive erythroblasts were located with cells positive for PCNA, a cell proliferation marker. C-kit-positive cells were detected and mRNAs related to hematopoiesis were expressed in these structures. Moreover, TER119-positive erythroblasts emerged and formed clusters and hematopoiesis-related factors were detected in the omentum of mice treated with NBP and PHZ. The levels of G-CSF in the serum and hematopoietic progenitor cells (HPCs) in the peripheral blood were increased upon treatment with both NBP and PHZ. These results suggest that the induced hematopoietic structures act as the sites of erythropoiesis and that NBP-induced G-CSF production causes HPC mobilization, homing and colonization in the omentum because they constitutively express some factors, including SDF-1; thus, the newly discovered hematopoietic structure in this study might be formed.

  17. Progenitors of Secondary Crest Myofibroblasts are Developmentally Committed in Early Lung Mesoderm

    PubMed Central

    Li, Changgong; Li, Min; Li, Sha; Xing, Yiming; Yang, Chang-Yo; Li, Aimin; Borok, Zea; De Langhe, Stijn; Minoo, Parviz

    2015-01-01

    Development of the mammalian lung is predicated on cross-communications between two highly interactive tissues, the endodermally-derived epithelium and the mesodermally-derived pulmonary mesenchyme. While much attention has been paid the lung epithelium, the pulmonary mesenchyme, partly due to lack of specific tractable markers remains under-investigated. The lung mesenchyme is derived from the lateral plate mesoderm and is the principal recipient of Hedgehog (Hh) signaling, a morphogenetic network that regulates multiple aspects of embryonic development. Using the Hh-responsive Gli1-creERT2 mouse line, we identified the mesodermal targets of Hh signaling at various time points during embryonic and postnatal lung development. Cell lineage analysis showed these cells serve as progenitors to contribute to multiple lineages of mesodermally-derived differentiated cell types that include parenchymal or interstitial myofibroblasts, parabronchial and perivascular smooth muscle as well as rare populations of cells within the mesothelium. Most importantly, Gli1-creERT2 identified the progenitors of secondary crest myofibroblasts, a hitherto intractable cell type that plays a key role in alveolar formation, a vital process about which little is currently known. Transcriptome analysis of Hh-targeted progenitor cells transitioning from the pseudoglandular to the saccular phase of lung development revealed important modulations of key signaling pathways. Amongst these, there was significant down-regulation of canonical WNT signaling. Ectopic stabilization of β-Catenin via inactivation of Apc by Gli1-creERT2 expanded the Hh-targeted progenitor pools, which caused the formation of fibroblastic masses within the lung parenchyma. The Gli1-creERT2 mouse line represents a novel tool in the analysis of mesenchymal cell biology and alveolar formation during lung development. PMID:25448080

  18. Extramedullary Myelopoiesis in Malaria Depends on Mobilization of Myeloid-Restricted Progenitors by IFN-γ Induced Chemokines

    PubMed Central

    Belyaev, Nikolai N.; Biró, Judit; Langhorne, Jean; Potocnik, Alexandre J.

    2013-01-01

    Resolution of a variety of acute bacterial and parasitic infections critically relies on the stimulation of myelopoiesis leading in cases to extramedullary hematopoiesis. Here, we report the isolation of the earliest myeloid-restricted progenitors in acute infection with the rodent malaria parasite, Plasmodium chabaudi. The rapid disappearance of these infection-induced myeloid progenitors from the bone marrow (BM) equated with contraction of the functional myeloid potential in that organ. The loss of BM myelopoiesis was not affected by the complete genetic inactivation of toll-like receptor signaling. De-activation of IFN-γ signaling completely abrogated the contraction of BM myeloid progenitors. Radiation chimeras of Ifngr1-null and control BM revealed that IFN-γ signaling in an irradiation-resistant stromal compartment was crucial for the loss of early myeloid progenitors. Systemic IFN-γ triggered the secretion of C-C motif ligand chemokines CCL2 and CCL7 leading to the egress of early, myeloid-committed progenitors from the bone marrow mediated by their common receptor CCR2. The mobilization of myeloid progenitors initiated extramedullary myelopoiesis in the spleen in a CCR2-dependent manner resulting in augmented myelopoiesis during acute malaria. Consistent with the lack of splenic myelopoiesis in the absence of CCR2 we observed a significant persistence of parasitemia in malaria infected CCR2-deficient hosts. Our findings reveal how the activated immune system mobilizes early myeloid progenitors out of the BM thereby transiently establishing myelopoiesis in the spleen in order to contain and resolve the infection locally. PMID:23762028

  19. Functional Traits Differ between Cereal Crop Progenitors and Other Wild Grasses Gathered in the Neolithic Fertile Crescent

    PubMed Central

    Cunniff, Jennifer; Wilkinson, Sarah; Charles, Michael; Jones, Glynis; Rees, Mark; Osborne, Colin P.

    2014-01-01

    The reasons why some plant species were selected as crops and others were abandoned during the Neolithic emergence of agriculture are poorly understood. We tested the hypothesis that the traits of Fertile Crescent crop progenitors were advantageous in the fertile, disturbed habitats surrounding early settlements and in cultivated fields. We screened functional traits related to competition and disturbance in a group of grass species that were increasingly exploited by early plant gatherers, and that were later domesticated (crop progenitors); and in a set of grass species for which there is archaeological evidence of gathering, but which were never domesticated (wild species). We hypothesised that crop progenitors would have greater seed mass, growth rate, height and yield than wild species, as these traits are indicative of greater competitive ability, and that crop progenitors would be more resilient to defoliation. Our results show that crop progenitors have larger seed mass than wild species, germinate faster and have greater seedling size. Increased seed size is weakly but positively correlated with a higher growth rate, which is primarily driven by greater biomass assimilation per unit leaf area. Crop progenitors also tend to have a taller stature, greater grain yield and higher resilience to defoliation. Collectively, the data are consistent with the hypothesis that adaptations to competition and disturbance gave crop progenitors a selective advantage in the areas surrounding early human settlements and in cultivated environments, leading to their adoption as crops through processes of unconscious selection. PMID:24489941

  20. Proximity-Based Differential Single-Cell Analysis of the Niche to Identify Stem/Progenitor Cell Regulators.

    PubMed

    Silberstein, Lev; Goncalves, Kevin A; Kharchenko, Peter V; Turcotte, Raphael; Kfoury, Youmna; Mercier, Francois; Baryawno, Ninib; Severe, Nicolas; Bachand, Jacqueline; Spencer, Joel A; Papazian, Ani; Lee, Dongjun; Chitteti, Brahmananda Reddy; Srour, Edward F; Hoggatt, Jonathan; Tate, Tiffany; Lo Celso, Cristina; Ono, Noriaki; Nutt, Stephen; Heino, Jyrki; Sipilä, Kalle; Shioda, Toshihiro; Osawa, Masatake; Lin, Charles P; Hu, Guo-Fu; Scadden, David T

    2016-10-06

    Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on the differential single-cell gene expression analysis of mesenchymal osteolineage cells close to, and further removed from, hematopoietic stem/progenitor cells (HSPCs) to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. We functionally examined, among the genes that were preferentially expressed in proximal cells, three secreted or cell-surface molecules not previously connected to HSPC biology-the secreted RNase angiogenin, the cytokine IL18, and the adhesion molecule Embigin-and discovered that all of these factors are HSPC quiescence regulators. Therefore, our proximity-based differential single-cell approach reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance the understanding of microenvironmental regulation of stem cell function. Copyright © 2016 Elsevier Inc. All rights reserved.