Wnt5a Regulates Hematopoietic Stem Cell Proliferation and Repopulation Through the Ryk Receptor

PubMed Central

Povinelli, Benjamin J.; Nemeth, Michael J.

2017-01-01

Proper regulation of the balance between hematopoietic stem cell (HSC) proliferation, self-renewal, and differentiation is necessary to maintain hematopoiesis throughout life. The Wnt family of ligands has been implicated as critical regulators of these processes through a network of signaling pathways. Previously, we have demonstrated that the Wnt5a ligand can induce HSC quiescence through a noncanonical Wnt pathway, resulting in an increased ability to reconstitute hematopoiesis. In this study, we tested the hypothesis that the Ryk protein, a Wnt ligand receptor that can bind the Wnt5a ligand, regulated the response of HSCs to Wnt5a. We observed that inhibiting Ryk blocked the ability of Wnt5a to induce HSC quiescence and enhance short-term and long-term hematopoietic repopulation. We found that Wnt5a suppressed production of reactive oxygen species, a known inducer of HSC proliferation. The ability of Wnt5a to inhibit ROS production was also regulated by Ryk. From these data, we propose that Wnt5a regulates HSC quiescence and hematopoietic repopulation through the Ryk receptor and that this process is mediated by suppression of reactive oxygen species. PMID:23939973

Wnt5a regulates hematopoietic stem cell proliferation and repopulation through the Ryk receptor.

PubMed

Povinelli, Benjamin J; Nemeth, Michael J

2014-01-01

Proper regulation of the balance between hematopoietic stem cell (HSC) proliferation, self-renewal, and differentiation is necessary to maintain hematopoiesis throughout life. The Wnt family of ligands has been implicated as critical regulators of these processes through a network of signaling pathways. Previously, we have demonstrated that the Wnt5a ligand can induce HSC quiescence through a noncanonical Wnt pathway, resulting in an increased ability to reconstitute hematopoiesis. In this study, we tested the hypothesis that the Ryk protein, a Wnt ligand receptor that can bind the Wnt5a ligand, regulated the response of HSCs to Wnt5a. We observed that inhibiting Ryk blocked the ability of Wnt5a to induce HSC quiescence and enhance short-term and long-term hematopoietic repopulation. We found that Wnt5a suppressed production of reactive oxygen species, a known inducer of HSC proliferation. The ability of Wnt5a to inhibit ROS production was also regulated by Ryk. From these data, we propose that Wnt5a regulates HSC quiescence and hematopoietic repopulation through the Ryk receptor and that this process is mediated by suppression of reactive oxygen species. © 2013 AlphaMed Press.

Second Cancers After Fractionated Radiotherapy: Stochastic Population Dynamics Effects

NASA Technical Reports Server (NTRS)

Sachs, Rainer K.; Shuryak, Igor; Brenner, David; Fakir, Hatim; Hahnfeldt, Philip

2007-01-01

When ionizing radiation is used in cancer therapy it can induce second cancers in nearby organs. Mainly due to longer patient survival times, these second cancers have become of increasing concern. Estimating the risk of solid second cancers involves modeling: because of long latency times, available data is usually for older, obsolescent treatment regimens. Moreover, modeling second cancers gives unique insights into human carcinogenesis, since the therapy involves administering well characterized doses of a well studied carcinogen, followed by long-term monitoring. In addition to putative radiation initiation that produces pre-malignant cells, inactivation (i.e. cell killing), and subsequent cell repopulation by proliferation can be important at the doses relevant to second cancer situations. A recent initiation/inactivation/proliferation (IIP) model characterized quantitatively the observed occurrence of second breast and lung cancers, using a deterministic cell population dynamics approach. To analyze ifradiation-initiated pre-malignant clones become extinct before full repopulation can occur, we here give a stochastic version of this I I model. Combining Monte Carlo simulations with standard solutions for time-inhomogeneous birth-death equations, we show that repeated cycles of inactivation and repopulation, as occur during fractionated radiation therapy, can lead to distributions of pre-malignant cells per patient with variance >> mean, even when pre-malignant clones are Poisson-distributed. Thus fewer patients would be affected, but with a higher probability, than a deterministic model, tracking average pre-malignant cell numbers, would predict. Our results are applied to data on breast cancers after radiotherapy for Hodgkin disease. The stochastic IIP analysis, unlike the deterministic one, indicates: a) initiated, pre-malignant cells can have a growth advantage during repopulation, not just during the longer tumor latency period that follows; b) weekend treatment gaps during radiotherapy, apart from decreasing the probability of eradicating the primary cancer, substantially increase the risk of later second cancers.

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.

Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells

PubMed Central

Himburg, Heather A; Muramoto, Garrett G; Daher, Pamela; Meadows, Sarah K; Russell, J Lauren; Doan, Phuong; Chi, Jen-Tsan; Salter, Alice B; Lento, William E; Reya, Tannishtha; Chao, Nelson; Chute, John P

2013-01-01

Hematopoietic stem cell (HSC) self-renewal is regulated by both intrinsic and extrinsic signals. Although some of the pathways that regulate HSC self-renewal have been uncovered, it remains largely unknown whether these pathways can be triggered by deliverable growth factors to induce HSC growth or regeneration. Here we show that pleiotrophin, a neurite outgrowth factor with no known function in hematopoiesis, efficiently promotes HSC expansion in vitro and HSC regeneration in vivo. Treatment of mouse bone marrow HSCs with pleiotrophin caused a marked increase in long-term repopulating HSC counts in culture, as measured in competitive repopulating assays. Treatment of human cord blood CD34+CDCD38−Lin− cells with pleiotrophin also substantially increased severe combined immunodeficient (SCID)-repopulating cell counts in culture, compared to input and cytokine-treated cultures. Systemic administration of pleiotrophin to irradiated mice caused a pronounced expansion of bone marrow stem and progenitor cells in vivo, indicating that pleiotrophin is a regenerative growth factor for HSCs. Mechanistically, pleiotrophin activated phosphoinositide 3-kinase (PI3K) signaling in HSCs; antagonism of PI3K or Notch signaling inhibited pleiotrophin-mediated expansion of HSCs in culture. We identify the secreted growth factor pleiotrophin as a new regulator of both HSC expansion and regeneration PMID:20305662

Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells.

PubMed

Himburg, Heather A; Muramoto, Garrett G; Daher, Pamela; Meadows, Sarah K; Russell, J Lauren; Doan, Phuong; Chi, Jen-Tsan; Salter, Alice B; Lento, William E; Reya, Tannishtha; Chao, Nelson J; Chute, John P

2010-04-01

Hematopoietic stem cell (HSC) self-renewal is regulated by both intrinsic and extrinsic signals. Although some of the pathways that regulate HSC self-renewal have been uncovered, it remains largely unknown whether these pathways can be triggered by deliverable growth factors to induce HSC growth or regeneration. Here we show that pleiotrophin, a neurite outgrowth factor with no known function in hematopoiesis, efficiently promotes HSC expansion in vitro and HSC regeneration in vivo. Treatment of mouse bone marrow HSCs with pleiotrophin caused a marked increase in long-term repopulating HSC numbers in culture, as measured in competitive repopulating assays. Treatment of human cord blood CD34(+)CDCD38(-)Lin(-) cells with pleiotrophin also substantially increased severe combined immunodeficient (SCID)-repopulating cell counts in culture, compared to input and cytokine-treated cultures. Systemic administration of pleiotrophin to irradiated mice caused a pronounced expansion of bone marrow stem and progenitor cells in vivo, indicating that pleiotrophin is a regenerative growth factor for HSCs. Mechanistically, pleiotrophin activated phosphoinositide 3-kinase (PI3K) signaling in HSCs; antagonism of PI3K or Notch signaling inhibited pleiotrophin-mediated expansion of HSCs in culture. We identify the secreted growth factor pleiotrophin as a new regulator of both HSC expansion and regeneration.

Spontaneous circulation of myeloid-lymphoid-initiating cells and SCID-repopulating cells in sickle cell crisis.

PubMed

Lamming, Christopher E D; Augustin, Lance; Blackstad, Mark; Lund, Troy C; Hebbel, Robert P; Verfaillie, Catherine M

2003-03-01

The only curative therapy for sickle cell disease (SCD) is allogeneic hematopoietic stem cell (HSC) transplantation. Gene therapy approaches for autologous HSC transplantation are being developed. Although earlier engraftment is seen when cells from GCSF-mobilized blood are transplanted than when bone marrow is transplanted, administration of GCSF to patients with SCD can cause significant morbidity. We tested whether primitive hematopoietic progenitors are spontaneously mobilized in the blood of patients with SCD during acute crisis (AC-SCD patients). The frequency of myeloid-lymphoid-initiating cells (ML-ICs) and SCID-repopulating cells (SRCs) was significantly higher in blood from AC-SCD patients than in blood from patients with steady-state SCD or from normal donors. The presence of SRCs in peripheral blood was not associated with detection of long-term culture-initiating cells, consistent with the notion that SRCs are more primitive than long-term culture-initiating cells. As ML-ICs and SRCs were both detected in blood of AC-SCD patients only, these assays may both measure primitive progenitors. The frequency of ML-ICs also correlated with increases in stem cell factor, GCSF, and IL-8 levels in AC-SCD compared with steady-state SCD and normal-donor sera. Because significant numbers of ML-ICs and SRCs are mobilized in the blood without exogenous cytokine treatment during acute crisis of SCD, collection of peripheral blood progenitors during crisis may yield a source of autologous HSCs suitable for ex-vivo correction by gene therapy approaches and subsequent transplantation.

Large Animal Models for Foamy Virus Vector Gene Therapy

PubMed Central

Trobridge, Grant D.; Horn, Peter A.; Beard, Brian C.; Kiem, Hans-Peter

2012-01-01

Foamy virus (FV) vectors have shown great promise for hematopoietic stem cell (HSC) gene therapy. Their ability to efficiently deliver transgenes to multi-lineage long-term repopulating cells in large animal models suggests they will be effective for several human hematopoietic diseases. Here, we review FV vector studies in large animal models, including the use of FV vectors with the mutant O6-methylguanine-DNA methyltransferase, MGMTP140K to increase the number of genetically modified cells after transplantation. In these studies, FV vectors have mediated efficient gene transfer to polyclonal repopulating cells using short ex vivo transduction protocols designed to minimize the negative effects of ex vivo culture on stem cell engraftment. In this regard, FV vectors appear superior to gammaretroviral vectors, which require longer ex vivo culture to effect efficient transduction. FV vectors have also compared favorably with lentiviral vectors when directly compared in the dog model. FV vectors have corrected leukocyte adhesion deficiency and pyruvate kinase deficiency in the dog large animal model. FV vectors also appear safer than gammaretroviral vectors based on a reduced frequency of integrants near promoters and also near proto-oncogenes in canine repopulating cells. Together, these studies suggest that FV vectors should be highly effective for several human hematopoietic diseases, including those that will require relatively high percentages of gene-modified cells to achieve clinical benefit. PMID:23223198

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

Chertkov, J.L.; Drize, N.J.; Gurevitch, O.A.

Intravenously injected bone marrow cells do not participate in the regeneration of hemopoietic stromal progenitors in irradiated mice, nor in the curetted parts of the recipient's marrow. The hemopoietic stromal progenitors in allogeneic chimeras are of recipient origin. The adherent cell layer (ACL) of long-term cultures of allogeneic chimera bone marrow contains only recipient hemopoietic stromal progenitors. However, in ectopic hemopoietic foci produced by marrow implantation under the renal capsule and repopulated by the recipient hemopoietic cells after irradiation and reconstitution by syngeneic hemopoietic cells, the stromal progenitors were of implant donor origin, as were stromal progenitors of the ACLmore » in long-term cultures of hemopoietic cells from ectopic foci. Our results confirm that the stromal and hemopoietic progenitors differ in origin and that hemopoietic stromal progenitors are not transplantable by the intravenous route in mice.« less

Spontaneous circulation of myeloid-lymphoid–initiating cells and SCID-repopulating cells in sickle cell crisis

PubMed Central

Lamming, Christopher E.D.; Augustin, Lance; Blackstad, Mark; Lund, Troy C.; Hebbel, Robert P.; Verfaillie, Catherine M.

2003-01-01

The only curative therapy for sickle cell disease (SCD) is allogeneic hematopoietic stem cell (HSC) transplantation. Gene therapy approaches for autologous HSC transplantation are being developed. Although earlier engraftment is seen when cells from GCSF-mobilized blood are transplanted than when bone marrow is transplanted, administration of GCSF to patients with SCD can cause significant morbidity. We tested whether primitive hematopoietic progenitors are spontaneously mobilized in the blood of patients with SCD during acute crisis (AC-SCD patients). The frequency of myeloid-lymphoid–initiating cells (ML-ICs) and SCID-repopulating cells (SRCs) was significantly higher in blood from AC-SCD patients than in blood from patients with steady-state SCD or from normal donors. The presence of SRCs in peripheral blood was not associated with detection of long-term culture–initiating cells, consistent with the notion that SRCs are more primitive than long-term culture–initiating cells. As ML-ICs and SRCs were both detected in blood of AC-SCD patients only, these assays may both measure primitive progenitors. The frequency of ML-ICs also correlated with increases in stem cell factor, GCSF, and IL-8 levels in AC-SCD compared with steady-state SCD and normal-donor sera. Because significant numbers of ML-ICs and SRCs are mobilized in the blood without exogenous cytokine treatment during acute crisis of SCD, collection of peripheral blood progenitors during crisis may yield a source of autologous HSCs suitable for ex-vivo correction by gene therapy approaches and subsequent transplantation. PMID:12639987

Mammary stem cells and the differentiation hierarchy: current status and perspectives

PubMed Central

Visvader, Jane E.; Stingl, John

2014-01-01

The mammary epithelium is highly responsive to local and systemic signals, which orchestrate morphogenesis of the ductal tree during puberty and pregnancy. Based on transplantation and lineage tracing studies, a hierarchy of stem and progenitor cells has been shown to exist among the mammary epithelium. Lineage tracing has highlighted the existence of bipotent mammary stem cells (MaSCs) in situ as well as long-lived unipotent cells that drive morphogenesis and homeostasis of the ductal tree. Moreover, there is accumulating evidence for a heterogeneous MaSC compartment comprising fetal MaSCs, slow-cycling cells, and both long-term and short-term repopulating cells. In parallel, diverse luminal progenitor subtypes have been identified in mouse and human mammary tissue. Elucidation of the normal cellular hierarchy is an important step toward understanding the “cells of origin” and molecular perturbations that drive breast cancer. PMID:24888586

HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation.

PubMed

Antonchuk, J; Sauvageau, G; Humphries, R K

2001-09-01

Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, we have shown that overexpression of HOXB4 in mouse bone marrow can greatly enhance the level of hematopoietic stem cell (HSC) regeneration achieved at late times (> 4 months) posttransplantation. The objective of this study was to resolve if HOXB4 increases the rate and/or duration of HSC regeneration, and also to see if this enhancement was associated with impaired production of end cells or would lead to competitive reconstitution of all compartments. Retroviral vectors were generated with the GFP reporter gene +/- HOXB4 to enable the isolation and direct tracking of transduced cells in culture or following transplantation. Stem cell recovery was measured by limit dilution assay for long-term competitive repopulating cells (CRU). HOXB4-overexpressing cells have enhanced growth in vitro, as demonstrated by their rapid dominance in mixed cultures and their shortened population doubling time. Furthermore, HOXB4-transduced cells have a marked competitive repopulating advantage in vivo in both primitive and mature compartments. CRU recovery in HOXB4 recipients was extremely rapid, reaching 25% of normal by 14 days posttransplant or some 80-fold greater than control transplant recipients, and attaining normal numbers by 12 weeks. Mice transplanted with even higher numbers of HOXB4-transduced CRU regenerated up to but not beyond the normal CRU levels. HOXB4 is a potent enhancer of primitive hematopoietic cell growth, likely by increasing self-renewal probability but without impairing homeostatic control of HSC population size or the rate of production and maintenance of mature end cells.

Repopulating hematopoietic stem cells from steady-state blood before and after ex vivo culture are enriched in CD34+CD133+CXCR4low fraction.

PubMed

Lapostolle, Véronique; Chevaleyre, Jean; Duchez, Pascale; Rodriguez, Laura; Vlaski-Lafarge, Marija; Sandvig, Ioanna; Brunet de la Grange, Philippe; Ivanovic, Zoran

2018-06-01

Feasibility of ex vivo expansion allows us to consider the steady-state peripheral blood as an alternative source of hematopoietic stem progenitor cells for transplantation when growth factor-induced cell mobilization is contraindicated or inapplicable. Ex vivo expansion dramatically enhances the in vivo reconstituting cell population from steady-state blood. In order to investigate phenotype and the expression of homing molecules, CD34, CD133, CD90, CD45RA, CD26 and CD9 expression was determined on sorted CD34+ cells according to CXCR4 (neg, low, bright) and CD133 expression before and after ex vivo expansion. Hematopoietic stem cell activity was determined in vivo on the basis of hematopoietic repopulation of primary and secondary recipients - NSG immuno-deficient mice. In vivo reconstituting cells in steady-state blood CD34+ cell fraction before expansion belong to the CD133+ population and are CXCR4low or, to a lesser extent, CXCR4neg, while after ex vivo expansion they are contained in only the CD133+CXCR4low cells. The failure of CXCR4bright population to engraft is probably due to the exclusive expression of CD26 by these cells. The limiting-dilution analysis showed that both repopulating cell number and individual proliferative capacity were enhanced by ex vivo expansion. Thus, steady-state peripheral blood cells exhibit a different phenotype compared to mobilized and cord blood ones, as well as to those issued from the bone marrow. This data represent the first phenotypic characterization of steady-state blood cells exhibiting short and long term hematopoietic reconstituting potential, which can be expanded ex vivo, a sine qua non for their subsequent use for transplantation. Copyright © 2018, Ferrata Storti Foundation.

In vivo selection of hematopoietic progenitor cells and temozolomide dose intensification in rhesus macaques through lentiviral transduction with a drug resistance gene

PubMed Central

Larochelle, Andre; Choi, Uimook; Shou, Yan; Naumann, Nora; Loktionova, Natalia A.; Clevenger, Joshua R.; Krouse, Allen; Metzger, Mark; Donahue, Robert E.; Kang, Elizabeth; Stewart, Clinton; Persons, Derek; Malech, Harry L.; Dunbar, Cynthia E.; Sorrentino, Brian P.

2009-01-01

Major limitations to gene therapy using HSCs are low gene transfer efficiency and the inability of most therapeutic genes to confer a selective advantage on the gene-corrected cells. One approach to enrich for gene-modified cells in vivo is to include in the retroviral vector a drug resistance gene, such as the P140K mutant of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT*). We transplanted 5 rhesus macaques with CD34+ cells transduced with lentiviral vectors encoding MGMT* and a fluorescent marker, with or without homeobox B4 (HOXB4), a potent stem cell self-renewal gene. Transgene expression and common integration sites in lymphoid and myeloid lineages several months after transplantation confirmed transduction of long-term repopulating HSCs. However, all animals showed only a transient increase in gene-marked lymphoid and myeloid cells after O6-benzylguanine (BG) and temozolomide (TMZ) administration. In 1 animal, cells transduced with MGMT* lentiviral vectors were protected and expanded after multiple courses of BG/TMZ, providing a substantial increase in the maximum tolerated dose of TMZ. Additional cycles of chemotherapy using 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in similar increases in gene marking levels, but caused high levels of nonhematopoietic toxicity. Inclusion of HOXB4 in the MGMT* vectors resulted in no substantial increase in gene marking or HSC amplification after chemotherapy treatment. Our data therefore suggest that lentivirally mediated gene transfer in transplanted HSCs can provide in vivo chemoprotection of progenitor cells, although selection of long-term repopulating HSCs was not seen. PMID:19509470

Thrombopoietin treatment of one graft in a double cord blood transplant provides early platelet recovery while contributing to long-term engraftment in NSG mice.

PubMed

van der Garde, Mark; van Hensbergen, Yvette; Brand, Anneke; Slot, Manon C; de Graaf-Dijkstra, Alice; Mulder, Arend; Watt, Suzanne M; Zwaginga, Jaap Jan

2015-01-01

Human cord blood (CB) hematopoietic stem cell (HSC) transplants demonstrate delayed early neutrophil and platelet recovery and delayed longer term immune reconstitution compared to bone marrow and mobilized peripheral blood transplants. Despite advances in enhancing early neutrophil engraftment, platelet recovery after CB transplantation is not significantly altered when compared to contemporaneous controls. Recent studies have identified a platelet-biased murine HSC subset, maintained by thrombopoietin (TPO), which has enhanced capacity for short- and long-term platelet reconstitution, can self-renew, and can give rise to myeloid- and lymphoid-biased HSCs. In previous studies, we have shown that transplantation of human CB CD34(+) cells precultured in TPO as a single graft accelerates early platelet recovery as well as yielding long-term repopulation in immune-deficient mice. In this study, using a double CB murine transplant model, we investigated whether TPO cultured human CB CD34(+) cells have a competitive advantage or disadvantage over untreated human CB CD34(+) cells in terms of (1) short-term and longer term platelet recovery and (2) longer term hematological recovery. Our studies demonstrate that the TPO treated graft shows accelerated early platelet recovery without impairing the platelet engraftment of untreated CD34(+) cells. Notably, this was followed by a dominant contribution to platelet production through the untreated CD34(+) cell graft over the intermediate to longer term. Furthermore, although the contribution of the TPO treated graft to long-term hematological engraftment was reduced, the TPO treated and untreated grafts both contributed significantly to long-term chimerism in vivo.

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.

Umbilical cord blood expansion with nicotinamide provides long-term multilineage engraftment.

PubMed

Horwitz, Mitchell E; Chao, Nelson J; Rizzieri, David A; Long, Gwynn D; Sullivan, Keith M; Gasparetto, Cristina; Chute, John P; Morris, Ashley; McDonald, Carolyn; Waters-Pick, Barbara; Stiff, Patrick; Wease, Steven; Peled, Amnon; Snyder, David; Cohen, Einat Galamidi; Shoham, Hadas; Landau, Efrat; Friend, Etty; Peleg, Iddo; Aschengrau, Dorit; Yackoubov, Dima; Kurtzberg, Joanne; Peled, Tony

2014-07-01

Delayed hematopoietic recovery is a major drawback of umbilical cord blood (UCB) transplantation. Transplantation of ex vivo-expanded UCB shortens time to hematopoietic recovery, but long-term, robust engraftment by the expanded unit has yet to be demonstrated. We tested the hypothesis that a UCB-derived cell product consisting of stem cells expanded for 21 days in the presence of nicotinamide and a noncultured T cell fraction (NiCord) can accelerate hematopoietic recovery and provide long-term engraftment. In a phase I trial, 11 adults with hematologic malignancies received myeloablative bone marrow conditioning followed by transplantation with NiCord and a second unmanipulated UCB unit. Safety, hematopoietic recovery, and donor engraftment were assessed and compared with historical controls. No adverse events were attributable to the infusion of NiCord. Complete or partial neutrophil and T cell engraftment derived from NiCord was observed in 8 patients, and NiCord engraftment remained stable in all patients, with a median follow-up of 21 months. Two patients achieved long-term engraftment with the unmanipulated unit. Patients transplanted with NiCord achieved earlier median neutrophil recovery (13 vs. 25 days, P < 0.001) compared with that seen in historical controls. The 1-year overall and progression-free survival rates were 82% and 73%, respectively. UCB-derived hematopoietic stem and progenitor cells expanded in the presence of nicotinamide and transplanted with a T cell-containing fraction contain both short-term and long-term repopulating cells. The results justify further study of NiCord transplantation as a single UCB graft. If long-term safety is confirmed, NiCord has the potential to broaden accessibility and reduce the toxicity of UCB transplantation. Clinicaltrials.gov NCT01221857. Gamida Cell Ltd.

Absence of CD34 on some human SCID-repopulating cells.

PubMed

Dick, J E

1999-04-30

The availability of in vivo repopulation assays has greatly aided the study of human hematopoietic stem cells. Here, I shall review recent data that has identified a novel class of human repopulating cells that do not express classical stem cell markers including CD34 but still retain the ability to repopulate nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice.

Identifying States along the Hematopoietic Stem Cell Differentiation Hierarchy with Single Cell Specificity via Raman Spectroscopy.

PubMed

Ilin, Yelena; Choi, Ji Sun; Harley, Brendan A C; Kraft, Mary L

2015-11-17

A major challenge for expanding specific types of hematopoietic cells ex vivo for the treatment of blood cell pathologies is identifying the combinations of cellular and matrix cues that direct hematopoietic stem cells (HSC) to self-renew or differentiate into cell populations ex vivo. Microscale screening platforms enable minimizing the number of rare HSCs required to screen the effects of numerous cues on HSC fate decisions. These platforms create a strong demand for label-free methods that accurately identify the fate decisions of individual hematopoietic cells at specific locations on the platform. We demonstrate the capacity to identify discrete cells along the HSC differentiation hierarchy via multivariate analysis of Raman spectra. Notably, cell state identification is accurate for individual cells and independent of the biophysical properties of the functionalized polyacrylamide gels upon which these cells are cultured. We report partial least-squares discriminant analysis (PLS-DA) models of single cell Raman spectra enable identifying four dissimilar hematopoietic cell populations across the HSC lineage specification. Successful discrimination was obtained for a population enriched for long-term repopulating HSCs (LT-HSCs) versus their more differentiated progeny, including closely related short-term repopulating HSCs (ST-HSCs) and fully differentiated lymphoid (B cells) and myeloid (granulocytes) cells. The lineage-specific differentiation states of cells from these four subpopulations were accurately identified independent of the stiffness of the underlying biomaterial substrate, indicating subtle spectral variations that discriminated these populations were not masked by features from the culture substrate. This approach enables identifying the lineage-specific differentiation stages of hematopoietic cells on biomaterial substrates of differing composition and may facilitate correlating hematopoietic cell fate decisions with the extrinsic cues that elicited them.

[Liver engineering as a new source of donor organs : A systematic review].

PubMed

Mußbach, F; Dahmen, U; Dirsch, O; Settmacher, U

2016-06-01

Organ engineering is a new strategy to cope with the shortage of donor organs. A functional scaffold from explanted organs is prepared by removing all cellular components (decellularization) and the reseeding (repopulation) of the organ scaffold to generate a functional organ in vitro for transplantation. This technique was also applied to the liver (liver engineering). Outline of the current state of the art and resulting approaches for future research strategies. Systematic review according to the PRISMA guidelines: a PubMed-based literature search (search terms liver, decellularization), selection of relevant articles based on predetermined criteria for relevance (e.g. decellularization, repopulation and transplantation), extraction and critical appraisal of data and results concerning the conditions for decellularization, repopulation and transplantation. Decellularization was successfully performed in small and large animal models. Hepatocytes as well as stem cells and hepatic cell lines were applied for repopulation and 7 publications could show the successful transplantation of acellular and repopulated organ scaffolds. The current scientific need for further studies concerning the source of donor organs, optimization of the decellularization process, the cell type for the reseeding process and the establishment of the optimal conditions for the repopulation of the scaffold is still tremendous. For successful recellularization of the liver three goals need to be achieved: (1) reseeding of the organ scaffold with a sufficient amount of parenchymal cells, (2) endothelialization of the vascular tree to ensure the supply of oxygen and nutrients to parenchymal cells and (3) an appropriate epithelialization of the biliary tree. In order to progress to clinical trials a suitable transplantation model to verify the function of the organ constructs must be established. Liver engineering using biological cell-free organ scaffolds represents a scientific and ethical challenge. The existing results emphasize the potential of this new and promising strategy to create organs for transplantation in the future.

Bat-mouse bone marrow chimera: a novel animal model for dissecting the uniqueness of the bat immune system.

PubMed

Yong, Kylie Su Mei; Ng, Justin Han Jia; Her, Zhisheng; Hey, Ying Ying; Tan, Sue Yee; Tan, Wilson Wei Sheng; Irac, Sergio Erdal; Liu, Min; Chan, Xue Ying; Gunawan, Merry; Foo, Randy Jee Hiang; Low, Dolyce Hong Wen; Mendenhall, Ian Hewitt; Chionh, Yok Teng; Dutertre, Charles-Antoine; Chen, Qingfeng; Wang, Lin-Fa

2018-03-16

Bats are an important animal model with long lifespans, low incidences of tumorigenesis and an ability to asymptomatically harbour pathogens. Currently, in vivo studies of bats are hampered due to their low reproduction rates. To overcome this, we transplanted bat cells from bone marrow (BM) and spleen into an immunodeficient mouse strain NOD-scid IL-2R -/- (NSG), and have successfully established stable, long-term reconstitution of bat immune cells in mice (bat-mice). Immune functionality of our bat-mouse model was demonstrated through generation of antigen-specific antibody response by bat cells following immunization. Post-engraftment of total bat BM cells and splenocytes, bat immune cells survived, expanded and repopulated the mouse without any observable clinical abnormalities. Utilizing bat's remarkable immunological functions, this novel model has a potential to be transformed into a powerful platform for basic and translational research.

Mammary stem cells have myoepithelial cell properties

PubMed Central

Prater, Michael D.; Petit, Valérie; Russell, I. Alasdair; Giraddi, Rajshekhar; Shehata, Mona; Menon, Suraj; Schulte, Reiner; Kalajzic, Ivo; Rath, Nicola; Olson, Michael F.; Metzger, Daniel; Faraldo, Marisa M.; Deugnier, Marie-Ange; Glukhova, Marina A.; Stingl, John

2014-01-01

Contractile myoepithelial cells dominate the basal layer of the mammary epithelium and are considered to be differentiated cells. However, we observe that up to 54% of single basal cells can form colonies when seeded into adherent culture in the presence of agents that disrupt acin-myosin interactions, and on average, 65% of the single-cell-derived basal colonies can repopulate a mammary gland when transplanted in vivo. This indicates that a high proportion of basal myoepithelial cells can give rise to a mammary repopulating unit (MRU). We demonstrate that myoepithelial cells, flow-sorted using 2 independent myoepithelial-specific reporter strategies, have MRU capacity. Using an inducible lineage tracing approach we follow the progeny of α-smooth muscle actin-expressing myoepithelial cells and show that they function as long-lived lineage-restricted stem cells in the virgin state and during pregnancy. PMID:25173976

Sequential treatment of CD34+ cells from patients with primary myelofibrosis with chromatin-modifying agents eliminate JAK2V617F-positive NOD/SCID marrow repopulating cells

PubMed Central

Wang, Xiaoli; Zhang, Wei; Tripodi, Joseph; Lu, Min; Xu, Mingjiang; Najfeld, Vesna; Li, Yan

2010-01-01

Because primary myelofibrosis (PMF) originates at the level of the pluripotent hematopoietic stem cell (HSC), we examined the effects of various therapeutic agents on the in vitro and in vivo behavior of PMF CD34+ cells. Treatment of PMF CD34+ cells with chromatin-modifying agents (CMAs) but not hydroxyurea, Janus kinase 2 (JAK2) inhibitors, or low doses of interferon-α led to the generation of greater numbers of CD34+ chemokine (C-X-C motif) receptor (CXCR)4+ cells, which were capable of migrating in response to chemokine (C-X-C motif) ligand (CXCL)12 and resulted in a reduction in the proportion of hematopoietic progenitor cells (HPCs) that were JAK2V617F+. Furthermore, sequential treatment of PMF CD34+ cells but not normal CD34+ cells with decitabine (5-aza-2′-deoxycytidine [5azaD]), followed by suberoylanilide hydroxamic acid (SAHA; 5azaD/SAHA), or trichostatin A (5azaD/TSA) resulted in a higher degree of apoptosis. Two to 6 months after the transplantation of CMAs treated JAK2V617F+ PMF CD34+ cells into nonobese diabetic/severe combined immunodeficient (SCID)/IL-2Rγnull mice, the percentage of JAK2V617F/JAK2total in human CD45+ marrow cells was dramatically reduced. These findings suggest that both PMF HPCs, short-term and long-term SCID repopulating cells (SRCs), are JAK2V617F+ and that JAK2V617F+ HPCs and SRCs can be eliminated by sequential treatment with CMAs. Sequential treatment with CMAs, therefore, represents a possible effective means of treating PMF at the level of the malignant SRC. PMID:20858855

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.

Berberine inhibits the chemotherapy-induced repopulation by suppressing the arachidonic acid metabolic pathway and phosphorylation of FAK in ovarian cancer.

PubMed

Zhao, Yawei; Cui, Lianzhi; Pan, Yue; Shao, Dan; Zheng, Xiao; Zhang, Fan; Zhang, Hansi; He, Kan; Chen, Li

2017-12-01

Cytotoxic chemotherapy is an effective and traditional treatment of ovarian cancer. However, chemotherapy-induced apoptosis may also trigger and ultimately accelerate the repopulation of the small number of adjacent surviving cells. This study mainly focused on the tumour cell repopulation caused by chemotherapy in ovarian cancer and the adjunctive/synergistic effect of Berberine on the prevention of tumour repopulation. The transwell system was used to mimic the co-culture of surviving ovarian cancer cells in the microenvironment of cytotoxic chemotherapy-treated dying cells. Tumour cell proliferation was observed by crystal violet staining. AA and PGE 2 levels were measured by ELISA, and changes of protein expression were analysed by Western blot. Chemotherapy drug VP16 treatment triggered AA pathway, leading to the elevated PGE 2 level, and ultimately enhanced the repopulation of ovarian cancer cells. Berberine can block the caspase 3-iPLA 2 -AA-COX-2-PGE 2 pathway by inhibiting the expression of iPLA 2 and COX-2. Berberine can also reverse the increased phosphorylation of FAK caused by abnormal PGE 2 level and thus reverse the repopulation of ovarian cancer cells after VP16 treatment. Our observation suggested that Berberine could inhibit the chemotherapy-induced repopulation of ovarian cancer cells by suppressing the AA pathway and phosphorylation of FAK. And these findings implicated a novel combined use of Berberine and chemotherapeutics, which might prevent ovarian cancer recurrence by abrogating early tumour repopulation. © 2017 John Wiley & Sons Ltd.

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

Rituximab monitoring and redosing in pediatric neuromyelitis optica spectrum disorder

PubMed Central

Nosadini, Margherita; Alper, Gulay; Riney, Catherine J.; Benson, Leslie A.; Mohammad, Shekeeb S.; Ramanathan, Sudarshini; Nolan, Melinda; Appleton, Richard; Leventer, Richard J.; Deiva, Kumaran; Brilot, Fabienne; Gorman, Mark P.; Waldman, Amy T.; Banwell, Brenda

2016-01-01

Objective: To study rituximab in pediatric neuromyelitis optica (NMO)/NMO spectrum disorders (NMOSD) and the relationship between rituximab, B cell repopulation, and relapses in order to improve rituximab monitoring and redosing. Methods: Multicenter retrospective study of 16 children with NMO/NMOSD receiving ≥2 rituximab courses. According to CD19 counts, events during rituximab were categorized as “repopulation,” “depletion,” or “depletion failure” relapses (repopulation threshold CD19 ≥10 × 106 cells/L). Results: The 16 patients (14 girls; mean age 9.6 years, range 1.8–15.3) had a mean of 6.1 events (range 1–11) during a mean follow-up of 6.1 years (range 1.6–13.6) and received a total of 76 rituximab courses (mean 4.7, range 2–9) in 42.6-year cohort treatment. Before rituximab, 62.5% had received azathioprine, mycophenolate mofetil, or cyclophosphamide. Mean time from rituximab to last documented B cell depletion and first repopulation was 4.5 and 6.8 months, respectively, with large interpatient variability. Earliest repopulations occurred with the lowest doses. Significant reduction between pre- and post-rituximab annualized relapse rate (ARR) was observed (p = 0.003). During rituximab, 6 patients were relapse-free, although 21 relapses occurred in 10 patients, including 13 “repopulation,” 3 “depletion,” and 4 “depletion failure” relapses. Of the 13 “repopulation” relapses, 4 had CD19 10–50 × 106 cells/L, 10 had inadequate monitoring (≤1 CD19 in the 4 months before relapses), and 5 had delayed redosing after repopulation detection. Conclusion: Rituximab is effective in relapse prevention, but B cell repopulation creates a risk of relapse. Redosing before B cell repopulation could reduce the relapse risk further. Classification of evidence: This study provides Class IV evidence that rituximab significantly reduces ARR in pediatric NMO/NMOSD. This study also demonstrates a relationship between B cell repopulation and relapses. PMID:26819962

Endothelial cell repopulation after stenting determines in-stent neointima formation: effects of bare-metal vs. drug-eluting stents and genetic endothelial cell modification.

PubMed

Douglas, Gillian; Van Kampen, Erik; Hale, Ashley B; McNeill, Eileen; Patel, Jyoti; Crabtree, Mark J; Ali, Ziad; Hoerr, Robert A; Alp, Nicholas J; Channon, Keith M

2013-11-01

Understanding endothelial cell repopulation post-stenting and how this modulates in-stent restenosis is critical to improving arterial healing post-stenting. We used a novel murine stent model to investigate endothelial cell repopulation post-stenting, comparing the response of drug-eluting stents with a primary genetic modification to improve endothelial cell function. Endothelial cell repopulation was assessed en face in stented arteries in ApoE(-/-) mice with endothelial-specific LacZ expression. Stent deployment resulted in near-complete denudation of endothelium, but was followed by endothelial cell repopulation, by cells originating from both bone marrow-derived endothelial progenitor cells and from the adjacent vasculature. Paclitaxel-eluting stents reduced neointima formation (0.423 ± 0.065 vs. 0.240 ± 0.040 mm(2), P = 0.038), but decreased endothelial cell repopulation (238 ± 17 vs. 154 ± 22 nuclei/mm(2), P = 0.018), despite complete strut coverage. To test the effects of selectively improving endothelial cell function, we used transgenic mice with endothelial-specific overexpression of GTP-cyclohydrolase 1 (GCH-Tg) as a model of enhanced endothelial cell function and increased NO production. GCH-Tg ApoE(-/-) mice had less neointima formation compared with ApoE(-/-) littermates (0.52 ± 0.08 vs. 0.26 ± 0.09 mm(2), P = 0.039). In contrast to paclitaxel-eluting stents, reduced neointima formation in GCH-Tg mice was accompanied by increased endothelial cell coverage (156 ± 17 vs. 209 ± 23 nuclei/mm(2), P = 0.043). Drug-eluting stents reduce not only neointima formation but also endothelial cell repopulation, independent of strut coverage. In contrast, selective targeting of endothelial cell function is sufficient to improve endothelial cell repopulation and reduce neointima formation. Targeting endothelial cell function is a rational therapeutic strategy to improve vascular healing and decrease neointima formation after stenting.

Temporary microglia-depletion after cosmic radiation modifies phagocytic activity and prevents cognitive deficits.

PubMed

Krukowski, Karen; Feng, Xi; Paladini, Maria Serena; Chou, Austin; Sacramento, Kristen; Grue, Katherine; Riparip, Lara-Kirstie; Jones, Tamako; Campbell-Beachler, Mary; Nelson, Gregory; Rosi, Susanna

2018-05-18

Microglia are the main immune component in the brain that can regulate neuronal health and synapse function. Exposure to cosmic radiation can cause long-term cognitive impairments in rodent models thereby presenting potential obstacles for astronauts engaged in deep space travel. The mechanism/s for how cosmic radiation induces cognitive deficits are currently unknown. We find that temporary microglia depletion, one week after cosmic radiation, prevents the development of long-term memory deficits. Gene array profiling reveals that acute microglia depletion alters the late neuroinflammatory response to cosmic radiation. The repopulated microglia present a modified functional phenotype with reduced expression of scavenger receptors, lysosome membrane protein and complement receptor, all shown to be involved in microglia-synapses interaction. The lower phagocytic activity observed in the repopulated microglia is paralleled by improved synaptic protein expression. Our data provide mechanistic evidence for the role of microglia in the development of cognitive deficits after cosmic radiation exposure.

Nuclease-free Adeno-Associated Virus-Mediated Il2rg Gene Editing in X-SCID Mice.

PubMed

Hiramoto, Takafumi; Li, Li B; Funk, Sarah E; Hirata, Roli K; Russell, David W

2018-05-02

X-linked severe combined immunodeficiency (X-SCID) has been successfully treated by hematopoietic stem cell (HSC) transduction with retroviral vectors expressing the interleukin-2 receptor subunit gamma gene (IL2RG), but several patients developed malignancies due to vector integration near cellular oncogenes. This adverse side effect could in principle be avoided by accurate IL2RG gene editing with a vector that does not contain a functional promoter or IL2RG gene. Here, we show that adeno-associated virus (AAV) gene editing vectors can insert a partial Il2rg cDNA at the endogenous Il2rg locus in X-SCID murine bone marrow cells and that these ex vivo-edited cells repopulate transplant recipients and produce CD4 + and CD8 + T cells. Circulating, edited lymphocytes increased over time and appeared in secondary transplant recipients, demonstrating successful editing in long-term repopulating cells. Random vector integration events were nearly undetectable, and malignant transformation of the transplanted cells was not observed. Similar editing frequencies were observed in human hematopoietic cells. Our results demonstrate that therapeutically relevant HSC gene editing can be achieved by AAV vectors in the absence of site-specific nucleases and suggest that this may be a safe and effective therapy for hematopoietic diseases where in vivo selection can increase edited cell numbers. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Exonuclease 1 is a critical mediator of survival during DNA double strand break repair in nonquiescent hematopoietic stem and progenitor cells.

PubMed

Desai, Amar; Qing, Yulan; Gerson, Stanton L

2014-02-01

Hematopoietic stem cell (HSC) populations require DNA repair pathways to maintain their long-term survival and reconstitution capabilities, but mediators of these processes are still being elucidated. Exonuclease 1 (Exo1) participates in homologous recombination (HR) and Exo1 loss results in impaired 5' HR end resection. We use cultured Exo1(mut) fibroblasts and bone marrow to demonstrate that loss of Exo1 function results in defective HR in cycling cells. Conversely, in Exo1(mut) mice HR is not required for maintenance of quiescent HSCs at steady state, confirming the steady state HSC reliance on nonhomologous end joining (NHEJ). Exo1(mut) mice sustained serial repopulation, displayed no defect in competitive repopulation or niche occupancy, and exhibited no increased sensitivity to whole body ionizing radiation. However, when Exo1(mut) HSCs were pushed into cell cycle in vivo with 5-fluorouracil or poly IC, the hematopoietic population became hypersensitive to IR, resulting in HSC defects and animal death. We propose Exo1-mediated HR is dispensable for stem cell function in quiescent HSC, whereas it is essential to HSC response to DNA damage processing after cell cycle entry, and its loss is not compensated by intact NHEJ. In HSCs, the maintenance of stem cell function after DNA damage is dependent on the DNA repair capacity, segregated by active versus quiescent points in cell cycle. © AlphaMed Press.

Hepatic stem/progenitor cells and stem-cell transplantation for the treatment of liver disease.

PubMed

Kakinuma, Sei; Nakauchi, Hiromitsu; Watanabe, Mamoru

2009-01-01

Allogeneic liver transplantation is still the only effective treatment available to patients with liver failure. However, because there is a serious shortage of liver donors, an alternative therapeutic approach is needed. Transplantation of mature hepatocytes has been evaluated in clinical trials, but the long-term efficacy remains unclear and the paucity of donor cells limits this strategy. Stem-cell transplantation is a more promising alternative approach. Several studies have provided information about the mechanism underlying the proliferation and differentiation of hepatic stem/progenitor cells. Moreover, in experimental models of liver disease, transplantation of hepatic stem/progenitor cells or hepatocyte-like cells derived from multipotent stem cells led to donor cell-mediated repopulation of the liver and improved survival rates. However, before stem-cell transplantation can be applied in the clinic to treat liver failure in humans, it will be necessary to overcome several difficulties associated with the technique.

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

System for tracking transplanted limbal epithelial stem cells in the treatment of corneal stem cell deficiency (Conference Presentation)

NASA Astrophysics Data System (ADS)

Boadi, Joseph; Matcher, Stephen; MacNeil, Sheila; Sangwan, Virender S.

2016-04-01

The prevailing hypothesis for the existence and healing of the avascular corneal epithelium is that this layer of cells are continually produced by stem cells in the limbus and transported onto the cornea to mature into corneal epithelium. In the event that the cornea is damaged and the limbal stem cell population is severely reduced, this condition known as Limbal Stem Cell Deficiency and can lead to blindness. There are numerous treatments but most have high long term failure rates. Most treatment methods include the transplantation of limbal stem cells into damaged limbus with hope of repopulating the region and regenerating at healthy corneal epithelium. Optical Coherence Tomography (OCT) is well known for its high resolution in vivo images. A bespoke OCT has been built to investigate the trajectories of these limbal stem cells after transplantation to see whether if they do repopulate the damaged limbus or not. In the experimentation magneto-labelling was used to track the limbal stem cells. For the magneto-labelling a mixture of limbal stem cells and cornea epithelium are cultured with super paramagnetic iron (Fe3O4) nanoparticles (20-30nm in size) for 24hours, to allow for uptake. The cells are then transplanted onto the denuded cornea. The transplanted cell mixture with the encapsulated magnetic nanoparticles is actuated with an external magnetic field 0.08T leading to a phase modulation on the signal. A Phase sensitive Magneto-motive OCT is used to locate the transplanted cells. The location of the cells with embed SPIOs were located both in 2D and 3D.

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

Distinct bone marrow blood vessels differentially regulate haematopoiesis.

PubMed

Itkin, Tomer; Gur-Cohen, Shiri; Spencer, Joel A; Schajnovitz, Amir; Ramasamy, Saravana K; Kusumbe, Anjali P; Ledergor, Guy; Jung, Yookyung; Milo, Idan; Poulos, Michael G; Kalinkovich, Alexander; Ludin, Aya; Kollet, Orit; Shakhar, Guy; Butler, Jason M; Rafii, Shahin; Adams, Ralf H; Scadden, David T; Lin, Charles P; Lapidot, Tsvee

2016-04-21

Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols.

Identification of CD34+ and CD34− leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia

PubMed Central

Aoki, Yuki; Watanabe, Takashi; Saito, Yoriko; Kuroki, Yoko; Hijikata, Atsushi; Takagi, Masatoshi; Tomizawa, Daisuke; Eguchi, Mariko; Eguchi-Ishimae, Minenori; Kaneko, Akiko; Ono, Rintaro; Sato, Kaori; Suzuki, Nahoko; Fujiki, Saera; Koh, Katsuyoshi; Ishii, Eiichi; Shultz, Leonard D.; Ohara, Osamu; Mizutani, Shuki

2015-01-01

Translocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. We characterized leukemia-initiating cells (LICs) in primary infant MLL-rearranged leukemia using a xenotransplantation model. In MLL-AF4 patients, CD34+CD38+CD19+ and CD34−CD19+ cells initiated leukemia, and in MLL-AF9 patients, CD34−CD19+ cells were LICs. In MLL-ENL patients, either CD34+ or CD34− cells were LICs, depending on the pattern of CD34 expression. In contrast, in patients with these MLL translocations, CD34+CD38−CD19−CD33− cells were enriched for normal hematopoietic stem cells (HSCs) with in vivo long-term multilineage hematopoietic repopulation capacity. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34+CD38−CD19−CD33− cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4+ single positive (SP), CD8+ SP, and CD4+CD8+ double-positive (DP) T cells. Global gene expression profiling revealed that CD9, CD32, and CD24 were over-represented in MLL-AF4, MLL-AF9, and MLL-ENL LICs compared with normal HSCs. In patient samples, these molecules were expressed in CD34+CD38+ and CD34− LICs but not in CD34+CD38−CD19−CD33− HSCs. Identification of LICs and LIC-specific molecules in primary human MLL-rearranged acute lymphoblastic leukemia may lead to improved therapeutic strategies for MLL-rearranged leukemia. PMID:25538041

Functional dissection of hematopoietic stem cell populations with a stemness-monitoring system based on NS-GFP transgene expression.

PubMed

Ali, Mohamed A E; Fuse, Kyoko; Tadokoro, Yuko; Hoshii, Takayuki; Ueno, Masaya; Kobayashi, Masahiko; Nomura, Naho; Vu, Ha Thi; Peng, Hui; Hegazy, Ahmed M; Masuko, Masayoshi; Sone, Hirohito; Arai, Fumio; Tajima, Atsushi; Hirao, Atsushi

2017-09-12

Hematopoietic stem cells (HSCs) in a steady state can be efficiently purified by selecting for a combination of several cell surface markers; however, such markers do not consistently reflect HSC activity. In this study, we successfully enriched HSCs with a unique stemness-monitoring system using a transgenic mouse in which green florescence protein (GFP) is driven by the promoter/enhancer region of the nucleostemin (NS) gene. We found that the phenotypically defined long-term (LT)-HSC population exhibited the highest level of NS-GFP intensity, whereas NS-GFP intensity was strongly downregulated during differentiation in vitro and in vivo. Within the LT-HSC population, NS-GFP high cells exhibited significantly higher repopulating capacity than NS-GFP low cells. Gene expression analysis revealed that nine genes, including Vwf and Cdkn1c (p57), are highly expressed in NS-GFP high cells and may represent a signature of HSCs, i.e., a stemness signature. When LT-HSCs suffered from remarkable stress, such as transplantation or irradiation, NS-GFP intensity was downregulated. Finally, we found that high levels of NS-GFP identified HSC-like cells even among CD34 + cells, which have been considered progenitor cells without long-term reconstitution ability. Thus, high NS-GFP expression represents stem cell characteristics in hematopoietic cells, making this system useful for identifying previously uncharacterized HSCs.

Anti-retroviral therapy fails to restore the severe Th-17: Tc-17 imbalance observed in peripheral blood during simian immunodeficiency virus infection.

PubMed

Kader, M; Bixler, S; Piatak, M; Lifson, J; Mattapallil, J J

2009-10-01

Human immuno deficiency virus and simian immunodeficiency virus infections are characterized by a severe loss of Th-17 cells (IL-17(+)CD4(+) T cells) that has been associated with disease progression and systemic dissemination of bacterial infections. Anti-retroviral therapy (ART) has led to repopulation of CD4(+) T cells in peripheral tissues with little sustainable repopulation in mucosal tissues. Given the central importance of Th-17 cells in mucosal homeostasis, it is not known if the failure of ART to permanently repopulate mucosal tissues is associated with a failure to restore Th-17 cells that are lost during infection. Dynamics of alpha4(+)beta7(hi) CD4(+) T cells in peripheral blood of SIV infected rhesus macaques were evaluated and compared to animals that were treated with ART. The frequency of Th-17 and Tc-17 cells was determined following infection and after therapy. Relative expression of IL-21, IL-23, and TGFbeta was determined using Taqman PCR. Treatment of SIV infected rhesus macaques with anti-retroviral therapy was associated with a substantial repopulation of mucosal homing alpha4(+)beta7(hi)CD4(+) T cells in peripheral blood. This repopulation, however, was not accompanied by a restoration of Th-17 responses. Interestingly, SIV infection was associated with an increase in Tc-17 responses (IL-17(+)CD8(+) T cells) suggesting to a skewing in the ratio of Th-17: Tc-17 cells from a predominantly Th-17 phenotype to a predominantly Tc-17 phenotype. Surprisingly, Tc-17 responses remained high during the course of therapy suggesting that ART failed to correct the imbalance in Th-17 : Tc-17 responses induced following SIV infection. ART was associated with substantial repopulation of alpha4(+)beta7(hi) CD4(+) T cells in peripheral blood with little or no rebound of Th-17 cells. On the other hand, repopulation of alpha4(+)beta7(hi) CD4(+) T cells was accompanied by persistence of high levels of Tc-17 cells in peripheral blood. The dysregulation of Th-17 and Tc-17 responses likely plays a role in disease progression.

Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells.

PubMed

Masiuk, Katelyn E; Brown, Devin; Laborada, Jennifer; Hollis, Roger P; Urbinati, Fabrizia; Kohn, Donald B

2017-09-06

Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1 × 10 8 to 1 × 10 9 CD34 + cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34 + cells uses LV inefficiently, because the majority of CD34 + cells are short-term progenitors with a limited post-transplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34 + CD38 - cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34 + CD38 - cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34 + purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34 + CD38 - cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38 + cells. Importantly, LV modification and transplantation of IB-purified CD34 + CD38 - cells/non-modified CD38 + cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34 + cells, while utilizing ∼7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Monte Carlo radiotherapy simulations of accelerated repopulation and reoxygenation for hypoxic head and neck cancer

PubMed Central

Harriss-Phillips, W M; Bezak, E; Yeoh, E K

2011-01-01

Objective A temporal Monte Carlo tumour growth and radiotherapy effect model (HYP-RT) simulating hypoxia in head and neck cancer has been developed and used to analyse parameters influencing cell kill during conventionally fractionated radiotherapy. The model was designed to simulate individual cell division up to 108 cells, while incorporating radiobiological effects, including accelerated repopulation and reoxygenation during treatment. Method Reoxygenation of hypoxic tumours has been modelled using randomised increments of oxygen to tumour cells after each treatment fraction. The process of accelerated repopulation has been modelled by increasing the symmetrical stem cell division probability. Both phenomena were onset immediately or after a number of weeks of simulated treatment. Results The extra dose required to control (total cell kill) hypoxic vs oxic tumours was 15–25% (8–20 Gy for 5×2 Gy per week) depending on the timing of accelerated repopulation onset. Reoxygenation of hypoxic tumours resulted in resensitisation and reduction in total dose required by approximately 10%, depending on the time of onset. When modelled simultaneously, accelerated repopulation and reoxygenation affected cell kill in hypoxic tumours in a similar manner to when the phenomena were modelled individually; however, the degree was altered, with non-additive results. Simulation results were in good agreement with standard linear quadratic theory; however, differed for more complex comparisons where hypoxia, reoxygenation as well as accelerated repopulation effects were considered. Conclusion Simulations have quantitatively confirmed the need for patient individualisation in radiotherapy for hypoxic head and neck tumours, and have shown the benefits of modelling complex and dynamic processes using Monte Carlo methods. PMID:21933980

Proliferation of multipotent hematopoietic cells controlled by a truncated erythropoietin receptor transgene.

PubMed Central

Kirby, S L; Cook, D N; Walton, W; Smithies, O

1996-01-01

The long-term efficacy of gene therapy using bone marrow transplantation requires the engraftment of genetically altered totipotent hematopoietic stem cells (THSCs). Ex vivo expansion of corrected THSCs is one way to increase the efficiency of the procedure. Similarly, selective in vivo expansion of the therapeutic THSCs rather than the endogenous THSCs could favor the transplant. To test whether a conferred proliferative advantage gene can facilitate the in vitro and in vivo expansion of hematopoietic stem cells, we have generated transgenic mice expressing a truncated receptor for the growth factor erythropoietin. These mice are phenotypically normal, but when treated in vivo with exogenous erythropoietin they exhibit a marked increase in multipotent, clonogenic hematopoietic cells [colony-forming units in the spleen (CFU-S) and CFUs that give rise to granulocytes, erythroid cells, macrophages, and megakaryocytes within the same colony (CFU-GEMM)] in comparison with the wild-type mice. In addition, long-term in vitro culture of tEpoR transgenic bone marrow in the presence of erythropoietin induces exponential expansion of trilineage hematopoietic stem cells not seen with wild-type bone marrow. Thus, the truncated erythropoietin receptor gene shows promise as a means for obtaining cytokine-inducible hematopoietic stem cell proliferation to facilitate the direct targeting of THSCs and to provide a competitive repopulation advantage for transplanted therapeutic stem cells. Images Fig. 3 PMID:8790342

Age-Dependent Netrin-1 Signaling Regulates NG2+ Glial Cell Spatial Homeostasis in Normal Adult Gray Matter

PubMed Central

Birey, Fikri

2015-01-01

Neuron–glial antigen 2-positive (NG2+) glial cells are the most proliferative glia type in the adult CNS, and their tile-like arrangement in adult gray matter is under tight regulation. However, little is known about the cues that govern this unique distribution. To this end, using a NG2+ glial cell ablation model in mice, we examined the repopulation dynamics of NG2+ glial cells in the mature and aged mice gray matter. We found that some resident NG2+ glial cells that escaped depletion rapidly enter the cell cycle to repopulate the cortex with altered spatial distribution. We reveal that netrin-1 signaling is involved in the NG2+ glial cell early proliferative, late repopulation, and distribution response after ablation in the gray matter. However, ablation of NG2+ glial cell in older animals failed to stimulate a similar repopulation response, possibly because of a decrease in the sensitivity to netrin-1. Our findings indicate that endogenous netrin-1 plays a role in NG2+ glial cell homeostasis that is distinct from its role in myelination. PMID:25926469

Liver repopulation by c-Met-positive stem/progenitor cells isolated from the developing rat liver.

PubMed

Suzuki, Atsushi; Zheng, Yun-wen; Fukao, Katashi; Nakauchi, Hiromitsu; Taniguchi, Hideki

2004-01-01

Self-renewing stem cells responsible for tissue or organ development and regeneration have been recently described. To isolate such cells using flow cytometry, it should be required to find molecules expressing on their cell surfaces. We have previously reported that, on cells fulfilling the criteria for hepatic stem cells, the hepatocyte growth factor receptor c-Met is expressed specifically in the developing mouse liver. In this study, to determine whether c-Met is an essential marker for hepatic stem cells in other animal strains, we examined the potential for in vivo liver-repopulation in sorted fetal rat-derived c-Met+ cells using the retrorsine model. Using flow cytometry and monoclonal antibodies for c-Met and leukocyte common antigen CD45, fetal rat liver cells were fractionated according to the expression of these molecules. Then, cells in each cell subpopulation were sorted and transplanted into the retrorsine-treated adult rats with two-third hepatectomy. At 9 months post transplant, frequency of liver-repopulation was examined by qualitative and quantitative analyses. When we transplanted c-Met+ CD45- sorted cells, many donor-derived cells formed colonies that included mature hepatocytes expressing albumin and containing abundant glycogen in their cytoplasm. In contrast, c-Met- cells and CD45+ cells could not repopulate damaged recipient livers. High enrichment of liver-repopulating cells was conducted by sorting of c-Met+ cells from the developing rat liver. This result suggests that c-Met/HGF interaction plays a crucial role for stem cell growth, differentiation, and self-renewal in rat liver organogenesis. Since the c-Met is also expressed in the fetal mouse-derived hepatic stem cells, this molecule could be expected to be an essential marker for such cell population in the various animal strains, including human.

Specific allogeneic unresponsiveness in the adult host: present-day experimental models

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

Rapaport, F.T.; Bachvaroff, R.J.; Cronkite, E.

As part of a long-term intensive effort to apply the induction of adult allogensic unresponsiveness to the transplantation problem, two techniques to control the variability in the persistence of immunologically competent postthymic cells iin the treated host and/or the inoculum of autologous marrow returned to the host after irradiation are described. The first consisted of exposing the peripheral blood of prospective recipients to a 5-week course of extra-corporeal irradiation (ECIB), the other of exposing the stored autologous marrow scheduled to repopulate a given recipient to methyl-prednisolone (MPd) and DNase prior to renifusion into the recipient. Serial analysis of bone marrowmore » cell samples at various intervals before and after treatment was undertaken. The significance of the disappearance of a particular population of nonnuclear cells from the samples, and the association of such disappearance with increased success in the induction of allogeneic unresponsiveness is discussed. (ACR)« less

Age-Dependent Netrin-1 Signaling Regulates NG2+ Glial Cell Spatial Homeostasis in Normal Adult Gray Matter.

PubMed

Birey, Fikri; Aguirre, Adan

2015-04-29

Neuron-glial antigen 2-positive (NG2(+)) glial cells are the most proliferative glia type in the adult CNS, and their tile-like arrangement in adult gray matter is under tight regulation. However, little is known about the cues that govern this unique distribution. To this end, using a NG2(+) glial cell ablation model in mice, we examined the repopulation dynamics of NG2(+) glial cells in the mature and aged mice gray matter. We found that some resident NG2(+) glial cells that escaped depletion rapidly enter the cell cycle to repopulate the cortex with altered spatial distribution. We reveal that netrin-1 signaling is involved in the NG2(+) glial cell early proliferative, late repopulation, and distribution response after ablation in the gray matter. However, ablation of NG2(+) glial cell in older animals failed to stimulate a similar repopulation response, possibly because of a decrease in the sensitivity to netrin-1. Our findings indicate that endogenous netrin-1 plays a role in NG2(+) glial cell homeostasis that is distinct from its role in myelination. Copyright © 2015 the authors 0270-6474/15/356946-06$15.00/0.

Natural Killer Cell Immunotherapy Targeting Cancer Stem Cells

PubMed Central

Luna, Jesus I; Grossenbacher, Steven K.; Murphy, William J; Canter, Robert J

2017-01-01

Introduction Standard cytoreductive cancer therapy, such as chemotherapy and radiotherapy, are frequently resisted by a small portion of cancer cells with “stem-cell” like properties including quiescence and repopulation. Immunotherapy represents a breakthrough modality for improving oncologic outcomes in cancer patients. Since the success of immunotherapy is not contingent on target cell proliferation, it may also be uniquely suited to address the problem of resistance and repopulation exerted by cancer stem cells (CSCs). Areas covered Natural killer (NK) cells have long been known for their ability to reject allogeneic hematopoietic stem cells, and there are increasing data demonstrating that NK cells can selectively identify and lyse CSCs. In this report, we review the current knowledge of CSCs and NK cells and highlight recent studies that support the concept that NK cells are capable of targeting CSC in solid tumors, especially in the context of combination therapy simultaneously targeting non-CSCs and CSCs. Expert Opinion Unlike cytotoxic cancer treatments, NK cells are able to target and eliminate quiescent/non-proliferating cells such as CSCs, and these enigmatic cells are an important source of relapse and metastasis. NK targeting of CSCs represents a novel and potentially high impact method to capitalize on the intrinsic therapeutic potential of NK cells. PMID:27960589

Substance P enhances proliferation and paracrine potential of adipose-derived stem cells in vitro

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

Kim, Suna; Piao, Jiyuan; Son, Youngsook

Stem cells have tremendous promise to treat intractable diseases. Notably, adipose-derived stem cells (ADSCs) are actively being investigated because of ease of sampling and high repopulation capacity in vitro. ADSCs can exert a therapeutic effect through differentiation and paracrine potential, and these actions have been proven in many diseases, including cutaneous and inflammatory diseases. Transplantation of ADSCs necessitates therapeutic quantities and thus, long term ex vivo culture of ADSCs. However, this procedure can impair the activity of ADSCs and provoke cellular senescence, leading to low efficacy in vivo. Accordingly, strategies to restore cellular activity and inhibit senescence of stem cells during ex vivo culturemore » are needed for stem cell-based therapies. This study evaluated a potential supplementary role of Substance P (SP) in ADSC ex vivo culture. After confirming that the ADSC cell cycle was damaged by passage 6 (p6), ADSCs at p6 were cultured with SP, and their proliferation rates, cumulative cell numbers, cytokine profiles, and impact on T/endothelial cells were assessed. Long-term culture weakened proliferation ability and secretion of the cytokines, transforming growth factor-beta 1 (TGF-beta1), vascular endothelial growth factor (VEGF), and stromal cell derived factor-1 alpha (SDF-1alpha) in ADSCs. However, SP treatment reduced the population doubling time (PDT), enabling gain of a sufficient number of ADSCs at early passages. In addition, SP restored cytokine secretion, enhancing the ADSC-mediated paracrine effect on T cell and human umbilical vein endothelial cells (HUVECs). Taken together, these results suggest that SP can retain the therapeutic effect of ADSCs by elevating their proliferative and paracrine potential in ex vivo culture. - Highlights: • Long-term culture of ADSCs leads to cell senescence. • Paracrine potential of ADSC decreases as passage number increases. • SP enhances the weakened proliferation capacity of ADSCs. • SP stimulates cytokine secretion from ADSC with impaired paracrine potential.« less

Is There Any Reason to Prefer Cord Blood Instead of Adult Donors for Hematopoietic Stem Cell Transplants?

PubMed

Beksac, Meral

2015-01-01

As cord blood (CB) enables rapid access and tolerance to HLA mismatches, a number of unrelated CB transplants have reached 30,000. Such transplant activity has been the result of international accreditation programs maintaining highly qualified cord blood units (CBUs) reaching more than 600,000 CBUs stored worldwide. Efforts to increase stem cell content or engraftment rate of the graft by ex vivo expansion, modulation by molecules such as fucose, prostaglandin E2 derivative, complement CD26 inhibitors, or CXCR4/CXCL12 axis have been able to accelerate engraftment speed and rate. Furthermore, introduction of reduced intensity conditioning protocols, better HLA matching, and recognition of the importance of HLA-C have improved CB transplants success by decreasing transplant-related mortality. CB progenitor/stem cell content has been compared with adult stem cells revealing higher long-term repopulating capacity compared to bone marrow-mesenchymal stromal cells and lesser oncogenic potential than progenitor-induced stem cells. This chapter summarizes the advantages and disadvantages of CB compared to adult stem cells within the context of stem cell biology and transplantation.

Tumour volume response, initial cell kill and cellular repopulation in B16 melanoma treated with cyclophosphamide and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.

PubMed Central

Stephens, T. C.; Peacock, J. H.

1977-01-01

The relationship between tumour volume response and cell kill in B16 melanoma following treatment in vivo with cyclophosphamide (CY) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was investigated. Tumour volume response, expressed as growth delay, was estimated from measurements of tumour dimensions. Depression of in vitro colony-forming ability of cells from treated tumours was used as the measure of tumour cell kill. The relationship between these parameters was clearly different for the two agents studied. CY produced more growth delay (7.5 days) per decade of tumour cell kill than CCNU (2 to 3.5 days). The possibility that this was due to a technical artefact was rejected in favour of an alternative explanation that different rates of cellular repopulation in tumours treated with CY and CCNU might be responsible. Cellular repopulation was measured directly, by performing cell-survival assays at various times after treatment with doses of CY and CCNU which produced about 3 decades of cell kill. The rate of repopulation by clonogenic cells was much slower after treatment with CY than with CCNU, and this appears to account for the longer duration of the growth delay obtained with CY. PMID:921888

Inhibiting the Aurora B Kinase Potently Suppresses Repopulation During Fractionated Irradiation of Human Lung Cancer Cell Lines

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

Sak, Ali, E-mail: ali.sak@uni-due.de; Stuschke, Martin; Groneberg, Michael

2012-10-01

Purpose: The use of molecular-targeted agents during radiotherapy of non-small-cell lung cancer (NSCLC) is a promising strategy to inhibit repopulation, thereby improving therapeutic outcome. We assessed the combined effectiveness of inhibiting Aurora B kinase and irradiation on human NSCLC cell lines in vitro. Methods and Materials: NSCLC cell lines were exposed to concentrations of AZD1152-hydroxyquinazoline pyrazol anilide (AZD1152-HQPA) inhibiting colony formation by 50% (IC50{sub clone}) in combination with single dose irradiation or different fractionation schedules using multiple 2-Gy fractions per day up to total doses of 4-40 Gy. The total irradiation dose required to control growth of 50% of themore » plaque monolayers (TCD50) was determined. Apoptosis, G2/M progression, and polyploidization were also analyzed. Results: TCD50 values after single dose irradiation were similar for the H460 and H661 cell lines with 11.4 {+-} 0.2 Gy and 10.7 {+-} 0.3 Gy, respectively. Fractionated irradiation using 3 Multiplication-Sign 2 Gy/day, 2 Multiplication-Sign 2 Gy/day, and 1 Multiplication-Sign 2 Gy/day schedules significantly increased TCD50 values for both cell lines grown as plaque monolayers with increasing radiation treatment time. This could be explained by a repopulation effect per day that counteracts 75 {+-} 8% and 27 {+-} 6% of the effect of a 2-Gy fraction in H460 and H661 cells, respectively. AZD1152-HQPA treatment concomitant to radiotherapy significantly decreased the daily repopulation effect (H460: 28 {+-} 5%, H661: 10 {+-} 4% of a 2-Gy fraction per day). Treatment with IC50{sub clone} AZD1152-HPQA did not induce apoptosis, prolong radiation-induced G2 arrest, or delay cell cycle progression before the spindle check point. However, polyploidization was detected, especially in cell lines without functional p53. Conclusions: Inhibition of Aurora B kinase with low AZD1152-HQPA concentrations during irradiation of NSCLC cell lines affects repopulation during radiotherapy. Thus, concomitant Aurora B kinase inhibition and irradiation may be a promising strategy for fast repopulating tumors, which are difficult to cure by dose escalation based on conventional fractionation.« less

Repopulation Kinetics and the Linear-Quadratic Model

NASA Astrophysics Data System (ADS)

O'Rourke, S. F. C.; McAneney, H.; Starrett, C.; O'Sullivan, J. M.

2009-08-01

The standard Linear-Quadratic (LQ) survival model for radiotherapy is used to investigate different schedules of radiation treatment planning for advanced head and neck cancer. We explore how these treament protocols may be affected by different tumour repopulation kinetics between treatments. The laws for tumour cell repopulation include the logistic and Gompertz models and this extends the work of Wheldon et al. [1], which was concerned with the case of exponential repopulation between treatments. Treatment schedules investigated include standarized and accelerated fractionation. Calculations based on the present work show, that even with growth laws scaled to ensure that the repopulation kinetics for advanced head and neck cancer are comparable, considerable variation in the survival fraction to orders of magnitude emerged. Calculations show that application of the Gompertz model results in a significantly poorer prognosis for tumour eradication. Gaps in treatment also highlight the differences in the LQ model with the effect of repopulation kinetics included.

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

Bertoncello, I.; Hodgson, G.S.; Bradley, T.R.

A multiparameter cell separative procedure is described that enables normal transplantable hemopoietic stem cells that preferentially home to the marrow of lethally irradiated mice to be enriched and separated from the majority of spleen colony-forming cells that are assayed 13 days after transplantation (CFU-S13). First, bone marrow cells are centrifuged in a discontinuous bovine serum albumin gradient. Low-density cells are harvested and labeled with the supravital cationic fluorochrome rhodamine 123 (Rh123). Labeled cells are analyzed using a fluorescence-activated cell sorter, and cells are sorted on the basis of relative Rh123 fluorescence within a predetermined forward versus 90 degrees red lightmore » scatter window that has been optimized for the recovery and enrichment of cells with marrow repopulating ability (MRA). Cells with MRA were characterized by relatively low Rh123 fluorescence and could be separated from a fraction that fluoresced more intensely and contained the majority of CFU-S13 but low MRA. Cells with platelet repopulating ability cofractionate with MRA whereas cells with erythroid repopulating ability remain associated with CFU-S13.« less

Latexin Inactivation Enhances Survival and Long-Term Engraftment of Hematopoietic Stem Cells and Expands the Entire Hematopoietic System in Mice.

PubMed

Liu, Yi; Zhang, Cuiping; Li, Zhenyu; Wang, Chi; Jia, Jianhang; Gao, Tianyan; Hildebrandt, Gerhard; Zhou, Daohong; Bondada, Subbarao; Ji, Peng; St Clair, Daret; Liu, Jinze; Zhan, Changguo; Geiger, Hartmut; Wang, Shuxia; Liang, Ying

2017-04-11

Natural genetic diversity offers an important yet largely untapped resource to decipher the molecular mechanisms regulating hematopoietic stem cell (HSC) function. Latexin (Lxn) is a negative stem cell regulatory gene identified on the basis of genetic diversity. By using an Lxn knockout mouse model, we found that Lxn inactivation in vivo led to the physiological expansion of the entire hematopoietic hierarchy. Loss of Lxn enhanced the competitive repopulation capacity and survival of HSCs in a cell-intrinsic manner. Gene profiling of Lxn-null HSCs showed altered expression of genes enriched in cell-matrix and cell-cell interactions. Thrombospondin 1 (Thbs1) was a potential downstream target with a dramatic downregulation in Lxn-null HSCs. Enforced expression of Thbs1 restored the Lxn inactivation-mediated HSC phenotypes. This study reveals that Lxn plays an important role in the maintenance of homeostatic hematopoiesis, and it may lead to development of safe and effective approaches to manipulate HSCs for clinical benefit. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Long-term hematopoietic stem cell damage in a murine model of the hematopoietic syndrome of the acute radiation syndrome.

PubMed

Chua, Hui Lin; Plett, P Artur; Sampson, Carol H; Joshi, Mandar; Tabbey, Rebeka; Katz, Barry P; MacVittie, Thomas J; Orschell, Christie M

2012-10-01

Residual bone marrow damage (RBMD) persists for years following exposure to radiation and is believed to be due to decreased self-renewal potential of radiation-damaged hematopoietic stem cells (HSC). Current literature has examined primarily sublethal doses of radiation and time points within a few months of exposure. In this study, the authors examined RBMD in mice surviving lethal doses of total body ionizing irradiation (TBI) in a murine model of the Hematopoietic Syndrome of the Acute Radiation Syndrome (H-ARS). Survivors were analyzed at various time points up to 19 mo post-TBI for hematopoietic function. The competitive bone marrow (BM) repopulating potential of 150 purified c-Kit+ Sca-1+ lineage- CD150+ cells (KSLCD150+) remained severely deficient throughout the study compared to KSLCD150+ cells from non-TBI age-matched controls. The minimal engraftment from these TBI HSCs is predominantly myeloid, with minimal production of lymphocytes both in vitro and in vivo. All classes of blood cells as well as BM cellularity were significantly decreased in TBI mice, especially at later time points as mice aged. Primitive BM hematopoietic cells (KSLCD150+) displayed significantly increased cell cycling in TBI mice at all time points, which may be a physiological attempt to maintain HSC numbers in the post-irradiation state. Taken together, these data suggest that the increased cycling among primitive hematopoietic cells in survivors of lethal radiation may contribute to long-term HSC exhaustion and subsequent RBMD, exacerbated by the added insult of aging at later time points.

In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase

PubMed Central

Gori, Jennifer L.; Tian, Xinghui; Swanson, Debra; Gunther, Roland; Shultz, Leonard D.; McIvor, R. Scott; Kaufman, Dan S.

2009-01-01

SUMMARY Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase engraftment of gene-modified hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR – GFP expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rγcnull (NSG) mice after injection of Tyr22-DHFR-derived cells significantly increases human CD34+ and CD45+ cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR-cells in vivo, and provides a novel approach for combined human cell and gene therapy. PMID:19829316

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)

Allograft integration in a rabbit transgenic model for anterior cruciate ligament reconstruction.

PubMed

Bachy, M; Sherifi, I; Zadegan, F; Petite, H; Vialle, R; Hannouche, D

2016-04-01

Tissue engineering strategies include both cell-based and cell homing therapies. Ligamentous tissues are highly specialized and constitute vital components of the musculoskeletal system. Their damage causes significant morbidity and loss in function. The aim of this study is to analyze tendinous graft integration, cell repopulation and ligamentization by using GFP+/- allografts in GFP+/- transgenic New Zealand white (NZW) rabbits. Graft implantation was designed to closely mimic anterior cruciate ligament (ACL) repair surgery. Allografts were implanted in 8 NZW rabbits and assessed at 5 days, 3 weeks and 6 weeks through: (1) arthroCT imaging, (2) morphological analysis of the transplanted allograft, (3) histological analysis, (4) collagen type I immunochemistry, and (5) GFP cell tracking. Collagen remodeling was appreciated at 3 and 6 weeks. Graft repopulation with host cells, chondrocyte-like cells at the tendon-bone interface and graft corticalization in the bone tunnels were noticed at 3 weeks. By contrast we noticed a central necrosis aspect in the allografts intra-articularly at 6 weeks with a cell migration towards the graft edge near the synovium. Our study has served to gain a better understanding of tendinous allograft bone integration, ligamentization and allograft repopulation. We believe that both cell-based therapies and cell homing therapies are beneficial in ligament tissue engineering. Future studies may elucidate whether cell repopulation occurs with pre-differentiated or progenitor cells. We believe that both cell-based therapies and cell homing therapies are beneficial in ligament tissue engineering. Level V (animal study). Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Substance P enhances proliferation and paracrine potential of adipose-derived stem cells in vitro.

PubMed

Kim, Suna; Piao, Jiyuan; Son, Youngsook; Hong, Hyun Sook

2017-03-25

Stem cells have tremendous promise to treat intractable diseases. Notably, adipose-derived stem cells (ADSCs) are actively being investigated because of ease of sampling and high repopulation capacity in vitro. ADSCs can exert a therapeutic effect through differentiation and paracrine potential, and these actions have been proven in many diseases, including cutaneous and inflammatory diseases. Transplantation of ADSCs necessitates therapeutic quantities and thus, long term ex vivo culture of ADSCs. However, this procedure can impair the activity of ADSCs and provoke cellular senescence, leading to low efficacy in vivo. Accordingly, strategies to restore cellular activity and inhibit senescence of stem cells during ex vivo culture are needed for stem cell-based therapies. This study evaluated a potential supplementary role of Substance P (SP) in ADSC ex vivo culture. After confirming that the ADSC cell cycle was damaged by passage 6 (p6), ADSCs at p6 were cultured with SP, and their proliferation rates, cumulative cell numbers, cytokine profiles, and impact on T/endothelial cells were assessed. Long-term culture weakened proliferation ability and secretion of the cytokines, transforming growth factor-beta 1 (TGF-beta1), vascular endothelial growth factor (VEGF), and stromal cell derived factor-1 alpha (SDF-1alpha) in ADSCs. However, SP treatment reduced the population doubling time (PDT), enabling gain of a sufficient number of ADSCs at early passages. In addition, SP restored cytokine secretion, enhancing the ADSC-mediated paracrine effect on T cell and human umbilical vein endothelial cells (HUVECs). Taken together, these results suggest that SP can retain the therapeutic effect of ADSCs by elevating their proliferative and paracrine potential in ex vivo culture. Copyright © 2017 Elsevier Inc. All rights reserved.

Deletion of the Imprinted Gene Grb10 Promotes Hematopoietic Stem Cell Self-Renewal and Regeneration.

PubMed

Yan, Xiao; Himburg, Heather A; Pohl, Katherine; Quarmyne, Mamle; Tran, Evelyn; Zhang, Yurun; Fang, Tiancheng; Kan, Jenny; Chao, Nelson J; Zhao, Liman; Doan, Phuong L; Chute, John P

2016-11-01

Imprinted genes are differentially expressed by adult stem cells, but their functions in regulating adult stem cell fate are incompletely understood. Here we show that growth factor receptor-bound protein 10 (Grb10), an imprinted gene, regulates hematopoietic stem cell (HSC) self-renewal and regeneration. Deletion of the maternal allele of Grb10 in mice (Grb10 m/+ mice) substantially increased HSC long-term repopulating capacity, as compared to that of Grb10 +/+ mice. After total body irradiation (TBI), Grb10 m/+ mice demonstrated accelerated HSC regeneration and hematopoietic reconstitution, as compared to Grb10 +/+ mice. Grb10-deficient HSCs displayed increased proliferation after competitive transplantation or TBI, commensurate with upregulation of CDK4 and Cyclin E. Furthermore, the enhanced HSC regeneration observed in Grb10-deficient mice was dependent on activation of the Akt/mTORC1 pathway. This study reveals a function for the imprinted gene Grb10 in regulating HSC self-renewal and regeneration and suggests that the inhibition of Grb10 can promote hematopoietic regeneration in vivo. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

In vitro study of the adverse effect of nicotine and physical strain on human gingival fibroblasts as a model of the healing of wounds commonly found in the military.

PubMed

Dinos, Michael E; Borke, James L; Swiec, Gary D; McPherson, James C; Goodin, Jeremy L; Chuang, Augustine H

2015-03-01

Significant adverse effects on fibroblast growth and metabolism are observed with nicotine. We investigated the synergistic effects of nicotine and cyclical mechanical strain (CMS) on human gingival fibroblasts (HGFs) in a wound-healing model. HGFs were isolated and grown in Dulbecco's modified Eagle's medium. Three-millimeter wounds were created on a confluent cell monolayer grown in a media containing 0, 1, 2, or 4 mM nicotine, with or without CMS. The applied deformation regimen remains constant for 6 days. On days 1, 2, 4, and 6, the cells were stained with hematoxylin and eosin Y for the evaluation of wound repopulation. The application of CMS alone demonstrates a biphasic response, with an initial stimulatory effect on wound repopulation (days 1-2) and less repopulation during the later phase (days 4-6). The addition of nicotine clearly demonstrated a time and inverse dose-dependent relationship on wound repopulation, with no effect during the early phase and reduced wound repopulation during the later phase. Initial treatment of HGF wounds with CMS resulted in faster wound repopulation regardless of nicotine presence. By day 6, wound healing of HGF exposed to both nicotine and CMS is delayed. These findings suggest that CMS and nicotine may affect fibroblasts and delay wound healing at other sites in the body as well. Reprint & Copyright © 2015 Association of Military Surgeons of the U.S.

Evaluation of stem cell reserve using serial bone marrow transplantation and competitive repopulation in a murine model of chronic hemolytic anemia

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

Maggio-Price, L.; Wolf, N.S.; Priestley, G.V.

1988-09-01

Serial transplantation and competitive repopulation were used to evaluate any loss of self-replicative capacity of bone marrow stem cells in a mouse model with increased and persistent hemopoietic demands. Congenic marrows from old control and from young and old mice with hereditary spherocytic anemia (sphha/sphha) were serially transplanted at 35-day intervals into normal irradiated recipients. Old anemic marrow failed or reverted to recipient karyotype at a mean of 3.5 transplants, and young anemic marrow reverted at a mean of 4.0 transplants, whereas controls did so at a mean of 5.0 transplants. In a competitive assay in which a mixture ofmore » anemic and control marrow was transplanted, the anemic marrow persisted to 10 months following transplantation; anemic marrow repopulation was greater if anemic marrow sex matched with the host. It is possible that lifelong stress of severe anemia decreases stem cell reserve in the anemic sphha/sphha mouse marrow. However, marginal differences in serial transplantation number and the maintenance of anemic marrow in a competition assay would suggest that marrow stem cells, under prolonged stress, are capable of exhibiting good repopulating and self-replicating abilities.« less

A role for GPx3 in activity of normal and leukemia stem cells

PubMed Central

Herault, Olivier; Hope, Kristin J.; Deneault, Eric; Mayotte, Nadine; Chagraoui, Jalila; Wilhelm, Brian T.; Cellot, Sonia; Sauvageau, Martin; Andrade-Navarro, Miguel A.; Hébert, Josée

2012-01-01

The determinants of normal and leukemic stem cell self-renewal remain poorly characterized. We report that expression of the reactive oxygen species (ROS) scavenger glutathione peroxidase 3 (GPx3) positively correlates with the frequency of leukemia stem cells (LSCs) in Hoxa9+Meis1-induced leukemias. Compared with a leukemia with a low frequency of LSCs, a leukemia with a high frequency of LSCs showed hypomethylation of the Gpx3 promoter region, and expressed high levels of Gpx3 and low levels of ROS. LSCs and normal hematopoietic stem cells (HSCs) engineered to express Gpx3 short hairpin RNA (shRNA) were much less competitive in vivo than control cells. However, progenitor cell proliferation and differentiation was not affected by Gpx3 shRNA. Consistent with this, HSCs overexpressing Gpx3 were significantly more competitive than control cells in long-term repopulation experiments, and overexpression of the self-renewal genes Prdm16 or Hoxb4 boosted Gpx3 expression. In human primary acute myeloid leukemia samples, GPX3 expression level directly correlated with adverse prognostic outcome, revealing a potential novel target for the eradication of LSCs. PMID:22508837

Unaltered repopulation properties of mouse hematopoietic stem cells transduced with lentiviral vectors

PubMed Central

Gonzalez-Murillo, Africa; Lozano, M. Luz; Montini, Eugenio; Bueren, Juan A.

2008-01-01

Recent studies of retroviral-mediated gene transfer have shown that retroviral integrations themselves may trigger nonmalignant clonal expansion of hematopoietic stem cells (HSCs) in transplant recipients. These observations suggested that previous conclusions of HSC dynamics based on gamma-retroviral gene marking should be confirmed with improved vectors having a more limited capacity to transactivate endogenous genes. Because of the low trans-activation activity of self-inactivating lentiviral vectors (LVs), we have investigated whether the LV marking of mouse HSCs induces a competitive repopulation advantage in recipients of serially transplants. As deduced from analyses conducted in primary and secondary recipients, we concluded that lentivirally transduced HSCs have no competitive repopulation advantages over untransduced HSCs. By linear amplification-mediated polymerase chain reaction (LAM-PCR) analysis, we characterized LV-targeted genes in HSC clones that engrafted up to quaternary recipients. Although 9 clones harbored integrations close to defined retroviral insertion sites, none was characterized as a common integration site, and none was present in HSC clones repopulating quaternary recipients. Taken together, our results show unaltered repopulation properties of HSCs transduced with LVs, and confirm early studies suggesting the natural capacity of a few HSC clones to generate a monoclonal or oligoclonal hematopoiesis in transplant recipients. PMID:18684860

Bacterial repopulation of drinking water pipe walls after chlorination.

PubMed

Mathieu, Laurence; Francius, Grégory; El Zein, Racha; Angel, Edith; Block, Jean-Claude

2016-09-01

The short-term kinetics of bacterial repopulation were evaluated after chlorination of high-density polyethylene (HDPE) colonized with drinking water biofilms and compared with bare HDPE surfaces. The effect of chlorination was partial as a residual biofilm persisted and was time-limited as repopulation occurred immediately after water resupply. The total number of bacteria reached the same levels on both the bare and chlorinated biofilm-fouled HDPE after a seven-day exposure to drinking water. Due to the presence of a residual biofilm, the hydrophobicity of chlorinated biofilm-fouled surface exhibited much lower adhesion forces (2.1 nN) compared to bare surfaces (8.9 nN). This could explain the rapid repopulation after chlorination, with a twofold faster bacterial accumulation rate on the bare HDPE surface. γ-Proteobacteria dominated the early stages of repopulation of both surfaces and a shift in the dominance occurred over the colonization time. Such observations define a timescale for cleaning frequency in industrial environments and guidelines for a rinsing procedure using drinking water.

Influence of anaesthetics on tumour-cell kill and repopulation in B16 melanoma treated with melphalan.

PubMed Central

Peacock, J. H.; Stephens, T. C.

1978-01-01

The influence of anaesthetics on the in vivo response of B16 melanoma to melphalan was studied using an in vitro cell-survival assay. Three anaesthetics were used, Saffan (Althesin) Sagatal (Nembutal) and Hypnorm. When Saffan was administered to tumour-bearing animals before melphalan there was a significant increase in tumour-cell kill. This effect was not observed with Sagatal or Hypnorm. Maximum increase in tumour-cell kill was achieved when Saffan was administered about 1 h before melphalan, and was dependent on Saffan dose. Clonogenic tumour-cell repopulation after melphalan was rapid (TD - 1 day) and the rate was similar from 2 levels of cell kill. When Saffan was combined with melphalan the repopulation rate was the same as with melphalan alone, and the increased cell kill was reflected in increased growth delay. The in vitro response of B16 melanoma cells to melphalan was unaltered by pretreatment with, or simultaneous exposure to Saffan. The results suggest that the mechanism of the enhanced cell kill in vivo is probably due to an indirect systemic effect, rather than a direct effect on the tumour cells. PMID:743490

Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells

PubMed Central

Kobayashi, Hideki; Butler, Jason M.; O'Donnell, Rebekah; Kobayashi, Mariko; Ding, Bi-Sen; Bonner, Bryant; Chiu, Vi K.; Nolan, Daniel J.; Shido, Koji; Benjamin, Laura; Rafii, Shahin

2010-01-01

Endothelial cells establish an instructive vascular niche that reconstitutes haematopoietic stem and progenitor cells (HSPCs) through release of specific paracrine growth factors, known as angiocrine factors. However, the mechanism by which endothelial cells balance the rate of proliferation and lineage-specific differentiation of HSPCs is unknown. Here, we demonstrate that Akt activation in endothelial cells, through recruitment of mTOR, but not the FoxO pathway, upregulates specific angiocrine factors that support expansion of CD34−Flt3− KLS HSPCs with long-term haematopoietic stem cell (LT-HSC) repopulation capacity. Conversely, co-activation of Akt-stimulated endothelial cells with p42/44 MAPK shifts the balance towards maintenance and differentiation of the HSPCs. Selective activation of Akt1 in the endothelial cells of adult mice increased the number of colony forming units in the spleen and CD34−Flt3− KLS HSPCs with LT-HSC activity in the bone marrow, accelerating haematopoietic recovery. Therefore, the activation state of endothelial cells modulates reconstitution of HSPCs through the upregulation of angiocrine factors, with Akt–mTOR-activated endothelial cells supporting the self-renewal of LT-HSCs and expansion of HSPCs, whereas MAPK co-activation favours maintenance and lineage-specific differentiation of HSPCs. PMID:20972423

Cladribine treatment of multiple sclerosis is associated with depletion of memory B cells.

PubMed

Ceronie, Bryan; Jacobs, Benjamin M; Baker, David; Dubuisson, Nicolas; Mao, Zhifeng; Ammoscato, Francesca; Lock, Helen; Longhurst, Hilary J; Giovannoni, Gavin; Schmierer, Klaus

2018-05-01

The mechanism of action of oral cladribine, recently licensed for relapsing multiple sclerosis, is unknown. To determine whether cladribine depletes memory B cells consistent with our recent hypothesis that effective, disease-modifying treatments act by physical/functional depletion of memory B cells. A cross-sectional study examined 40 people with multiple sclerosis at the end of the first cycle of alemtuzumab or injectable cladribine. The relative proportions and absolute numbers of peripheral blood B lymphocyte subsets were measured using flow cytometry. Cell-subtype expression of genes involved in cladribine metabolism was examined from data in public repositories. Cladribine markedly depleted class-switched and unswitched memory B cells to levels comparable with alemtuzumab, but without the associated initial lymphopenia. CD3 + T cell depletion was modest. The mRNA expression of metabolism genes varied between lymphocyte subsets. A high ratio of deoxycytidine kinase to group I cytosolic 5' nucleotidase expression was present in B cells and was particularly high in mature, memory and notably germinal centre B cells, but not plasma cells. Selective B cell cytotoxicity coupled with slow repopulation kinetics results in long-term, memory B cell depletion by cladribine. These may offer a new target, possibly with potential biomarker activity, for future drug development.

Experimental Model for Successful Liver Cell Therapy by Lenti TTR-YapERT2 Transduced Hepatocytes with Tamoxifen Control of Yap Subcellular Location

PubMed Central

Yovchev, Mladen; Jaber, Fadi L.; Lu, Zhonglei; Patel, Shachi; Locker, Joseph; Rogler, Leslie E.; Murray, John W.; Sudol, Marius; Dabeva, Mariana D.; Zhu, Liang; Shafritz, David A.

2016-01-01

Liver repopulation by transplanted hepatocytes has not been achieved previously in a normal liver microenvironment. Here we report that adult rat hepatocytes transduced ex vivo with a lentivirus expressing a human YapERT2 fusion protein (hYapERT2) under control of the hepatocyte-specific transthyretin (TTR) promoter repopulate normal rat liver in a tamoxifen-dependent manner. Transplanted hepatocytes expand very slowly but progressively to produce 10% repopulation at 6 months, showing clusters of mature hepatocytes that are fully integrated into hepatic parenchyma, with no evidence for dedifferentiation, dysplasia or malignant transformation. Thus, we have developed the first vector designed to regulate the growth control properties of Yap that renders it capable of producing effective cell therapy. The level of liver repopulation achieved has significant translational implications, as it is 2-3x the level required to cure many monogenic disorders of liver function that have no underlying hepatic pathology and is potentially applicable to diseases of other tissues and organs. PMID:26763940

Kinetics of depopulation, repopulation and host cell infiltration in the rhabdomyosarcoma R1H after 14 MeV neutron irradiation.

PubMed

Brammer, I; Zywietz, F; Beck-Bornholdt, H P; Jung, H

1992-05-01

The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H in the rat was studied following irradiation with 5 Gy of 14 MeV neutrons. Several parameters were sequentially measured over a time period of 4 weeks after irradiation: the tumour volume was assessed by in situ caliper measurements; the numerical density of tumour cells was obtained by morphometry; the clonogenic fraction of tumour cells was derived from in vitro colony assay; and the numerical ratio of host to tumour cells was determined by flow cytometry. From these primary parameters the number of clonogenic tumour cells, non-clonogenic tumour cells, and nucleated host cells per tumour, as well as their variation with time, were derived. The results were compared with two sets of data obtained previously for the same tumour exposed to 15 Gy of 200 kVp X-rays. Survival of tumour cells was reduced to 5.5 +/- 0.5% by 5 Gy neutrons and to 4.5 +/- 0.5% by 15 Gy X-rays, i.e. an RBE of close to 3. There was a lag period before the onset of repopulation (4.9 +/- 0.4 days and 4.9 +/- 0.5 days, respectively), followed by a high initial rate of repopulation corresponding to a doubling time of 2.0 +/- 0.2 days for neutrons and 2.1 +/- 0.2 days for X-rays. The rate of depopulation was significantly different for the two treatment modalities; the halving time for the number of non-clonogenic tumour cells was 11 +/- 4 days for neutrons and 2.8 +/- 0.5 days for X-rays.(ABSTRACT TRUNCATED AT 250 WORDS)

A majority of mice show long-term expression of a human. beta. -globin gene after retrovirus transfer into hematopoietic stem cells

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

Bender, M.A.; Gelinas, R.E.; Miller

1989-04-01

Murine bone marrow was infected with a high-titer retrovirus vector containing the human {beta}-globin and neomycin phosphotransferase genes. Anemic W/W/sup v/ mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human {beta}-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human {beta}-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human {beta}-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide.more » DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active {beta}-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.« less

Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers.

PubMed Central

Sutherland, H J; Lansdorp, P M; Henkelman, D H; Eaves, A C; Eaves, C J

1990-01-01

A major goal of current hematopoiesis research is to develop in vitro methods suitable for the measurement and characterization of stem cells with long-term in vivo repopulating potential. Previous studies from several centers have suggested the presence in normal human or murine marrow of a population of very primitive cells that are biologically, physically, and pharmacologically different from cells detectable by short-term colony assays and that can give rise to the latter in long-term cultures (LTCs) containing a competent stromal cell layer. In this report, we show that such cultures can be used to provide a quantitative assay for human "LTC-initiating cells" based on an assessment of the number of clonogenic cells present after 5-8 weeks. Production of derivative clonogenic cells is shown to be absolutely dependent on the presence of a stromal cell feeder. When this requirement is met, the clonogenic cell output (determined by assessment of 5-week-old cultures) is linearly related to the input cell number over a wide range of cell concentrations. Using limiting dilution analysis techniques, we have established the frequency of LTC-initiating cells in normal human marrow to be approximately 1 per 2 X 10(4) cells and in a highly purified CD34-positive subpopulation to be approximately 1 per 50-100 cells. The proliferative capacity exhibited by individual LTC-initiating cells cultured under apparently identical culture conditions was found to be highly variable. Values for the number of clonogenic cells per LTC-initiating cell in 5-week-old cultures ranged from 1 to 30 (the average being 4) with similar levels being detected in positive 8-week-old cultures. Some LTC-initiating cells are multipotent as evidenced by their generation of erythroid as well as granulopoietic progeny. The availability of a system for quantitative analysis of the proliferative and differentiative behavior of this newly defined compartment of primitive human hematopoietic cells should facilitate future studies of specific genetic or microenvironmental parameters involved in the regulation of these cells. Images PMID:2333304

The Delayed Effects of Acute Radiation Syndrome: Evidence of Long-Term Functional Changes in the Clonogenic Cells of the Small Intestine.

PubMed

Booth, Catherine; Tudor, Gregory L; Katz, Barry P; MacVittie, Thomas J

2015-11-01

Long term or residual damage post-irradiation has been described for many tissues. In hematopoietic stem cells (HSC), this is only revealed when the HSC are stressed and required to regenerate and repopulate a myeloablated host. Such an assay cannot be used to assess the recovery potential of previously irradiated intestinal stem cells (ISC) due to their incompatibility with transplantation. The best approximation to the HSC assay is the crypt microcolony assay, also based on clonogen survival. In the current study, the regenerative capacity of intestinal clonogenic cells in mice that had survived 13 Gy irradiation (with 5% bone marrow shielding to allow survival through the hematopoietic syndrome) and were then aged for 200 d was compared to previously unirradiated age-matched controls. Interestingly, at 200 d following 13 Gy, there remained a statistically significant reduction in crypts present in the various small intestinal regions (illustrating that the gastrointestinal epithelium had not fully recovered despite the 200-d interval). However, upon re-irradiation on day 196, those mice previously irradiated had improved crypt survival and regeneration compared to the age-matched controls. This was evident in all regions of the small intestine following 11-13 Gy re-exposure. Thus, there were either more clonogens per crypt within those previously irradiated and/or those that were present were more radioresistant (possibly because a subpopulation was more quiescent). This is contrary to the popular belief that previously irradiated animals may have an impaired/delayed regenerative response and be more radiosensitive.

Taxonomic and developmental aspects of radiosensitivity

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

Harrison, F.L.; Anderson, S.L.

1996-11-01

Considerable information is available on the effects of radioactivity on adult and early life stages of organisms. The preponderance of data is on mortality after a single irradiation with relatively high doses. Unfortunately, because experiments were carried out under different conditions and for different time periods, the validity of comparing the results from different laxonomic groups is questionable. In general, the conclusions are that there is a relationship (1) between radioresistance to high doses of acute radiation and taxonomy of the organism, primitive forms being more radioresistant than complex vertebrates and (2) between radiosensitivity and developmental stage, early life stagesmore » being more sensitive than later stages. The first conclusion may be related to the capability of the organism to repopulate cells and to differentiate and redifferentiate them; the second to the rate of cellular division and to the degree of differentiation. In question, however, is the relevance of the responses from high levels of acute radiation to that of the responses to long-term exposure to low levels of radiation, which are ecologically of more interest. Data from studies of the effects of acute and chronic exposure on development of gametes and zygotes indicate that, for some fishes and invertebrates, responses at the cellular and molecular levels show effect levels comparable to those observed in some mammals. Acute doses between 0,05 and 0.5Cy and dose rates between 0.02 to 0.2mCy/h appear to define critical ranges in which detrimental effects on fertility are first observed in a variety of radiosensitive organisms. To better understand inherent radiosensitivity, we need more information on the ability of cells to repopulate and differentiate and to prevent or repair damage to biological critical molecules, such as DNA, because these factors may alter significantly organisms` responses to radiation.« less

Effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro.

PubMed

Lee, J S; Kim, J M; Hong, E K; Kim, S-O; Yoo, Y-J; Cha, J-H

2009-02-01

A growing amount of attention has been placed on periodontal regeneration and wound healing for periodontal therapy. This study was conducted in an effort to determine the effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro. Human periodontal ligament cells were acquired from explant tissue of human healthy periodontal ligament. After the wounding of periodontal ligament cells, the change in expression of heparin-binding epidermal growth factor-like growth factor and epidermal growth factor receptors 1-4 mRNA was assessed. The effects of heparin-binding epidermal growth factor-like growth factor on periodontal ligament cell proliferation and repopulation were assessed in vitro via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and by photographing the injuries, respectively. Extracellular signal-regulated kinase (Erk)1/2, p38 and Akt phosphorylation was characterized via western blotting. Scratch wounding resulted in a significant up-regulation of heparin-binding epidermal growth factor-like growth factor mRNA expression, whereas wounding had no effect on the expression levels of epidermal growth factor receptors 1-4. Interestingly, no expression of epidermal growth factor receptors 2 and 4 was detectable prior to or after wounding. Heparin-binding epidermal growth factor-like growth factor treatment promoted the proliferation and repopulation of periodontal ligament cells. The scratch wounding also stimulated the phosphorylation of Erk1/2 and p38, but not of Akt, in periodontal ligament cells, and heparin-binding epidermal growth factor-like growth factor treatment applied after wounding amplified and extended the activations of Erk1/2 and p38, but not of Akt. Furthermore, Erk1/2 inhibition blocked the process of cell repopulation induced by heparin-binding epidermal growth factor-like growth factor, whereas the inhibition of p38 delayed the process. These results indicate that heparin-binding epidermal growth factor-like growth factor may constitute a critical factor in the wound healing of human periodontal ligament cells by a mechanism that requires the activation of Erk1/2 via specific interaction with epidermal growth factor receptor 1.

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

Aerts-Kaya, Fatima S.F.; Visser, Trudi P.; Arshad, Shazia

Purpose: 5-Androstene-3{beta},17{beta}-diol (5-AED) stimulates recovery of hematopoiesis after exposure to radiation. To elucidate its cellular targets, the effects of 5-AED alone and in combination with (pegylated) granulocyte colony-stimulating factor and thrombopoietin (TPO) on immature hematopoietic progenitor cells were evaluated following total body irradiation. Methods and Materials: BALB/c mice were exposed to radiation delivered as a single or as a fractionated dose, and recovery of bone marrow progenitors and peripheral blood parameters was assessed. Results: BALB/c mice treated with 5-AED displayed accelerated multilineage blood cell recovery and elevated bone marrow (BM) cellularity and numbers of progenitor cells. The spleen colony-forming unitmore » (CFU-S) assay, representing the life-saving short-term repopulating cells in BM of irradiated donor mice revealed that combined treatment with 5-AED plus TPO resulted in a 20.1-fold increase in CFU-S relative to that of placebo controls, and a 3.7 and 3.1-fold increase in comparison to 5-AED and TPO, whereas no effect was seen of Peg-G-CSF with or without 5-AED. Contrary to TPO, 5-AED also stimulated reconstitution of the more immature marrow repopulating (MRA) cells. Conclusions: 5-AED potently counteracts the hematopoietic effects of radiation-induced myelosuppression and promotes multilineage reconstitution by stimulating immature bone marrow cells in a pattern distinct from, but synergistic with TPO.« less

Angiocrine functions of organ-specific endothelial cells

PubMed Central

Rafii, Shahin; Butler, Jason M; Ding, Bi-Sen

2016-01-01

Preface Endothelial cells lining blood vessel capillaries are not just passive conduits for delivering blood. Tissue-specific endothelium establish specialized vascular niches that deploy specific sets of growth factors, known as angiocrine factors, which actively participate in inducing, specifying, patterning, and guiding organ regeneration and maintaining homeostasis and metabolism. Angiocrine factors upregulated in response to injury orchestrates self-renewal and differentiation of tissue-specific repopulating resident stem and progenitor cells into functional organs. Uncovering the precise mechanisms whereby physiological-levels of angiocrine factors are spatially and temporally produced, and distributed by organotypic endothelium to repopulating cells, will lay the foundation for driving organ repair without scarring. PMID:26791722

Revised radiobiological modelling of the contribution of synchronous chemotherapy to the rate of grades 3-4 mucositis in head and neck cancer.

PubMed

Meade, Sara; McConkey, Chris; Sanghera, Paul; Mehanna, Hisham; Hartley, Andrew

2013-12-01

Biological effective dose (BED) calculations modelled on reduced accelerated repopulation when synchronous chemotherapy is delivered significantly correlate with observed differences in local control in randomised trials of platinum-based chemoradiation. The purpose of this study was to examine whether a similar relationship existed in the context of grades 3-4 mucositis. Biological effective dose from radiotherapy and synchronous chemotherapy was calculated using three different models: AB using the additional BED attributable to chemotherapy and standard repopulation parameters; zero repopulation (ZRP) using zero correction for repopulation; and variable t(p) (Vt(p)) using a variable doubling time for mucosal stem cell repopulation. The correlation between the percentage change in biological effective dose between trial arms, and the observed percentage change in the rate of grades 3-4 mucositis was examined by using the Pearson product-moment correlation. With the AB model, there were no significant correlations with observed differences in rates of grades 3-4 mucositis. With either the ZRP or Vt(p) models, significant correlations were observed. A value of 5 days for the doubling time during repopulation (T(p)) was associated with the most significant correlation (P = 0.002). Models where the dose lost due to accelerated repopulation is reduced imply a therapeutic loss from the use of synchronous chemotherapy when only local control and the rate of acute grades 3-4 mucositis are considered. © 2013 The Royal Australian and New Zealand College of Radiologists.

Murine bone marrow cells cultured ex vivo in the presence of multiple cytokine combinations lose radioprotective and long-term engraftment potential.

PubMed

Von Drygalski, A; Alespeiti, G; Ren, L; Adamson, J W

2004-02-01

The desire to improve engraftment following transplantation of limited numbers of hematopoietic stem cells (HSC) has spurred the investigation of ex vivo stem cell expansion techniques. While surrogate outcomes, such as an increase in SCID-repopulating cells, suggest successful stem cell expansion in some studies, it is not clear that such assays predict outcomes using a more clinically relevant approach (e.g., myeloablation). We have addressed this by testing three cytokine combinations for their ability to increase the radioprotective and long-term marrow reconstitution capacity of hematopoietic cells cultured ex vivo. Low numbers of light-density (LD) mouse bone marrow (BM) cells or their expanded product were injected into lethally irradiated (9 Gy) congenic recipients. Survival rates and percent donor engraftment were compared at 2, 5, and 7 months post-transplant. The three cytokine combinations used were: (i) kit-ligand (L), thrombopoietin (Tpo), Flt-3 L; (ii) cytokines in (i) plus interleukin-11 (IL-11); (iii) cytokines in (ii) plus IL-3. At 7 months post-transplant, LD cell doses of 10(4), 2-2.5 x 10(4), and 0.5-1.0 x 10(5) gave predictable survivals of 20-30%, 40-70%, and 100%, respectively. Mean percent donor engraftments were 54.9% (SEM 36%), 55.7% (SEM 36%), and 76.3% (SEM 21%), respectively. When cells expanded for 3 or 5-7 days with the various cytokine combinations were transplanted into different groups of mice, survival rates and percent donor engraftment were almost uniformly poorer than results obtained with unmanipulated cells, and cells expanded for 5-7 days led to poorer outcomes than cells expanded for 3 days. Overall, ex vivo expansion of LD BM cells with the cytokine combinations chosen failed to improve transplant outcomes in this model.

Leukemogenesis Induced by an Activating β-catenin mutation in Osteoblasts

PubMed Central

Kode, Aruna; Manavalan, John S.; Mosialou, Ioanna; Bhagat, Govind; Rathinam, Chozha V.; Luo, Na; Khiabanian, Hossein; Lee, Albert; Vundavalli, Murty; Friedman, Richard; Brum, Andrea; Park, David; Galili, Naomi; Mukherjee, Siddhartha; Teruya-Feldstein, Julie; Raza, Azra; Rabadan, Raul; Berman, Ellin; Kousteni, Stavroula

2014-01-01

Summary Cells of the osteoblast lineage affect homing, 1, 2 number of long term repopulating hematopoietic stem cells (HSCs) 3, 4, HSC mobilization and lineage determination and B lymphopoiesis 5-8. More recently osteoblasts were implicated in pre-leukemic conditions in mice 9, 10. Yet, it has not been shown that a single genetic event taking place in osteoblasts can induce leukemogenesis. We show here that in mice, an activating mutation of β-catenin in osteoblasts alters the differentiation potential of myeloid and lymphoid progenitors leading to development of acute myeloid leukemia (AML) with common chromosomal aberrations and cell autonomous progression. Activated β-catenin stimulates expression of the Notch ligand Jagged-1 in osteoblasts. Subsequent activation of Notch signaling in HSC progenitors induces the malignant changes. Demonstrating the pathogenetic role of the Notch pathway, genetic or pharmacological inhibition of Notch signaling ameliorates AML. Nuclear accumulation and increased β-catenin signaling in osteoblasts was also identified in 38% of patients with MDS/AML. These patients showed increased Notch signaling in hematopoietic cells. These findings demonstrate that genetic alterations in osteoblasts can induce AML, identify molecular signals leading to this transformation and suggest a potential novel pharmacotherapeutic approach to AML. PMID:24429522

PAR1 signaling regulates the retention and recruitment of EPCR-expressing bone marrow hematopoietic stem cells

PubMed Central

Gur-Cohen, Shiri; Itkin, Tomer; Chakrabarty, Sagarika; Graf, Claudine; Kollet, Orit; Ludin, Aya; Golan, Karin; Kalinkovich, Alexander; Ledergor, Guy; Wong, Eitan; Niemeyer, Elisabeth; Porat, Ziv; Erez, Ayelet; Sagi, Irit; Esmon, Charles T; Ruf, Wolfram; Lapidot, Tsvee

2016-01-01

Retention of long-term repopulating hematopoietic stem cells (LT-HSCs) in the bone marrow is essential for hematopoiesis and for protection from myelotoxic injury. We report that signaling cascades that are traditionally viewed as coagulation-related also control retention of EPCR+ LT-HSCs in the bone marrow and their recruitment to the blood via two different protease activated receptor 1 (PAR1)-mediated pathways. Thrombin-PAR1 signaling induces nitric oxide (NO) production, leading to TACE-mediated EPCR shedding, enhanced CXCL12-CXCR4-induced motility, and rapid stem and progenitor cell mobilization. Conversely, bone marrow blood vessels provide a microenvironment enriched with protein C that retain EPCR+ LT-HSCs by limiting NO generation, reducing Cdc42 activity and enhancing VLA4 affinity and adhesion. Inhibition of NO production by activated protein C (aPC)-EPCR-PAR1 signaling reduces progenitor cell egress, increases NOlow bone marrow EPCR+ LT-HSCs retention and protects mice from chemotherapy-induced hematological failure and death. Our study reveals new roles for PAR1 and EPCR that control NO production to balance maintenance and recruitment of bone marrow EPCR+ LT-HSCs with clinical relevance. PMID:26457757

Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia.

PubMed

Heckl, Dirk; Wicke, Daniel C; Brugman, Martijn H; Meyer, Johann; Schambach, Axel; Büsche, Guntram; Ballmaier, Matthias; Baum, Christopher; Modlich, Ute

2011-04-07

Thpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl(-/-)) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl(-/-) bone marrow cells into Mpl(-/-) mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl(-/-) cells had increased long-term repopulating potential, with a marked increase in lineage(-)Sca1(+)cKit(+) cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage(-)Sca1(+)cKit(+) cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.

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.

Mitomycin C in combination with radiotherapy as a potent inhibitor of tumour cell repopulation in a human squamous cell carcinoma

PubMed Central

Budach, W; Paulsen, F; Welz, S; Classen, J; Scheithauer, H; Marini, P; Belka, C; Bamberg, M

2002-01-01

The potential of Mitomycin C in combination with fractionated irradiation to inhibit tumour cell repopulation of a fast growing squamous cell carcinoma after fractionated radiotherapy was investigated in vivo. A rapidly growing human squamous cell carcinoma (FaDudd) was used for the study. For experiments, NMRI (nu/nu) mice with subcutaneously growing tumours were randomly allocated to no treatment, Mitomycin C, fractionated irradiation (ambient: 11x4.5 Gy in 15 days), or fractionated irradiation combined with Mitomycin C. Graded top up doses (clamped blood flow: 0–57 Gy) were given at day 16, 23, 30 or 37. End point of the study was the time to local tumour progression. Data were examined by multiple regression analysis (Cox). Mitomycin C alone resulted in a median time to local tumour progression of 23 (95% confidence limits: 17–43) days, fractionated irradiation in 31 (25–35) days and combined Mitomycin C plus fractionated irradiation in 65 (58–73) days (P=0.02). Mitomycin C decreased the relative risk of local recurrence by 94% (P<<0.001) equivalent to 31.7 Gy top up dose. Repopulation accounted for 1.33 (0.95–1.72) Gy per day top up dose after fractionated irradiation alone and for 0.68 (0.13–1.22) Gy per day after fractionated irradiation+Mitomycin C (P=0.018). Mitomycin C significantly reduces the risk of local recurrence and inhibits tumour cell repopulation in combination with fractionated irradiation in vivo in the tested tumour model. British Journal of Cancer (2002) 86, 470–476. DOI: 10.1038/sj/bjc/6600081 www.bjcancer.com © 2002 The Cancer Research Campaign PMID:11875717

Continuous in vivo infusion of interferon-gamma (IFN-γ) enhances engraftment of syngeneic wild-type cells in Fanca–/– and Fancg–/– mice

PubMed Central

Si, Yue; Ciccone, Samantha; Yang, Feng-Chun; Yuan, Jin; Zeng, Daisy; Chen, Shi; van de Vrugt, Henri J.; Critser, John; Arwert, Fre; Haneline, Laura S.; Clapp, D. Wade

2006-01-01

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc–/– cells to interferon-gamma (IFN-γ), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc–/– mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca–/– and Fancg–/– mice are hypersensitive to IFN-γ and that in vivo infusion of IFN-γ at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-γ conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients. PMID:16946306

Continuous in vivo infusion of interferon-gamma (IFN-gamma) enhances engraftment of syngeneic wild-type cells in Fanca-/- and Fancg-/- mice.

PubMed

Si, Yue; Ciccone, Samantha; Yang, Feng-Chun; Yuan, Jin; Zeng, Daisy; Chen, Shi; van de Vrugt, Henri J; Critser, John; Arwert, Fre; Haneline, Laura S; Clapp, D Wade

2006-12-15

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc-/- cells to interferon-gamma (IFN-gamma), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc-/- mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca-/- and Fancg-/- mice are hypersensitive to IFN-gamma and that in vivo infusion of IFN-gamma at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-gamma conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients.

Evaluation of hematopoietic potential generated by transplantation of muscle-derived stem cells in mice.

PubMed

Farace, Francoise; Prestoz, Laetitita; Badaoui, Sabrina; Guillier, Martine; Haond, Celine; Opolon, Paule; Thomas, Jean-Leon; Zalc, Bernard; Vainchenker, William; Turhan, Ali G

2004-02-01

Muscle tissue of adult mice has been shown to contain stem cells with hematopoietic repopulation ability in vivo. To determine the functional characteristics of stem cells giving rise to this hematopoietic activity, we have performed hematopoietic reconstitution experiments by the use of muscle versus marrow transplantation in lethally irradiated mice and followed the fate of transplanted cells by Y-chimerism using PCR and fluorescence in situ hybridization (FISH) analysis. We report here that transplantation of murine muscle generate a major hematopoietic chimerism at the level of CFU-C, CFU-S, and terminally-differentiated cells in three generations of lethally irradiated mice followed up to 1 year after transplantation. This potential is totally abolished when muscle grafts were performed by the use of muscle from previously irradiated mice. As compared to marrow transplantation, muscle transplants were able to generate similar potencies to give rise to myeloid, T, B, and natural killer (NK) cells. Interestingly, marrow stem cells that have been generated in primary and then in secondary recipients were able to contribute efficiently to myofibers in the muscle tissue of tertiary recipients. Altogether, our data demonstrate that muscle-derived stem cells present a major hematopoietic repopulating ability with evidence of self-replication in vivo. They are radiation-sensitive and similar to marrow-derived stem cells in terms of their ability to generate multilineage hematopoiesis. Finally, our data demonstrate that muscle-derived hematopoietic stem cells do not lose their ability to contribute to myofiber generation after at least two rounds of serial transplantation, suggesting a potential that is probably equivalent to that generated by marrow transplantation.

Reversing drug resistance of soft tumor-repopulating cells by tumor cell-derived chemotherapeutic microparticles

PubMed Central

Ma, Jingwei; Zhang, Yi; Tang, Ke; Zhang, Huafeng; Yin, Xiaonan; Li, Yong; Xu, Pingwei; Sun, Yanling; Ma, Ruihua; Ji, Tiantian; Chen, Junwei; Zhang, Shuang; Zhang, Tianzhen; Luo, Shunqun; Jin, Yang; Luo, Xiuli; Li, Chengyin; Gong, Hongwei; Long, Zhixiong; Lu, Jinzhi; Hu, Zhuowei; Cao, Xuetao; Wang, Ning; Yang, Xiangliang; Huang, Bo

2016-01-01

Developing novel approaches to reverse the drug resistance of tumor-repopulating cells (TRCs) or stem cell-like cancer cells is an urgent clinical need to improve outcomes of cancer patients. Here we show an innovative approach that reverses drug resistance of TRCs using tumor cell-derived microparticles (T-MPs) containing anti-tumor drugs. TRCs, by virtue of being more deformable than differentiated cancer cells, preferentially take up T-MPs that release anti-tumor drugs after entering cells, which in turn lead to death of TRCs. The underlying mechanisms include interfering with drug efflux and promoting nuclear entry of the drugs. Our findings demonstrate the importance of tumor cell softness in uptake of T-MPs and effectiveness of a novel approach in reversing drug resistance of TRCs with promising clinical applications. PMID:27167569

Chromatin insulator bodies are nuclear structures that form in response to osmotic stress and cell death

PubMed Central

Schoborg, Todd; Rickels, Ryan; Barrios, Josh

2013-01-01

Chromatin insulators assist in the formation of higher-order chromatin structures by mediating long-range contacts between distant genomic sites. It has been suggested that insulators accomplish this task by forming dense nuclear foci termed insulator bodies that result from the coalescence of multiple protein-bound insulators. However, these structures remain poorly understood, particularly the mechanisms triggering body formation and their role in nuclear function. In this paper, we show that insulator proteins undergo a dramatic and dynamic spatial reorganization into insulator bodies during osmostress and cell death in a high osmolarity glycerol–p38 mitogen-activated protein kinase–independent manner, leading to a large reduction in DNA-bound insulator proteins that rapidly repopulate chromatin as the bodies disassemble upon return to isotonicity. These bodies occupy distinct nuclear territories and contain a defined structural arrangement of insulator proteins. Our findings suggest insulator bodies are novel nuclear stress foci that can be used as a proxy to monitor the chromatin-bound state of insulator proteins and provide new insights into the effects of osmostress on nuclear and genome organization. PMID:23878275

Lineage-specific T-cell reconstitution following in vivo CD4+ and CD8+ lymphocyte depletion in nonhuman primates.

PubMed

Engram, Jessica C; Cervasi, Barbara; Borghans, Jose A M; Klatt, Nichole R; Gordon, Shari N; Chahroudi, Ann; Else, James G; Mittler, Robert S; Sodora, Donald L; de Boer, Rob J; Brenchley, Jason M; Silvestri, Guido; Paiardini, Mirko

2010-08-05

Many features of T-cell homeostasis in primates are still unclear, thus limiting our understanding of AIDS pathogenesis, in which T-cell homeostasis is lost. Here, we performed experiments of in vivo CD4(+) or CD8(+) lymphocyte depletion in 2 nonhuman primate species, rhesus macaques (RMs) and sooty mangabeys (SMs). Whereas RMs develop AIDS after infection with simian immunodeficiency virus (SIV), SIV-infected SMs are typically AIDS-resistant. We found that, in both species, most CD4(+) or CD8(+) T cells in blood and lymph nodes were depleted after treatment with their respective antibodies. These CD4(+) and CD8(+) lymphocyte depletions were followed by a largely lineage-specific CD4(+) and CD8(+) T-cell proliferation, involving mainly memory T cells, which correlated with interleukin-7 plasma levels. Interestingly, SMs showed a faster repopulation of naive CD4(+) T cells than RMs. In addition, in both species CD8(+) T-cell repopulation was faster than that of CD4(+) T cells, with CD8(+) T cells reconstituting a normal pool within 60 days and CD4(+) T cells remaining below baseline levels up to day 180 after depletion. While this study revealed subtle differences in CD4(+) T-cell repopulation in an AIDS-sensitive versus an AIDS-resistant species, such differences may have particular relevance in the presence of active SIV repli cation, where CD4(+) T-cell destruction is chronic.

Lineage-specific T-cell reconstitution following in vivo CD4+ and CD8+ lymphocyte depletion in nonhuman primates

PubMed Central

Engram, Jessica C.; Cervasi, Barbara; Borghans, Jose A. M.; Klatt, Nichole R.; Gordon, Shari N.; Chahroudi, Ann; Else, James G.; Mittler, Robert S.; Sodora, Donald L.; de Boer, Rob J.; Brenchley, Jason M.; Silvestri, Guido

2010-01-01

Many features of T-cell homeostasis in primates are still unclear, thus limiting our understanding of AIDS pathogenesis, in which T-cell homeostasis is lost. Here, we performed experiments of in vivo CD4+ or CD8+ lymphocyte depletion in 2 nonhuman primate species, rhesus macaques (RMs) and sooty mangabeys (SMs). Whereas RMs develop AIDS after infection with simian immunodeficiency virus (SIV), SIV-infected SMs are typically AIDS-resistant. We found that, in both species, most CD4+ or CD8+ T cells in blood and lymph nodes were depleted after treatment with their respective antibodies. These CD4+ and CD8+ lymphocyte depletions were followed by a largely lineage-specific CD4+ and CD8+ T-cell proliferation, involving mainly memory T cells, which correlated with interleukin-7 plasma levels. Interestingly, SMs showed a faster repopulation of naive CD4+ T cells than RMs. In addition, in both species CD8+ T-cell repopulation was faster than that of CD4+ T cells, with CD8+ T cells reconstituting a normal pool within 60 days and CD4+ T cells remaining below baseline levels up to day 180 after depletion. While this study revealed subtle differences in CD4+ T-cell repopulation in an AIDS-sensitive versus an AIDS-resistant species, such differences may have particular relevance in the presence of active SIV repli cation, where CD4+ T-cell destruction is chronic. PMID:20484087

Characterization of slow-cycling cells in the mouse cochlear lateral wall

PubMed Central

Ogawa, Kaoru

2017-01-01

Cochlear spiral ligament fibrocytes (SLFs) play essential roles in the physiology of hearing including ion recycling and the generation of endocochlear potential. In adult animals, SLFs can repopulate after damages, yet little is known about the characteristics of proliferating cells that support SLFs’ self-renewal. Here we report in detail about the characteristics of cycling cells in the spiral ligament (SL). Fifteen P6 mice and six noise-exposed P28 mice were injected with 5-bromo-2′-deoxyuridine (BrdU) for 7 days and we chased BrdU retaining cells for as long as 60 days. Immunohistochemistry revealed that the BrdU positive IB4 (an endotherial marker) negative cells expressed an early SLF marker Pou3f4 but negative for cleaved-Caspase 3. Marker studies revealed that type 3 SLFs displayed significantly higher percentage of BrdU+ cells compared to other subtypes. Notably, the cells retained BrdU until P72, demonstrating they were dividing slowly. In the noise-damaged mice, in contrast to the loss of the other types, the number of type 3 SLFs did not altered and the BrdU incorporating- phosphorylated Histone H3 positive type 3 cells were increased from day 1 to 14 after noise exposure. Furthermore, the cells repopulating type 1 area, where the cells diminished profoundly after damage, were positive for the type 3 SLF markers. Collectively, in the latral wall of the cochlea, type 3 SLFs have the stem cell capacity and may contribute to the endogenous regeneration of lateral wall spiral ligament. Manipulating type 3 cells may be employed for potential regenerative therapies. PMID:28632772

Elements of planetary protection against asteroid and comet hazard

NASA Astrophysics Data System (ADS)

Steklov, A. F.; Vidmachenko, A. P.; Dashkiev, G. N.; Zhilyaev, B. E.

2018-05-01

The principles of protection against asteroid-comet hazard should constitute the main priority of the modern Proto-cosmic civilization on the planet Earth. Any impact of a fairly large asteroid or cometary nucleus with a size of 1 to 20 or more kilometers will lead to a global catastrophe and, perhaps, to the death of Mankind. Forces in order to withstand such a blow of the cosmic body during large space invasions, we do not have and, most likely, will not be for a long time . We need as soon as possible to create technical facilities and systems for long-term comfortable living of large colonies of people on the Moon, Mars, Venus and Mercury, having arranged there some elements of the biosphere. In these colonies people should live in extraterrestrial space settlements, and should periodically and constantly "outplay" scenarios of reliable and guaranteed re-population of the planet Earth by people. Such periodic "exercises" on the actual modeling of the return to the "post-catastrophic" Earth should ensure the survival of humanity even in the worst versions of the consequences of possible dangerous space invasions. That is, we should always be ready for the repopulation on the Earth by people and for the reconstruction of the basic elements of the man's biosphere.

Growth promotion of genetically modified hematopoietic progenitors using an antibody/c-Mpl chimera.

PubMed

Kawahara, Masahiro; Chen, Jianhong; Sogo, Takahiro; Teng, Jinying; Otsu, Makoto; Onodera, Masafumi; Nakauchi, Hiromitsu; Ueda, Hiroshi; Nagamune, Teruyuki

2011-09-01

Thrombopoietin is a potent cytokine that exerts proliferation of hematopoietic stem cells (HSCs) through its cognate receptor, c-Mpl. Therefore, mimicry of c-Mpl signaling by a receptor recognizing an artificial ligand would be attractive to attain specific expansion of genetically modified HSCs. Here we propose a system enabling selective expansion of genetically modified cells using an antibody/receptor chimera that can be activated by a specific antigen. We constructed an antibody/c-Mpl chimera, in which single-chain Fv (ScFv) of an anti-fluorescein antibody was tethered to the extracellular D2 domain of the erythropoietin receptor and transmembrane/cytoplasmic domains of c-Mpl. When the chimera was expressed in interleukin (IL)-3-dependent pro-B cell line Ba/F3, genetically modified cells were selectively expanded in the presence of fluorescein-conjugated BSA (BSA-FL) as a specific antigen. Furthermore, highly purified mouse HSCs transduced with the retrovirus carrying antibody/c-Mpl chimera gene proliferated in vitro in response to BSA-FL, and the cells retained in vivo long-term repopulating abilities. These results demonstrate that the antibody/c-Mpl chimera is capable of signal transduction that mimics wild-type c-Mpl signaling. Copyright © 2011 Elsevier Ltd. All rights reserved.

Patterns of hemopoietic reconstitution in nonobese diabetic mice: dichotomy of allogeneic resistance versus competitive advantage of disease-resistant marrow.

PubMed

Kaufman, C L; Li, H; Ildstad, S T

1997-03-01

Complete replacement of the immune system via allogeneic bone marrow transplantation is sufficient to prevent diabetes in the nonobese diabetic (NOD) mouse model. In the present study we examined whether mixed allogeneic reconstitution would be sufficient to interrupt the autoimmune process with respect to occurrence of overt diabetes, as well as preexisting autoimmune insulitis. NOD mice were lethally irradiated and reconstituted with a mixture of NOD and B10.BR marrow. A relative resistance to allogeneic bone marrow engraftment was noted in NOD recipients of the mixed bone marrow inoculum, compared with disease-resistant controls. Moreover, unlike disease-resistant controls, all animals that initially repopulated as mixed donor/host chimeras became predominantly allogeneic by 4 mo, suggesting a competitive advantage for long term engraftment for disease-resistant marrow. All but one mouse in the group that engrafted with allogeneic marrow remained free of diabetes for the entire follow-up period (n = 22). Moreover, in all animals examined, virtually all islets were free of insulitis. In contrast, 74% of NOD mice that received similar conditioning and failed to engraft with donor marrow developed acute diabetes and intra-islet insulitis was present in all animals examined. These data suggest that NOD mice exhibit a relative resistance to engraftment compared with disease-resistant recipients. Conversely, animals that initially repopulated as a mixture of syngeneic and donor marrow become converted to virtually all donor by 4 mo. These data provide additional support that a defective stem cell is responsible for autoimmune diabetes in this experimental model.

The In Vivo PDGF Response During Remyelination in Mouse Spinal Cord Following Murine Hepatitis Virus Strain AS9-Induced Transient Demyelination

DTIC Science & Technology

1998-09-14

repopulation. These and other growth factors interacting with cell adhesion molecules andlor matrix molecules would be expected to mediate oligodendrocyte...oligodendrocyte lineage, along with a closely related CCHC zinc finger, is expressed in developing neurons in the mammalian central nervous system. J...repopulate and remyelinate demyelinated lesions. In vitro studies have shown that platelet- derived growth factor (PDGF) induces proliferation

Tumor-Repopulating Cells Induce PD-1 Expression in CD8+ T Cells by Transferring Kynurenine and AhR Activation.

PubMed

Liu, Yuying; Liang, Xiaoyu; Dong, Wenqian; Fang, Yi; Lv, Jiadi; Zhang, Tianzhen; Fiskesund, Roland; Xie, Jing; Liu, Jinyan; Yin, Xiaonan; Jin, Xun; Chen, Degao; Tang, Ke; Ma, Jingwei; Zhang, Huafeng; Yu, Jing; Yan, Jun; Liang, Huaping; Mo, Siqi; Cheng, Feiran; Zhou, Yabo; Zhang, Haizeng; Wang, Jing; Li, Jingnan; Chen, Yang; Cui, Bing; Hu, Zhuo-Wei; Cao, Xuetao; Xiao-Feng Qin, F; Huang, Bo

2018-03-12

Despite the clinical successes fostered by immune checkpoint inhibitors, mechanisms underlying PD-1 upregulation in tumor-infiltrating T cells remain an enigma. Here, we show that tumor-repopulating cells (TRCs) drive PD-1 upregulation in CD8 + T cells through a transcellular kynurenine (Kyn)-aryl hydrocarbon receptor (AhR) pathway. Interferon-γ produced by CD8 + T cells stimulates release of high levels of Kyn produced by TRCs, which is transferred into adjacent CD8 + T cells via the transporters SLC7A8 and PAT4. Kyn induces and activates AhR and thereby upregulates PD-1 expression. This Kyn-AhR pathway is confirmed in both tumor-bearing mice and cancer patients and its blockade enhances antitumor adoptive T cell therapy efficacy. Thus, we uncovered a mechanism of PD-1 upregulation with potential tumor immunotherapeutic applications. Copyright © 2018 Elsevier Inc. All rights reserved.

Antibody targeting KIT as pretransplantation conditioning in immunocompetent mice.

PubMed

Xue, Xingkui; Pech, Nancy K; Shelley, W Christopher; Srour, Edward F; Yoder, Mervin C; Dinauer, Mary C

2010-12-09

Inherited hematologic defects that lack an in vivo selective advantage following gene correction may benefit from effective yet minimally toxic cytoreduction of endogenous hematopoietic stem cells (HSCs) prior to transplantation of gene-modified HSCs. We studied the efficacy of administering a novel sequential treatment of parenteral ACK2, an antibody that blocks KIT, followed by low-dose irradiation (LD-IR) for conditioning of wild-type and X-linked chronic granulomatous disease (X-CGD) mice. In wild-type mice, combining ACK2 and LD-IR profoundly decreased endogenous competitive long-term HSC repopulating activity, and permitted efficient and durable donor-derived HSC engraftment after congenic transplantation. ACK2 alone was ineffective. The combination of ACK2 and LD-IR was also effective conditioning in X-CGD mice for engraftment of X-CGD donor HSCs transduced ex vivo with a lentiviral vector. We conclude that combining ACK2 with LD-IR is a promising approach to effectively deplete endogenous HSCs and facilitate engraftment of transplanted donor HSCs.

Importance of Sox2 in maintenance of cell proliferation and multipotency of mesenchymal stem cells in low-density culture.

PubMed

Yoon, D S; Kim, Y H; Jung, H S; Paik, S; Lee, J W

2011-10-01

This study has aimed to repopulate 'primitive' cells from late-passage mesenchymal stem cells (MSCs) of poor multipotentiality and low cell proliferation rate, by simply altering plating density. Effects of low density culture compared t high density culture on late-passage bone marrow (BM)-derived MSCs and pluripotency markers of multipotentiality were investigated. Cell proliferation, gene expression, RNA interference and differentiation potential were assayed. We repopulated 'primitive' cells by replating late-passage MSCs at low density (17 cells/cm(2) ) regardless of donor age. Repopulated MSCs from low-density culture were smaller cells with spindle shaped morphology compared to MSCs from high-density culture. The latter had enhanced colony-forming ability, proliferation rate, and adipogenic and chondrogenic potential. Strong expression of osteogenic-related genes (Cbfa1, Dlx5, alkaline phosphatase and type Ι collagen) in late-passage MSCs was reduced by replating at low density, whereas expression of three pluripotency markers (Sox2, Nanog and Oct-4), Osterix and Msx2 reverted to levels of early-passage MSCs. Knockdown of Sox2 and Msx2 but not Nanog, using RNA interference, showed significant decrease in colony-forming ability. Specifically, knockdown of Sox2 significantly inhibited multipotentiality and cell proliferation. Our data suggest that plating density should be considered to be a critical factor for enrichment of 'primitive' cells from heterogeneous BM and that replicative senescence and multipotentiality of MSCs during in vitro expansion may be predominantly regulated through Sox2. © 2011 Blackwell Publishing Ltd.

The Culture-Repopulating Ability Assays and Incubation in Low Oxygen: A Simple Way to Test Drugs on Leukaemia Stem or Progenitor Cells

PubMed Central

Cipolleschi, Maria Grazia; Rovida, Elisabetta; Sbarba, Persio Dello

2013-01-01

The Culture-Repopulating Ability (CRA) assays is a method to measure in vitro the bone marrow-repopulating potential of haematopoietic cells. The method was developed in our laboratory in the course of studies based on the use of growth factor-supplemented liquid cultures to study haematopoietic stem/progenitor cell resistance to, and selection at, low oxygen tensions in the incubation atmosphere. These studies led us to put forward the first hypothesis of the existence in vivo of haematopoietic stem cell niches where oxygen tension is physiologically lower than in other bone marrow areas. The CRA assays and incubation in low oxygen were later adapted to the study of leukaemias. Stabilized leukaemia cell lines, ensuring genetically homogeneous cells and enhancing repeatability of results, were found nevertheless phenotypically heterogeneous, comprising cell subsets exhibiting functional phenotypes of stem or progenitor cells. These subsets can be assayed separately, provided an experimental system capable to select one from another (such as different criteria for incubation in low oxygen) is established. On this basis, a two-step procedure was designed, including a primary culture of leukaemia cells in low oxygen for different times, where drug treatment is applied, followed by the transfer of residual cell population (CRA assay) to a drug-free secondary culture incubated at standard oxygen tension, where the expansion of population is allowed. The CRA assays, applied to cell lines first and then to primary cells, represent a simple and relatively rapid, yet accurate and reliable, method for the pre-screening of drugs potentially active on leukaemias which in our opinion could be adopted systematically before they are tested in vivo. PMID:23394087

β-Arrestin2 mediates progression of murine primary myelofibrosis.

PubMed

Rein, Lindsay Am; Wisler, James W; Kim, Jihee; Theriot, Barbara; Huang, LiYin; Price, Trevor; Yang, Haeyoon; Chen, Minyong; Chen, Wei; Sipkins, Dorothy; Fedoriw, Yuri; Walker, Julia Kl; Premont, Richard T; Lefkowitz, Robert J

2017-12-21

Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. β-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, β-arrestin2 (βarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between βarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor βarr2-knockout (βarr2-/-) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted βarr2-/- cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of βarr2-/- cells. In order to assess the effect of acute loss of βarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional βarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic βarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that βarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease.

Niche displacement of human leukemic stem cells uniquely allows their competitive replacement with healthy HSPCs

PubMed Central

Boyd, Allison L.; Campbell, Clinton J.V.; Hopkins, Claudia I.; Fiebig-Comyn, Aline; Russell, Jennifer; Ulemek, Jelena; Foley, Ronan; Leber, Brian; Xenocostas, Anargyros; Collins, Tony J.

2014-01-01

Allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) is currently the leading strategy to manage acute myeloid leukemia (AML). However, treatment-related morbidity limits the patient generalizability of HSCT use, and the survival of leukemic stem cells (LSCs) within protective areas of the bone marrow (BM) continues to lead to high relapse rates. Despite growing appreciation for the significance of the LSC microenvironment, it has remained unresolved whether LSCs preferentially situate within normal HSC niches or whether their niche requirements are more promiscuous. Here, we provide functional evidence that the spatial localization of phenotypically primitive human AML cells is restricted to niche elements shared with their normal counterparts, and that their intrinsic ability to initiate and retain occupancy of these niches can be rivaled by healthy hematopoietic stem and progenitor cells (HSPCs). When challenged in competitive BM repopulation assays, primary human leukemia-initiating cells (L-ICs) can be consistently outperformed by HSPCs for BM niche occupancy in a cell dose-dependent manner that ultimately compromises long-term L-IC renewal and subsequent leukemia-initiating capacity. The effectiveness of this approach could be demonstrated using cytokine-induced mobilization of established leukemia from the BM that facilitated the replacement of BM niches with transplanted HSPCs. These findings identify a functional vulnerability of primitive leukemia cells, and suggest that clinical development of these novel transplantation techniques should focus on the dissociation of L-IC–niche interactions to improve competitive replacement with healthy HSPCs during HSCT toward increased survival of patients. PMID:25180064

Wound Healing and Cancer Stem Cells: Inflammation as a Driver of Treatment Resistance in Breast Cancer

PubMed Central

Arnold, Kimberly M; Opdenaker, Lynn M; Flynn, Daniel; Sims-Mourtada, Jennifer

2015-01-01

The relationship between wound healing and cancer has long been recognized. The mechanisms that regulate wound healing have been shown to promote transformation and growth of malignant cells. In addition, chronic inflammation has been associated with malignant transformation in many tissues. Recently, pathways involved in inflammation and wound healing have been reported to enhance cancer stem cell (CSC) populations. These cells, which are highly resistant to current treatments, are capable of repopulating the tumor after treatment, causing local and systemic recurrences. In this review, we highlight proinflammatory cytokines and developmental pathways involved in tissue repair, whose deregulation in the tumor microenvironment may promote growth and survival of CSCs. We propose that the addition of anti-inflammatory agents to current treatment regimens may slow the growth of CSCs and improve therapeutic outcomes. PMID:25674014

[Anti-mouse CD122 antibody promotes the hematopoietic repopulating capacity of cord blood CD34⁺ cells in NOD/SCID mice].

PubMed

Sheng, Men-Yao; Shi, Hui; Xing, Wen; Wang, Wen-Jun; Si, Xiao-Hui; Bai, Jie; Yuan, Wei-Ping; Zhou, Yuan; Yang, Feng-Chun

2014-12-01

The study was aimed to investigate the effect of anti-mouse CD122 antibody on the hematopoietic repopulating capacity of cord blood CD34⁺ cells in a humanized murine model-non obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After sublethal irradiation with γ-ray, NOD/SCID mice were intraperitoneally injected with 200 µg mouse isotype control antibody or anti-mouse CD122 antibody. Human cord blood CD34⁺ cells or phosphate-buffered saline (PBS) were injected via the tail vein at 6-8 hours later. Cohort of the mice injected with anti-mice CD122 antibody or control antibody alone were sacrificed at different time point (at week 2, 3, and 4 weeks) after the injection, and the percentage of NK cells in the peripheral blood was analyzed by flow cytometry. To evaluate the effect of anti-mouse CD122 antibody on the repopulating capacity of cord blood CD34⁺ cells in the recipient mice, phenotype analysis was performed in the bone marrow at 6 and 8 weeks after the transplantation. The results showed that the proportion of NK cells in the peripheral blood were (4.6 ± 0.6)% and (5.7 ± 1.7)% at week 2 and 3 after anti-CD122 antibody injection respectively,which decreased by 60%, compared with the mice injected with isotype control antibody. After 6 and 8 weeks of cord blood CD34⁺ cell transplantation,the percentage of human CD45⁺ in the bone marrow of the recipient mice treated with anti-mice CD122 antibody was (63.0 ± 12.2)% and (53.2 ± 16.3)%,respectively,which were dramatically higher than that in the mice treated with isotype control antibody (7.7 ± 3.6)% and (6.1 ± 2.4)%. Moreover,at 8 weeks after transplantation,human CD34⁺ cells appeared significantly in the recipients treated with anti-CD122 antibody. It is concluded that the anti-mouse CD122 antibody enhances the hematopoietic repopulating capacity of cord blood CD34⁺ cells in the NOD/SCID mice through decreasing the proportion of NK cells.

Impact of alemtuzumab treatment on the survival and function of human regulatory T cells in vitro

PubMed Central

Havari, Evis; Turner, Michael J; Campos-Rivera, Juanita; Shankara, Srinivas; Nguyen, Tri-Hung; Roberts, Bruce; Siders, William; Kaplan, Johanne M

2014-01-01

Alemtuzumab is a humanized monoclonal antibody specific for the CD52 protein present at high levels on the surface of B and T lymphocytes. In clinical trials, alemtuzumab has shown a clinical benefit superior to that of interferon-β in relapsing–remitting multiple sclerosis patients. Treatment with alemtuzumab leads to the depletion of circulating lymphocytes followed by a repopulation process characterized by alterations in the number, proportions and properties of lymphocyte subsets. Of particular interest, an increase in the percentage of T cells with a regulatory phenotype (Treg cells) has been observed in multiple sclerosis patients after alemtuzumab. Since Treg cells play an important role in the control of autoimmune responses, the effect of alemtuzumab on Treg cells was further studied in vitro. Alemtuzumab effectively mediated complement-dependent cytolysis of human T lymphocytes and the remaining population was enriched in T cells with a regulatory phenotype. The alemtuzumab-exposed T cells displayed functional regulatory characteristics including anergy to stimulation with allogeneic dendritic cells and ability to suppress the allogeneic response of autologous T cells. Consistent with the observed increase in Treg cell frequency, the CD25hi T-cell population was necessary for the suppressive activity of alemtuzumab-exposed T cells. The mechanism of this suppression was found to be dependent on both cell–cell contact and interleukin-2 consumption. These findings suggest that an alemtuzumab-mediated increase in the proportion of Treg cells may play a role in promoting the long-term efficacy of alemtuzumab in patients with multiple sclerosis. PMID:24116901

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes.

PubMed

Ding, Jianqiang; Yannam, Govardhana R; Roy-Chowdhury, Namita; Hidvegi, Tunda; Basma, Hesham; Rennard, Stephen I; Wong, Ronald J; Avsar, Yesim; Guha, Chandan; Perlmutter, David H; Fox, Ira J; Roy-Chowdhury, Jayanta

2011-05-01

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z-expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%-98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z-expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.

A subset of virus-specific CD161+ T cells selectively express the multidrug transporter MDR1 and are resistant to chemotherapy in AML

PubMed Central

Alsuliman, Abdullah; Muftuoglu, Muharrem; Khoder, Ahmad; Ahn, Yong-Oon; Basar, Rafet; Verneris, Michael R.; Muranski, Pawel; Barrett, A. John; Liu, Enli; Li, Li; Stringaris, Kate; Armstrong-James, Darius; Shaim, Hila; Kondo, Kayo; Imahashi, Nobuhiko; Andersson, Borje; Marin, David; Champlin, Richard E.; Shpall, Elizabeth J.

2017-01-01

The establishment of long-lived pathogen-specific T cells is a fundamental property of the adaptive immune response. However, the mechanisms underlying long-term persistence of antigen-specific CD4+ T cells are not well-defined. Here we identify a subset of memory CD4+ T cells capable of effluxing cellular toxins, including rhodamine (Rho), through the multidrug efflux protein MDR1 (also known as P-glycoprotein and ABCB1). Drug-effluxing CD4+ T cells were characterized as CD161+CD95+CD45RA−CD127hiCD28+CD25int cells with a distinct chemokine profile and a Th1-polarized pro-inflammatory phenotype. CD4+CD161+Rho-effluxing T cells proliferated vigorously in response to stimulation with anti-CD3/CD28 beads and gave rise to CD161− progeny in vitro. These cells were also capable of self-renewal and maintained their phenotypic and functional characteristics when cultured with homeostatic cytokines. Multidrug-effluxing CD4+CD161+ T cells were enriched within the viral-specific Th1 repertoire of healthy donors and patients with acute myeloid leukemia (AML) and survived exposure to daunorubicin chemotherapy in vitro. Multidrug-effluxing CD4+CD161+ T cells also resisted chemotherapy-induced cytotoxicity in vivo and underwent significant expansion in AML patients rendered lymphopenic after chemotherapy, contributing to the repopulation of anti-CMV immunity. Finally, after influenza vaccination, the proportion of influenza-specific CD4+ T cells coexpressing CD161 was significantly higher after 2 years compared with 4 weeks after immunization, suggesting CD161 is a marker for long-lived antigen-specific memory T cells. These findings suggest that CD4+CD161+ T cells with rapid efflux capacity contribute to the maintenance of viral-specific memory T cells. These data provide novel insights into mechanisms that preserve antiviral immunity in patients undergoing chemotherapy and have implications for the development of novel immunotherapeutic approaches. PMID:27821506

Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation

PubMed Central

Mazza, Giuseppe; Rombouts, Krista; Rennie Hall, Andrew; Urbani, Luca; Vinh Luong, Tu; Al-Akkad, Walid; Longato, Lisa; Brown, David; Maghsoudlou, Panagiotis; Dhillon, Amar P.; Fuller, Barry; Davidson, Brian; Moore, Kevin; Dhar, Dipok; De Coppi, Paolo; Malago, Massimo; Pinzani, Massimo

2015-01-01

Liver synthetic and metabolic function can only be optimised by the growth of cells within a supportive liver matrix. This can be achieved by the utilisation of decellularised human liver tissue. Here we demonstrate complete decellularization of whole human liver and lobes to form an extracellular matrix scaffold with a preserved architecture. Decellularized human liver cubic scaffolds were repopulated for up to 21 days using human cell lines hepatic stellate cells (LX2), hepatocellular carcinoma (Sk-Hep-1) and hepatoblastoma (HepG2), with excellent viability, motility and proliferation and remodelling of the extracellular matrix. Biocompatibility was demonstrated by either omental or subcutaneous xenotransplantation of liver scaffold cubes (5 × 5 × 5 mm) into immune competent mice resulting in absent foreign body responses. We demonstrate decellularization of human liver and repopulation with derived human liver cells. This is a key advance in bioartificial liver development. PMID:26248878

Role of autophagy in the maintenance and function of cancer stem cells.

PubMed

Vitale, Ilio; Manic, Gwenola; Dandrea, Vito; De Maria, Ruggero

2015-01-01

Recent advances in experimental technologies and cancer models have made possible to demonstrate that the tumor is a dynamic system comprising heterogeneous populations of cancer cells organized in a hierarchical fashion with cancer stem cells (CSCs) at the apex. CSCs are immature cells characterized by self-renewal property and long-term repopulation potential. CSCs have been causally linked to cancer initiation, propagation, spreading, recurrence and relapse as well as to resistance to anticancer therapy. A growing body of evidence suggests that the function and physiology of CSCs may be influenced by genetic/epigenetic factors and tumor environment. In this context, macroautophagy is a lysosomal degradative process (herein referred to as autophagy) critical for the adaptive response to stress and the preservation of cellular and tissue homeostasis in all eukaryotes that may have a crucial role of in the origin, maintenance and invasiveness of CSCs. The activation of the autophagic machinery is also considered as an adaptive response of CSCs to perturbation of tumor microenvironment, caused for instance by anticancer therapy. Nevertheless, compelling preclinical and clinical evidence on the cytoprotective role of autophagy for CSCs is still missing. Here, we summarize the results on the contribution of autophagy in CSCs and how it impacts tumorigenesis and tumor progression. We also discuss the therapeutical potential of the modulation of autophagy as a means to eradicate CSCs.

Alkylating chemotherapeutic agents cyclophosphamide and melphalan cause functional injury to human bone marrow-derived mesenchymal stem cells.

PubMed

Kemp, Kevin; Morse, Ruth; Sanders, Kelly; Hows, Jill; Donaldson, Craig

2011-07-01

The adverse effects of melphalan and cyclophosphamide on hematopoietic stem cells are well-known; however, the effects on the mesenchymal stem cells (MSCs) residing in the bone marrow are less well characterised. Examining the effects of chemotherapeutic agents on patient MSCs in vivo is difficult due to variability in patients and differences in the drug combinations used, both of which could have implications on MSC function. As drugs are not commonly used as single agents during high-dose chemotherapy (HDC) regimens, there is a lack of data comparing the short- or long-term effects these drugs have on patients post treatment. To help address these problems, the effects of the alkylating chemotherapeutic agents cyclophosphamide and melphalan on human bone marrow MSCs were evaluated in vitro. Within this study, the exposure of MSCs to the chemotherapeutic agents cyclophosphamide or melphalan had strong negative effects on MSC expansion and CD44 expression. In addition, changes were seen in the ability of MSCs to support hematopoietic cell migration and repopulation. These observations therefore highlight potential disadvantages in the use of autologous MSCs in chemotherapeutically pre-treated patients for future therapeutic strategies. Furthermore, this study suggests that if the damage caused by chemotherapeutic agents to marrow MSCs is substantial, it would be logical to use cultured allogeneic MSCs therapeutically to assist or repair the marrow microenvironment after HDC.

Non-viral FoxM1 gene delivery to hepatocytes enhances liver repopulation

PubMed Central

Xiang, D; Liu, C-C; Wang, M-J; Li, J-X; Chen, F; Yao, H; Yu, B; Lu, L; Borjigin, U; Chen, Y-X; Zhong, L; Wangensteen, K J; He, Z-Y; Wang, X; Hu, Y-P

2014-01-01

Hepatocyte transplantation as a substitute strategy of orthotopic liver transplantation is being studied for treating end-stage liver diseases. Several technical hurdles must be overcome in order to achieve the therapeutic liver repopulation, such as the problem of insufficient expansion of the transplanted hepatocytes in recipient livers. In this study, we analyzed the application of FoxM1, a cell-cycle regulator, to enhance the proliferation capacity of hepatocytes. The non-viral sleeping beauty (SB) transposon vector carrying FoxM1 gene was constructed for delivering FoxM1 into the hepatocytes. The proliferation capacities of hepatocytes with FoxM1 expression were examined both in vivo and in vitro. Results indicated that the hepatocytes with FoxM1 expression had a higher proliferation rate than wild-type (WT) hepatocytes in vitro. In comparison with WT hepatocytes, the hepatocytes with FoxM1 expression had an enhanced level of liver repopulation in the recipient livers at both sub-acute injury (fumaryl acetoacetate hydrolase (Fah)–/– mice model) and acute injury (2/3 partial hepatectomy mice model). Importantly, there was no increased risk of tumorigenicity with FoxM1 expression in recipients even after serial transplantation. In conclusion, expression of FoxM1 in hepatocytes enhanced the capacity of liver repopulation without inducing tumorigenesis. FoxM1 gene delivered by non-viral SB vector into hepatocytes may be a viable approach to promote therapeutic repopulation after hepatocyte transplantation. PMID:24853430

Chemotherapeutic tumor microparticles combining low-dose irradiation reprogram tumor-promoting macrophages through a tumor-repopulating cell-curtailing pathway

PubMed Central

Sun, Yanling; Zheng, Zu'an; Zhang, Huafeng; Yu, Yuandong; Ma, Jingwei; Tang, Ke; Xu, Pingwei; Ji, Tiantian; Liang, Xiaoyu; Chen, Degao; Jin, Xun; Zhang, Tianzhen; Long, Zhixiong; Liu, Yuying; Huang, Bo

2017-01-01

ABSTRACT Stem cell-like tumor-repopulating cells (TRCs) have a critical role in establishing a tumor immunosuppressive microenvironment. However, means to enhance antitumor immunity by disrupting TRCs are absent. Our previous studies have shown that tumor cell-derived microparticles (T-MPs) preferentially abrogate TRCs by delivering antitumor drugs into nuclei of TRCs. Here, we show that low dose irradiation (LDI) enhances the effect of cisplatin-packaging T-MPs (Cis-MPs) on TRCs, leading to inhibiting tumor growth in different tumor models. This antitumor effect is not due to the direct killing of tumor cells but is T cell-dependent and relies on macrophages for their efficacy. The underlying mechanism is involved in therapeutic reprograming macrophages from tumor-promotion to tumor-inhibition by disrupting TRCs and curtailing their vicious education on macrophages. These findings provide a novel strategy to reset macrophage polarization and confer their function more like M1 than M2 types with highly promising potential clinical applications. PMID:28680743

Population Dynamics of Excited Atoms in Dissipative Cavities

NASA Astrophysics Data System (ADS)

Zou, Hong-Mei; Liu, Yu; Fang, Mao-Fa

2016-10-01

Population dynamics of excited atoms in dissipative cavities is investigated in this work. We present a method of controlling populations of excited atoms in dissipative cavities. For the initial state | e e> A B |00> a b , the repopulation of excited atoms can be obtained by using atom-cavity couplings and non-Markovian effects after the atomic excited energy decays to zero. For the initial state | g g> A B |11> a b , the two atoms can also be populated to the excited states from the initial ground states by using atom-cavity couplings and non-Markovian effects. And the stronger the atom-cavity coupling or the non-Markovian effect is, the larger the number of repopulation of excited atoms is. Particularly, when the atom-cavity coupling or the non-Markovian effect is very strong, the number of repopulation of excited atoms can be close to one in a short time and will tend to a steady value in a long time.

Optimizing autologous cell grafts to improve stem cell gene therapy.

PubMed

Psatha, Nikoletta; Karponi, Garyfalia; Yannaki, Evangelia

2016-07-01

Over the past decade, stem cell gene therapy has achieved unprecedented curative outcomes for several genetic disorders. Despite the unequivocal success, clinical gene therapy still faces challenges. Genetically engineered hematopoietic stem cells are particularly vulnerable to attenuation of their repopulating capacity once exposed to culture conditions, ultimately leading to low engraftment levels posttransplant. This becomes of particular importance when transduction rates are low or/and competitive transplant conditions are generated by reduced-intensity conditioning in the absence of a selective advantage of the transduced over the unmodified cells. These limitations could partially be overcome by introducing megadoses of genetically modified CD34(+) cells into conditioned patients or by transplanting hematopoietic stem cells hematopoietic stem cells with high engrafting and repopulating potential. On the basis of the lessons gained from cord blood transplantation, we summarize the most promising approaches to date of increasing either the numbers of hematopoietic stem cells for transplantation or/and their engraftability, as a platform toward the optimization of engineered stem cell grafts. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

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 differentiation and proliferation of lineage-committed CD34(-) cells.

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.

Identification of a population of cells with hematopoietic stem cell properties in mouse aorta-gonad-mesonephros cultures

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

Nobuhisa, Ikuo; Ohtsu, Naoki; Okada, Seiji

The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45{sup low} c-Kit{sup +} cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45{sup low} c-Kit{sup -} cells that showed a granulocyte morphology;more » CD45{sup high} c-Kit{sup low/-} that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45{sup low} c-Kit{sup +} cells from the AGM culture had the abilities to reproduce CD45{sup low} c-Kit{sup +} cells and differentiate into CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} cells, whereas CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} did not produce CD45{sup low} c-Kit{sup +} cells. Furthermore, CD45{sup low} c-Kit{sup +} cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45{sup low} c-Kit{sup +} cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.« less

CD8+ lymphocytes are required to maintain virus suppression in SIV-infected macaques treated with short-term antiretroviral therapy

PubMed Central

Cartwright, Emily K.; Spicer, Lori; Smith, S. Abigail; Lee, David; Fast, Randy; Paganini, Sara; Lawson, Benton O.; Nega, Melon; Easley, Kirk; Schmitz, Joern E.; Bosinger, Steven E.; Paiardini, Mirko; Chahroudi, Ann; Vanderford, Thomas H.; Estes, Jacob D.; Lifson, Jeffrey D.; Derdeyn, Cynthia A.; Silvestri, Guido

2016-01-01

Infection with HIV persists despite suppressive antiretroviral therapy (ART) and treatment interruption results in rapid viral rebound. Antibody-mediated CD8+ lymphocyte depletion in simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs) shows these cells contribute to virus control in untreated animals. However, the contribution of CD8+ lymphocytes to maintaining virus suppression under ART remains unknown. We showed that in SIV-infected RMs treated with short-term ART (i.e., 8-32 weeks), depletion of CD8+ lymphocytes resulted in increased plasma viremia in all animals, and that repopulation of CD8+ T cells was associated with prompt reestablishment of virus control. Although the number of SIV-DNA-positive cells remained unchanged after CD8 depletion and reconstitution, the frequency of SIV-infected CD4+ T cells pre-depletion positively correlated with both peak and area-under-the-curve of viremia post-depletion. These results suggest a role for CD8+ T cells in controlling virus production during ART, thus providing rationale to explore immunotherapeutic approaches in ART-treated HIV-infected individuals. PMID:27653601

Biology of high single doses of IORT: RBE, 5 R's, and other biological aspects.

PubMed

Herskind, Carsten; Ma, Lin; Liu, Qi; Zhang, Bo; Schneider, Frank; Veldwijk, Marlon R; Wenz, Frederik

2017-01-19

Intraoperative radiotherapy differs from conventional, fractionated radiotherapy in several aspects that may influence its biological effect. The radiation quality influences the relative biologic effectiveness (RBE), and the role of the five R's of radiotherapy (reassortment, repair, reoxygenation, repopulation, radiosensitivity) is different. Furthermore, putative special biological effects and the small volume receiving a high single dose may be important. The present review focuses on RBE, repair, and repopulation, and gives an overview of the other factors that potentially contribute to the efficacy. The increased RBE should be taken into account for low-energy X-rays while evidence of RBE < 1 for high-energy electrons at higher doses is presented. Various evidence supports a hypothesis that saturation of the primary DNA double-strand break (DSB) repair mechanisms leads to increasing use of an error-prone backup repair system leading to genomic instability that may contribute to inactivate tumour cells at high single doses. Furthermore, the elimination of repopulation of residual tumour cells in the tumour bed implies that some patients are likely to have very few residual tumour cells which may be cured even by low doses to the tumour bed. The highly localised dose distribution of IORT has the potential to inactivate tumour cells while sparing normal tissue by minimising the volume exposed to high doses. Whether special effects of high single doses also contribute to the efficacy will require further experimental and clinical studies.

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow.

PubMed

Suryawanshi, Gajendra W; Xu, Song; Xie, Yiming; Chou, Tom; Kim, Namshin; Chen, Irvin S Y; Kim, Sanggu

2017-06-14

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes

PubMed Central

Ding, Jianqiang; Yannam, Govardhana R.; Roy-Chowdhury, Namita; Hidvegi, Tunda; Basma, Hesham; Rennard, Stephen I.; Wong, Ronald J.; Avsar, Yesim; Guha, Chandan; Perlmutter, David H.; Fox, Ira J.; Roy-Chowdhury, Jayanta

2011-01-01

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z–expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%–98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z–expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals. PMID:21505264

β-Arrestin2 mediates progression of murine primary myelofibrosis

PubMed Central

Rein, Lindsay A.M.; Wisler, James W.; Kim, Jihee; Theriot, Barbara; Huang, LiYin; Price, Trevor; Yang, Haeyoon; Chen, Wei; Sipkins, Dorothy; Fedoriw, Yuri; Walker, Julia K.L.; Premont, Richard T.; Lefkowitz, Robert J.

2017-01-01

Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. β-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, β-arrestin2 (βarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between βarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor βarr2-knockout (βarr2–/–) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted βarr2–/– cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of βarr2–/– cells. In order to assess the effect of acute loss of βarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional βarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic βarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that βarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease. PMID:29263312

Hepatic progenitor cells of biliary origin with liver repopulation capacity

PubMed Central

Boulter, Luke; Tsuchiya, Atsunori; Cole, Alicia M; Hay, Trevor; Guest, Rachel V; Wojtacha, Davina; Man, Tak Yung; Mackinnon, Alison; Ridgway, Rachel A; Kendall, Timothy; Williams, Michael J; Jamieson, Thomas; Raven, Alex; Hay, David C; Iredale, John P; Clarke, Alan R; Sansom, Owen J; Forbes, Stuart J

2015-01-01

Summary Hepatocytes and cholangiocytes self renew following liver injury. Following severe injury hepatocytes are increasingly senescent, whether Hepatic Progenitor Cells (HPCs) then contribute to liver regeneration is unclear. Here, we describe a mouse model where Mdm2 is inducibly deleted in over 98% of hepatocytes, causing apoptosis, necrosis and senescence with nearly all hepatocytes expressing p21. This results in florid HPC activation, which is necessary for survival, followed by complete, functional liver reconstitution. HPCs isolated from genetically normal mice, using cell surface markers, were highly expandable and phenotypically stable in vitro. These HPCs were transplanted into adult mouse livers where hepatocyte Mdm2 was repeatedly deleted, creating a non-competitive repopulation assay. Transplanted HPCs contributed significantly to restoration of liver parenchyma, regenerating hepatocytes and biliary epithelia, highlighting their in vivo lineage potency. HPCs are therefore a potential future alternative to hepatocyte or liver transplantation for liver disease. PMID:26192438

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.

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

In vitro investigation of head and neck cancer stem cell proportions and their changes following X-ray irradiation as a function of HPV status.

PubMed

Reid, Paul; Wilson, Puthenparampil; Li, Yanrui; Marcu, Loredana G; Staudacher, Alexander H; Brown, Michael P; Bezak, Eva

2017-01-01

Some head and neck squamous cell carcinomas (HNSCC) have a distinct aetiology, which depends on the presence of oncogenic human papilloma virus (HPV). Also, HNSCC contains cancer stem cells (CSCs) that have greater radioresistance and capacity to change replication dynamics in response to irradiation compared to non-clonogenic cells. Since there is limited data on CSCs in HNSCC as a function of HPV status, better understanding of their radiobiology may enable improved treatment outcome. Baseline and post-irradiation changes in CSC proportions were investigated by flow cytometry in a HPV-negative (UM-SCC-1) and a HPV-positive (UM-SCC-47) HNSCC cell line, using fluorescent staining with CD44/ALDH markers. CSC proportions in both irradiated and unirradiated cultures were compared for the two cell lines at various times post-irradiation. To assess repopulation of CSCs, untreated cultures were depleted of CD44+/ALDH+ cells and re-cultured for 3 weeks before flow cytometry analysis. CSC proportions in untreated cell lines were 0.57% (UM-SCC-1) and 2.87% (UM-SCC-47). Untreated cell lines depleted of CD44+/ALDH+ repopulated this phenotype to a mean of 0.15% (UM-SCC-1) and 6.76% (UM-SCC-47). All UM-SCC-47 generations showed elevated CSC proportions after irradiation, with the most significant increase at 2 days post-irradiation. The highest elevation in UM-SCC-1 CSCs was observed at 1 day post-irradiation in the 2nd generation and at 3 days after irradiation in the 3rd generation. When measured after 10 days, only the 3rd generation of UM-SCC-1 showed elevated CSCs. CSC proportions in both cell lines were elevated after exposure and varied with time post irradiation. UM-SCC-47 displayed significant plasticity in repopulating the CSC phenotype in depleted cultures, which was not seen in UM-SCC-1.

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells.

PubMed

Codispoti, Bruna; Rinaldo, Nicola; Chiarella, Emanuela; Lupia, Michela; Spoleti, Cristina Barbara; Marafioti, Maria Grazia; Aloisio, Annamaria; Scicchitano, Stefania; Giordano, Marco; Nappo, Giovanna; Lucchino, Valeria; Moore, Malcolm A S; Zhou, Pengbo; Mesuraca, Maria; Bond, Heather Mandy; Morrone, Giovanni

2017-07-04

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery.Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes.

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells

PubMed Central

Codispoti, Bruna; Rinaldo, Nicola; Chiarella, Emanuela; Lupia, Michela; Spoleti, Cristina Barbara; Marafioti, Maria Grazia; Aloisio, Annamaria; Scicchitano, Stefania; Giordano, Marco; Nappo, Giovanna; Lucchino, Valeria; Moore, Malcolm A.S.; Zhou, Pengbo; Mesuraca, Maria

2017-01-01

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery. Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes. PMID:28187462

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.

Triggered by asphyxia neurogenesis seems not to be an endogenous repair mechanism, gliogenesis more like it.

PubMed

Keilhoff, G; John, R; Langnaese, K; Schweizer, H; Ebmeyer, U

2010-12-15

We analyzed the long-term consequences of asphyxial cardiac arrest for hippocampal cell proliferation in rats to evaluate if the ischaemia-induced degenerated CA1 region may be repopulated by endogenous (stem) cells. Studies were performed in an asphyxial cardiac arrest model with 5 minutes of asphyxiation and three different survival times: 7, 21, and 90 days. Sham-operated non-asphyxiated rats served as control. Cell proliferation was studied by labeling dividing cells with 5-bromo-2'-deoxy-uridine (BrdU). The neurodegenerative/regenerative pattern at single cell levels was monitored by immunohistochemistry. Alterations of gene expression were analyzed by real-time quantitative RT-PCR. Analysis of BrdU-incorporation demonstrated an increase at 7, 21 as well as 90 days after global ischaemia in the hippocampal CA1 pyramidal cell layer. Similar results were found in the dentate gyrus. Differentiation of BrdU-positive cells, investigated by cell phenotype-specific double fluorescent labeling, showed increased neurogenesis only in the dentate gyrus of animals surviving the cardiac arrest for 7 days. The majority of newcomers, especially in the damaged CA1 region, consisted of glial cells. Moreover, asphyxia seemed to be able to induce the migration of microglia and astroglia from adjacent areas into the damaged area and/or the activation of resident cells. In addition, we show microglia proliferation/activation even 90 days after cardiac arrest. This morphological finding was confirmed by PCR analysis. The results indicate that asphyxia triggers cell proliferation in general and gliogenesis in particular - a possible pro-reparative event. Furthermore, from the finding of microglia proliferation up to 90 days after insult we conclude that delayed cell death processes take place which should be considered for further therapy strategies. Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

MiR-17 Partly Promotes Hematopoietic Cell Expansion through Augmenting HIF-1α in Osteoblasts

PubMed Central

Yang, Yuxia; Ma, Wei; Wu, Dan; Huang, Yu; Li, Hongge; Zou, Junhua; Zhang, Yanju; Feng, Meifu; Luo, Jianyuan

2013-01-01

Background Hematopoietic stem cell (HSC) regulation is highly dependent on interactions with the marrow microenvironment, of which osteogenic cells play a crucial role. While evidence is accumulating for an important role of intrinsic miR-17 in regulating HSCs and HPCs, whether miR-17 signaling pathways are also necessary in the cell-extrinsic control of hematopoiesis hereto remains poorly understood. Methodology/Principal Findings Using the immortalized clone with the characteristics of osteoblasts, FBMOB-hTERT, in vitro expansion, long-term culture initiating cell (LTC-IC) and non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice repopulating cell (SRC) assay revealed that the ectopic expression of miR-17 partly promoted the ability of FBMOB-hTERT to support human cord blood (CB) CD34+ cell expansion and maintain their multipotency. It also seemed that osteoblastic miR-17 was prone to cause a specific expansion of the erythroid lineage. Conversely, deficient expression of miR-17 partly inhibited the hematopoietic supporting ability of FBMOB-hTERT. We further identified that HIF-1α is responsible for, at least in part, the promoted hematopoietic supporting ability of FBMOB-hTERT caused by miR-17. HIF-1α expression is markedly enhanced in miR-17 overexpressed FBMOB-hTERT upon interaction with CB CD34+ cells compared to other niche associated factors. More interestingly, the specific erythroid lineage expansion of CB CD34+ cells caused by osteoblastic miR-17 was abrogated by HIF-1α knock down. Conclusion/Significance Our data demonstrated that CB CD34+ cell expansion can be partly promoted by osteoblastic miR-17, and in particular, ectopic miR-17 can cause a specific expansion of the erythroid lineage through augmenting HIF-1α in osteoblasts. PMID:23936170

Application of biological effective dose (BED) to estimate the duration of symptomatic relief and repopulation dose equivalent in palliative radiotherapy and chemotherapy.

PubMed

Jones, Bleddyn; Cominos, Matilda; Dale, Roger G

2003-03-01

To investigate the potential for mathematic modeling in the assessment of symptom relief in palliative radiotherapy and cytotoxic chemotherapy. The linear quadratic model of radiation effect with the overall treatment time and the daily dose equivalent of repopulation is modified to include the regrowth time after completion of therapy. The predicted times to restore the original tumor volumes after treatment are dependent on the biological effective dose (BED) delivered and the repopulation parameter (K); it is also possible to estimate K values from analysis of palliative treatment response durations. Hypofractionated radiotherapy given at a low total dose may produce long symptom relief in slow-growing tumors because of their low alpha/beta ratios (which confer high fraction sensitivity) and their slow regrowth rates. Cancers that have high alpha/beta ratios (which confer low fraction sensitivity), and that are expected to repopulate rapidly during therapy, are predicted to have short durations of symptom control. The BED concept can be used to estimate the equivalent dose of radiotherapy that will achieve the same duration of symptom relief as palliative chemotherapy. Relatively simple radiobiologic modeling can be used to guide decision-making regarding the choice of the most appropriate palliative schedules and has important implications in the design of radiotherapy or chemotherapy clinical trials. The methods described provide a rationalization for treatment selection in a wide variety of tumors.

Regenerative periodontal therapy in mucogingival surgery for root coverage.

PubMed

Abitbol, T; Santi, E; Urbani, G

1997-02-01

This article illustrates the potential benefits of regenerative periodontal therapy in mucogingival surgery and esthetic dental treatment. Cases are described in which the treatment of soft-tissue recessions and root exposures are treated with surgical procedures where both clinical soft-tissue augmentation and the regeneration of periodontal attachment are obtained. Cases are also presented to illustrate the clinical application of guided tissue regeneration. Resorbable and nonresorbable barriers are placed over the root surface and bone and covered by the overlying flap, which allows the selective repopulation of the lesion by progenitor cells and the inhibition of a long junctional epithelium. Emphasis is placed on regenerative procedures in soft-tissue augmentation, particularly with respect to rationales, techniques, and indications.

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

Kleiner, J.B.; Amiel, D.; Harwood, F.L.

A rabbit model for anterior cruciate ligament (ACL) reconstruction using autogenous patellar tendon was utilized to study the early events of autograft cellular dynamics. Biochemical, autoradiographic, histological, and vascular injection techniques demonstrated that the native autograft cell population rapidly necroses. This repopulation occurs without a vascular contribution; cells entering the autograft are reliant upon synovial fluid nutrition.

Quiescent Oct4+ Neural Stem Cells (NSCs) Repopulate Ablated Glial Fibrillary Acidic Protein+ NSCs in the Adult Mouse Brain.

PubMed

Reeve, Rachel L; Yammine, Samantha Z; Morshead, Cindi M; van der Kooy, Derek

2017-09-01

Adult primitive neural stem cells (pNSCs) are a rare population of glial fibrillary acidic protein (GFAP) - Oct4 + cells in the mouse forebrain subependymal zone bordering the lateral ventricles that give rise to clonal neurospheres in leukemia inhibitory factor in vitro. pNSC neurospheres can be passaged to self-renew or give rise to GFAP + NSCs that form neurospheres in epidermal growth factor and fibroblast growth factor 2, which we collectively refer to as definitive NSCs (dNSCs). Label retention experiments using doxycycline-inducible histone-2B (H2B)-green fluorescent protein (GFP) mice and several chase periods of up to 1 year quantified the adult pNSC cell cycle time as 3-5 months. We hypothesized that while pNSCs are not very proliferative at baseline, they may exist as a reserve pool of NSCs in case of injury. To test this function of pNSCs, we obtained conditional Oct4 knockout mice, Oct4 fl/fl ;Sox1 Cre (Oct4 CKO ), which do not yield adult pNSC-derived neurospheres. When we ablated the progeny of pNSCs, namely all GFAP + dNSCs, in these Oct4 CKO mice, we found that dNSCs did not recover as they do in wild-type mice, suggesting that pNSCs are necessary for dNSC repopulation. Returning to the H2B-GFP mice, we observed that the cytosine β-d-arabinofuranoside ablation of proliferating cells including dNSCs-induced quiescent pNSCs to proliferate and significantly dilute their H2B-GFP label. In conclusion, we demonstrate that pNSCs are the most quiescent stem cells in the adult brain reported to date and that their lineage position upstream of GFAP + dNSCs allows them to repopulate a depleted neural lineage. Stem Cells 2017;35:2071-2082. © 2017 AlphaMed Press.

M1 and M2 macrophages differentially regulate hematopoietic stem cell self-renewal and ex vivo expansion

PubMed Central

Luo, Yi; Shao, Lijian; Chang, Jianhui; Feng, Wei; Liu, Y. Lucy; Cottler-Fox, Michele H.; Emanuel, Peter D.; Hauer-Jensen, Martin; Bernstein, Irwin D.; Liu, Lingbo; Chen, Xing; Zhou, Jianfeng; Murray, Peter J.

2018-01-01

Uncovering the cellular and molecular mechanisms by which hematopoietic stem cell (HSC) self-renewal is regulated can lead to the development of new strategies for promoting ex vivo HSC expansion. Here, we report the discovery that alternative (M2)-polarized macrophages (M2-MΦs) promote, but classical (M1)-polarized macrophages (M1-MΦs) inhibit, the self-renewal and expansion of HSCs from mouse bone marrow (BM) in vitro. The opposite effects of M1-MΦs and M2-MΦs on mouse BM HSCs were attributed to their differential expression of nitric oxide synthase 2 (NOS2) and arginase 1 (Arg1), because genetic knockout of Nos2 and Arg1 or inhibition of these enzymes with a specific inhibitor abrogated the differential effects of M1-MΦs and M2-MΦs. The opposite effects of M1-MΦs and M2-MΦs on HSCs from human umbilical cord blood (hUCB) were also observed when hUCB CD34+ cells were cocultured with M1-MΦs and M2-MΦs generated from hUCB CD34− cells. Importantly, coculture of hUCB CD34+ cells with human M2-MΦs for 8 days resulted in 28.7- and 6.6-fold increases in the number of CD34+ cells and long-term SCID mice–repopulating cells, respectively, compared with uncultured hUCB CD34+ cells. Our findings could lead to the development of new strategies to promote ex vivo hUCB HSC expansion to improve the clinical utility and outcome of hUCB HSC transplantation and may provide new insights into the pathogenesis of hematological dysfunctions associated with infection and inflammation that can lead to differential macrophage polarization. PMID:29666049

Loss of Angiotensin-Converting Enzyme 2 Exacerbates Diabetic Retinopathy by Promoting Bone Marrow Dysfunction.

PubMed

Duan, Yaqian; Beli, Eleni; Li Calzi, Sergio; Quigley, Judith L; Miller, Rehae C; Moldovan, Leni; Feng, Dongni; Salazar, Tatiana E; Hazra, Sugata; Al-Sabah, Jude; Chalam, Kakarla V; Le Phuong Trinh, Thao; Meroueh, Marya; Markel, Troy A; Murray, Matthew C; Vyas, Ruchi J; Boulton, Michael E; Parsons-Wingerter, Patricia; Oudit, Gavin Y; Obukhov, Alexander G; Grant, Maria B

2018-05-15

Angiotensin-converting enzyme 2 (ACE2) is the primary enzyme of the vasoprotective axis of the renin angiotensin system (RAS). We tested the hypothesis that loss of ACE2 would exacerbate diabetic retinopathy by promoting bone marrow dysfunction. ACE2 -/y were crossed with Akita mice, a model of type 1 diabetes. When comparing the bone marrow of the ACE2 -/y -Akita mice to that of Akita mice, we observed a reduction of both short-term and long-term repopulating hematopoietic stem cells, a shift of hematopoiesis towards myelopoiesis, and an impairment of lineage - c-kit + hematopoietic stem/progenitor cell (HS/PC) migration and proliferation. Migratory and proliferative dysfunction of these cells was corrected by exposure to angiotensin-1-7 (Ang-1-7), the protective peptide generated by ACE2. Over the duration of diabetes examined, ACE2 deficiency led to progressive reduction in electrical responses assessed by electroretinography and to increases in neural infarcts observed by fundus photography. Compared to Akita mice, ACE2 -/y -Akita at 9-months of diabetes showed an increased number of acellular capillaries indicative of more severe diabetic retinopathy. In diabetic and control human subjects, CD34 + cells, a key bone marrow HS/PC population, were assessed for changes in mRNA levels for MAS, the receptor for Ang-1-7. Levels were highest in CD34 + cells from diabetics without retinopathy. Higher serum Ang-1-7 levels predicted protection from development of retinopathy in diabetics. Treatment with Ang-1-7 or alamandine restored the impaired migration function of CD34 + cells from subjects with retinopathy. These data support that activation of the protective RAS within HS/PCs may represent a therapeutic strategy for prevention of diabetic retinopathy. This article is protected by copyright. All rights reserved. © 2018 AlphaMed Press.

Acute effects of alemtuzumab infusion in patients with active relapsing-remitting MS

PubMed Central

Thomas, Katja; Eisele, Judith; Rodriguez-Leal, Francisco Alejandro; Hainke, Undine

2016-01-01

Objective: Alemtuzumab exerts its clinical efficacy by its specific pattern of depletion and repopulation of different immune cells. Beyond long-term immunologic and clinical data, little is known about acute changes in immunologic and routine laboratory parameters and their clinical relevance during the initial alemtuzumab infusion. Methods: Fifteen patients with highly active MS were recruited. In addition to parameters including heart rate, blood pressure, body temperature, and monitoring of adverse events, complete blood cell count, liver enzymes, kidney function, acute-phase proteins, serum cytokine profile, complement activation, peripheral immune cell distribution, and their potential of cytokine release were investigated prior to and after methylprednisolone and after alemtuzumab on each day of alemtuzumab infusion. Results: After the first alemtuzumab infusion, both the total leukocyte and granulocyte counts markedly increased, whereas lymphocyte counts dramatically decreased. In addition to lymphocyte depletion, cell subtypes important for innate immunity also decreased within the first week after alemtuzumab infusion. Although patients reported feeling well, C-reactive protein and procalcitonin peaked at serum levels consistent with septic conditions. Increases in liver enzymes were detected, although kidney function remained stable. Proinflammatory serum cytokine levels clearly rose after the first alemtuzumab infusion. Alemtuzumab led to impaired cytokine release ex vivo in nondepleted cells. Normal clinical parameters and mild adverse events were presented. Conclusions: Dramatic immunologic effects were observed. Standardized infusion procedure and pretreatment management attenuated infusion-related reactions. Alemtuzumab-mediated effects led to artificially altered parameters in standard blood testing. We recommend clinical decision-making based on primarily clinical symptoms within the first alemtuzumab treatment week. PMID:27213173

HTR8/SVneo cells display trophoblast progenitor cell-like characteristics indicative of self-renewal, repopulation activity, and expression of "stemness-" associated transcription factors.

PubMed

Weber, Maja; Knoefler, Ilka; Schleussner, Ekkehard; Markert, Udo R; Fitzgerald, Justine S

2013-01-01

JEG3 is a choriocarcinoma--and HTR8/SVneo a transformed extravillous trophoblast--cell line often used to model the physiologically invasive extravillous trophoblast. Past studies suggest that these cell lines possess some stem or progenitor cell characteristics. Aim was to study whether these cells fulfill minimum criteria used to identify stem-like (progenitor) cells. In summary, we found that the expression profile of HTR8/SVneo (CDX2+, NOTCH1+, SOX2+, NANOG+, and OCT-) is distinct from JEG3 (CDX2+ and NOTCH1+) as seen only in human-serum blocked immunocytochemistry. This correlates with HTR8/SVneo's self-renewal capacities, as made visible via spheroid formation and multi-passagability in hanging drops protocols paralleling those used to maintain embryoid bodies. JEG3 displayed only low propensity to form and reform spheroids. HTR8/SVneo spheroids migrated to cover and seemingly repopulate human chorionic villi during confrontation cultures with placental explants in hanging drops. We conclude that HTR8/SVneo spheroid cells possess progenitor cell traits that are probably attained through corruption of "stemness-" associated transcription factor networks. Furthermore, trophoblastic cells are highly prone to unspecific binding, which is resistant to conventional blocking methods, but which can be alleviated through blockage with human serum.

Study of stem cell homing & self-renewal marker gene profile of ex vivo expanded human CD34+ cells manipulated with a mixture of cytokines & stromal cell-derived factor 1

PubMed Central

Kode, Jyoti; Khattry, Navin; Bakshi, Ashish; Amrutkar, Vasanti; Bagal, Bhausaheb; Karandikar, Rohini; Rane, Pallavi; Fujii, Nobutaka; Chiplunkar, Shubhada

2017-01-01

Background & objectives: Next generation transplantation medicine aims to develop stimulating cocktail for increased ex vivo expansion of primitive hematopoietic stem and progenitor cells (HSPC). The present study was done to evaluate the cocktail GF (Thrombopoietin + Stem Cell factor + Flt3-ligand) and homing-defining molecule Stromal cell-derived factor 1 (SDF1) for HSPC ex vivo expansion. Methods: Peripheral blood stem cell (n=74) harvests were analysed for CD34hi CD45lo HSPC. Immunomagnetically enriched HSPC were cultured for eight days and assessed for increase in HSPC, colony forming potential in vitro and in vivo engrafting potential by analyzing human CD45+ cells. Expression profile of genes for homing and stemness were studied using microarray analysis. Expression of adhesion/homing markers were validated by flow cytometry/ confocal microscopy. Results: CD34hi CD45lo HSPC expansion cultures with GF+SDF1 demonstrated increased nucleated cells (n=28, P< 0.001), absolute CD34+ cells (n=8, P=0.021) and increased colony forming units (cfu) compared to unstimulated and GF-stimulated HSPC. NOD-SCID mice transplanted with GF+SDF1-HSPC exhibited successful homing/engraftment (n=24, P< 0.001). Microarray analysis of expanded HSPC demonstrated increased telomerase activity and many homing-associated genes (35/49) and transcription factors for stemness/self-renewal (49/56) were significantly upregulated in GF+SDF1 stimulated HSPC when compared to GF-stimulated HSPC. Expression of CD44, CXCR4, CD26, CD14, CD45 and soluble IL-6 in expanded cultures were validated by flow cytometry and confocal microscopy. Interpretation & conclusions: Cocktail of cytokines and SDF1 showed good potential to successfully expand HSPC which exhibited enhanced ability to generate multilineage cells in short-term and long-term repopulation assay. This cocktail-mediated stem cell expansion has potential to obviate the need for longer and large volume apheresis procedure making it convenient for donors. PMID:29168461

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.

Immature doublecortin-positive hippocampal neurons are important for learning but not for remembering.

PubMed

Vukovic, Jana; Borlikova, Gilyana G; Ruitenberg, Marc J; Robinson, Gregory J; Sullivan, Robert K P; Walker, Tara L; Bartlett, Perry F

2013-04-10

It is now widely accepted that hippocampal neurogenesis underpins critical cognitive functions, such as learning and memory. To assess the behavioral importance of adult-born neurons, we developed a novel knock-in mouse model that allowed us to specifically and reversibly ablate hippocampal neurons at an immature stage. In these mice, the diphtheria toxin receptor (DTR) is expressed under control of the doublecortin (DCX) promoter, which allows for specific ablation of immature DCX-expressing neurons after administration of diphtheria toxin while leaving the neural precursor pool intact. Using a spatially challenging behavioral test (a modified version of the active place avoidance test), we present direct evidence that immature DCX-expressing neurons are required for successful acquisition of spatial learning, as well as reversal learning, but are not necessary for the retrieval of stored long-term memories. Importantly, the observed learning deficits were rescued as newly generated immature neurons repopulated the granule cell layer upon termination of the toxin treatment. Repeat (or cyclic) depletion of immature neurons reinstated behavioral deficits if the mice were challenged with a novel task. Together, these findings highlight the potential of stimulating neurogenesis as a means to enhance learning.

Repopulation of interacting tumor cells during fractionated radiotherapy: stochastic modeling of the tumor control probability.

PubMed

Fakir, Hatim; Hlatky, Lynn; Li, Huamin; Sachs, Rainer

2013-12-01

Optimal treatment planning for fractionated external beam radiation therapy requires inputs from radiobiology based on recent thinking about the "five Rs" (repopulation, radiosensitivity, reoxygenation, redistribution, and repair). The need is especially acute for the newer, often individualized, protocols made feasible by progress in image guided radiation therapy and dose conformity. Current stochastic tumor control probability (TCP) models incorporating tumor repopulation effects consider "stem-like cancer cells" (SLCC) to be independent, but the authors here propose that SLCC-SLCC interactions may be significant. The authors present a new stochastic TCP model for repopulating SLCC interacting within microenvironmental niches. Our approach is meant mainly for comparing similar protocols. It aims at practical generalizations of previous mathematical models. The authors consider protocols with complete sublethal damage repair between fractions. The authors use customized open-source software and recent mathematical approaches from stochastic process theory for calculating the time-dependent SLCC number and thereby estimating SLCC eradication probabilities. As specific numerical examples, the authors consider predicted TCP results for a 2 Gy per fraction, 60 Gy protocol compared to 64 Gy protocols involving early or late boosts in a limited volume to some fractions. In sample calculations with linear quadratic parameters α = 0.3 per Gy, α∕β = 10 Gy, boosting is predicted to raise TCP from a dismal 14.5% observed in some older protocols for advanced NSCLC to above 70%. This prediction is robust as regards: (a) the assumed values of parameters other than α and (b) the choice of models for intraniche SLCC-SLCC interactions. However, α = 0.03 per Gy leads to a prediction of almost no improvement when boosting. The predicted efficacy of moderate boosts depends sensitively on α. Presumably, the larger values of α are the ones appropriate for individualized treatment protocols, with the smaller values relevant only to protocols for a heterogeneous patient population. On that assumption, boosting is predicted to be highly effective. Front boosting, apart from practical advantages and a possible advantage as regards iatrogenic second cancers, also probably gives a slightly higher TCP than back boosting. If the total number of SLCC at the start of treatment can be measured even roughly, it will provide a highly sensitive way of discriminating between various models and parameter choices. Updated mathematical methods for calculating repopulation allow credible generalizations of earlier results.

Effective Hepatocyte Transplantation Using Rat Hepatocytes with Low Asialoglycoprotein Receptor Expression

PubMed Central

Ise, Hirohiko; Nikaido, Toshio; Negishi, Naoki; Sugihara, Nobuhiro; Suzuki, Fumitaka; Akaike, Toshihiro; Ikeda, Uichi

2004-01-01

Development of a reliable method of isolating highly proliferative potential hepatocytes provides information crucial to progress in the field of hepatocyte transplantation. The aim of this study was to develop reliable hepatocyte transplantation using highly proliferative, eg, progenitor-like hepatocytes, based on asialoglycoprotein receptor (ASGPR) expression levels for hepatocyte transplantation. We have previously reported that mouse hepatocytes with low ASGPR expression levels have highly proliferative potential and can be used as progenitor-like hepatocytes. We therefore fractionated F344 male rat hepatocytes expressing low and high levels of ASGPR and determined the liver repopulation capacity of hepatocytes according to low and high ASGPR expression in the liver. Next, 2 × 105 cells of each type were transplanted into female liver regenerative model dipeptidyl peptidase-deficient rats, and we estimated the rate of liver repopulation by the transplanted hepatocytes in the host liver, as determined by recognition of the Sry gene on the Y-chromosome. At 60 days after hepatocyte transplantation, the transplanted hepatocytes occupied ∼76% of the total hepatocyte mass in the case of the transplantation of hepatocytes with low ASGPR expression, but accounted for ∼12% and 17% of the mass in the case of the transplantation of hepatocytes with high ASGPR expression and unfractionated hepatocytes, respectively. In conclusion, these findings suggest that hepatocytes with low ASGPR expression can result in normal liver function and a high repopulation capacity in vivo. These results provide insight into development of a strategy for effective liver repopulation using transplanted hepatocytes. PMID:15277224

Effective hepatocyte transplantation using rat hepatocytes with low asialoglycoprotein receptor expression.

PubMed

Ise, Hirohiko; Nikaido, Toshio; Negishi, Naoki; Sugihara, Nobuhiro; Suzuki, Fumitaka; Akaike, Toshihiro; Ikeda, Uichi

2004-08-01

Development of a reliable method of isolating highly proliferative potential hepatocytes provides information crucial to progress in the field of hepatocyte transplantation. The aim of this study was to develop reliable hepatocyte transplantation using highly proliferative, eg, progenitor-like hepatocytes, based on asialoglycoprotein receptor (ASGPR) expression levels for hepatocyte transplantation. We have previously reported that mouse hepatocytes with low ASGPR expression levels have highly proliferative potential and can be used as progenitor-like hepatocytes. We therefore fractionated F344 male rat hepatocytes expressing low and high levels of ASGPR and determined the liver repopulation capacity of hepatocytes according to low and high ASGPR expression in the liver. Next, 2 x 10(5) cells of each type were transplanted into female liver regenerative model dipeptidyl peptidase-deficient rats, and we estimated the rate of liver repopulation by the transplanted hepatocytes in the host liver, as determined by recognition of the Sry gene on the Y-chromosome. At 60 days after hepatocyte transplantation, the transplanted hepatocytes occupied approximately 76% of the total hepatocyte mass in the case of the transplantation of hepatocytes with low ASGPR expression, but accounted for approximately 12% and 17% of the mass in the case of the transplantation of hepatocytes with high ASGPR expression and unfractionated hepatocytes, respectively. In conclusion, these findings suggest that hepatocytes with low ASGPR expression can result in normal liver function and a high repopulation capacity in vivo. These results provide insight into development of a strategy for effective liver repopulation using transplanted hepatocytes.

Lentiviral-Mediated Gene Therapy in Fanconi Anemia-A Mice Reveals Long-Term Engraftment and Continuous Turnover of Corrected HSCs.

PubMed

Molina-Estevez, F Javier; Nowrouzi, Ali; Lozano, M Luz; Galy, Anne; Charrier, Sabine; von Kalle, Christof; Guenechea, Guillermo; Bueren, Juan A; Schmidt, Manfred

2015-01-01

Fanconi anemia is a DNA repair-deficiency syndrome mainly characterized by cancer predisposition and bone marrow failure. Trying to restore the hematopoietic function in these patients, lentiviral vector-mediated gene therapy trials have recently been proposed. However, because no insertional oncogenesis studies have been conducted so far in DNA repair-deficiency syndromes such as Fanconi anemia, we have carried out a genome-wide screening of lentiviral insertion sites after the gene correction of Fanca(-/-) hematopoietic stem cells (HSCs), using LAM-PCR and 454-pyrosequencing. Our studies first demonstrated that transduction of Fanca(-/-) HSCs with a lentiviral vector designed for clinical application efficiently corrects the phenotype of Fanconi anemia repopulating cells without any sign of toxicity. The identification of more than 6,500 insertion sites in primary and secondary recipients showed a polyclonal pattern of reconstitution, as well as a continuous turnover of corrected Fanca(-/-) HSC clones, without evidences of selection towards specific common integration sites. Taken together our data show, for the first time in a DNA repair-deficiency syndrome, that lentiviral vector-mediated gene therapy efficiently corrects the phenotype of affected HSCs and promotes a healthy pattern of clonal turnover in vivo. These studies will have a particular impact in the development of new gene therapy trials in patients affected by DNA repair syndromes, particularly in Fanconi anemia.

Etanercept blocks inflammatory responses orchestrated by TNF-α to promote transplanted cell engraftment and proliferation in rat liver

PubMed Central

Viswanathan, Preeti; Kapoor, Sorabh; Kumaran, Vinay; Joseph, Brigid; Gupta, Sanjeev

2014-01-01

Engraftment of transplanted cells is critical for liver-directed cell therapy but most transplanted cells are rapidly cleared from liver sinusoids by proinflammatory cytokines/chemokines/receptors after activation of neutrophils or Kupffer cells. To define whether TNF-α served roles in cell-transplantation-induced hepatic inflammation, we used TNF-α antagonist, etanercept, for studies in syngeneic rat hepatocyte transplantation systems. After cell transplantation, multiple cytokines/chemokines/receptors were overexpressed, whereas etanercept prior to cell transplantation essentially normalized these responses. Moreover, ETN downregulated cell transplantation-induced intrahepatic release of secretory cytokines, such as high mobility group box 1. These effects of etanercept decreased cell transplantation-induced activation of neutrophils but not of Kupffer cells. Transplanted cell engraftment improved by several-fold in etanercept-treated animals. These gains in cell engraftment were repeatedly realized after pretreatment of animals with etanercept before multiple cell transplantation sessions. Transplanted cell numbers did not change over time indicating absence of cell proliferation after etanercept alone. By contrast, in animals preconditioned with retrorsine and partial hepatectomy, cell transplantation after etanercept pretreatment significantly accelerated liver repopulation compared with control rats. We concluded that TNF-α played a major role in orchestrating cell transplantation-induced inflammation through regulation of multiple cytokines/chemokines/receptor expression. As TNF-α antagonism by etanercept decreased transplanted cell clearance, improved cell engraftment and accelerated liver repopulation, this pharmacological approach to control hepatic inflammation will help optimize clinical strategies for liver cell therapy. PMID:24844924

Radiation-Induced Leukemia at Doses Relevant to Radiation Therapy: Modeling Mechanisms and Estimating Risks

NASA Technical Reports Server (NTRS)

Shuryak, Igor; Sachs, Rainer K.; Hlatky, Lynn; Mark P. Little; Hahnfeldt, Philip; Brenner, David J.

2006-01-01

Because many cancer patients are diagnosed earlier and live longer than in the past, second cancers induced by radiation therapy have become a clinically significant issue. An earlier biologically based model that was designed to estimate risks of high-dose radiation induced solid cancers included initiation of stem cells to a premalignant state, inactivation of stem cells at high radiation doses, and proliferation of stem cells during cellular repopulation after inactivation. This earlier model predicted the risks of solid tumors induced by radiation therapy but overestimated the corresponding leukemia risks. Methods: To extend the model to radiation-induced leukemias, we analyzed in addition to cellular initiation, inactivation, and proliferation a repopulation mechanism specific to the hematopoietic system: long-range migration through the blood stream of hematopoietic stem cells (HSCs) from distant locations. Parameters for the model were derived from HSC biologic data in the literature and from leukemia risks among atomic bomb survivors v^ ho were subjected to much lower radiation doses. Results: Proliferating HSCs that migrate from sites distant from the high-dose region include few preleukemic HSCs, thus decreasing the high-dose leukemia risk. The extended model for leukemia provides risk estimates that are consistent with epidemiologic data for leukemia risk associated with radiation therapy over a wide dose range. For example, when applied to an earlier case-control study of 110000 women undergoing radiotherapy for uterine cancer, the model predicted an excess relative risk (ERR) of 1.9 for leukemia among women who received a large inhomogeneous fractionated external beam dose to the bone marrow (mean = 14.9 Gy), consistent with the measured ERR (2.0, 95% confidence interval [CI] = 0.2 to 6.4; from 3.6 cases expected and 11 cases observed). As a corresponding example for brachytherapy, the predicted ERR of 0.80 among women who received an inhomogeneous low-dose-rate dose to the bone marrow (mean = 2.5 Gy) was consistent with the measured ERR (0.62, 95% Cl =-0.2 to 1.9). Conclusions: An extended, biologically based model for leukemia that includes HSC initiation, inactivation, proliferation, and, uniquely for leukemia, long-range HSC migration predicts, %Kith reasonable accuracy, risks for radiationinduced leukemia associated with exposure to therapeutic doses of radiation.

The Diverse Geographies of Rural Gentrification in Scotland

ERIC Educational Resources Information Center

Stockdale, Aileen

2010-01-01

Gentrification has for too long been investigated as an urban phenomenon. Only relatively recently has it been viewed as an avenue for fruitful rural research. This paper focuses on the repopulation of rural Scotland. Using survey and interview data it examines evidence of gentrification among in-migration flows and seeks to explore both the…

Deep drilling into the Chesapeake Bay impact structure

USGS Publications Warehouse

Gohn, G.S.; Koeberl, C.; Miller, K.G.; Reimold, W.U.; Browning, J.V.; Cockell, C.S.; Horton, J. Wright; Kenkmann, T.; Kulpecz, A.A.; Powars, D.S.; Sanford, W.E.; Voytek, M.A.

2008-01-01

Samples from a 1.76-kilometer-deep corehole drilled near the center of the late Eocene Chesapeake Bay impact structure (Virginia, USA) reveal its geologic, hydrologic, and biologic history. We conducted stratigraphic and petrologic analyses of the cores to elucidate the timing and results of impact-melt creation and distribution, transient-cavity collapse, and ocean-water resurge. Comparison of post-impact sedimentary sequences inside and outside the structure indicates that compaction of the crater fill influenced long-term sedimentation patterns in the mid-Atlantic region. Salty connate water of the target remains in the crater fill today, where it poses a potential threat to the regional groundwater resource. Observed depth variations in microbial abundance indicate a complex history of impact-related thermal sterilization and habitat modification, and subsequent post-impact repopulation.

Deep drilling into the Chesapeake Bay impact structure.

PubMed

Gohn, G S; Koeberl, C; Miller, K G; Reimold, W U; Browning, J V; Cockell, C S; Horton, J W; Kenkmann, T; Kulpecz, A A; Powars, D S; Sanford, W E; Voytek, M A

2008-06-27

Samples from a 1.76-kilometer-deep corehole drilled near the center of the late Eocene Chesapeake Bay impact structure (Virginia, USA) reveal its geologic, hydrologic, and biologic history. We conducted stratigraphic and petrologic analyses of the cores to elucidate the timing and results of impact-melt creation and distribution, transient-cavity collapse, and ocean-water resurge. Comparison of post-impact sedimentary sequences inside and outside the structure indicates that compaction of the crater fill influenced long-term sedimentation patterns in the mid-Atlantic region. Salty connate water of the target remains in the crater fill today, where it poses a potential threat to the regional groundwater resource. Observed depth variations in microbial abundance indicate a complex history of impact-related thermal sterilization and habitat modification, and subsequent post-impact repopulation.

Emv30null NOD-scid mice. An improved host for adoptive transfer of autoimmune diabetes and growth of human lymphohematopoietic cells.

PubMed

Serreze, D V; Leiter, E H; Hanson, M S; Christianson, S W; Shultz, L D; Hesselton, R M; Greiner, D L

1995-12-01

When used as hosts in passive transfer experiments, a stock of NOD/Lt mice congenic for the severe combined immunodeficiency (scid) mutation have provided great insight to the contributions of various T-cell populations in the pathogenesis of autoimmune insulin-dependent diabetes mellitus (IDDM). Moreover, NOD-scid mice support higher levels of human lymphohematopoietic cell growth than the C.B-17-scid strain in which the mutation originated. However, the ability to perform long-term lymphohematopoietic repopulation studies in the NOD-scid stock has been limited by the fact that most of these mice develop lethal thymic lymphomas beginning at 20 weeks of age. These thymic lymphomas are characterized by activation and subsequent genomic reintegrations of Emv30, an endogenous murine ecotropic retrovirus unique to the NOD genome. To test the role of this endogenous retrovirus in thymomagenesis, we produced a stock of Emv30null NOD-scid mice by congenic replacement of the proximal end of chromosome 11 with genetic material derived from the closely related NOR/Lt strain. Thymic lymphomas still initiate in Emv30null NOD-scid females, but their rate of progression is significantly retarded since the frequency of tumors weighing between 170 and 910 mg at 25 weeks of age was reduced to 20.8% vs. 76.2% in Emv30% segregants. The thymic lymphomas that did develop in Emv30null NOD-scid mice were not characterized by a compensatory increase in mink cell focus-forming proviral integrations, which initiate thymomagenesis in other susceptible mouse strains. Significantly, the ability of standard NOD T-cells to transfer IDDM to the Emv30null NOD-scid stock was not impaired.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficient extravasation of tumor-repopulating cells depends on cell deformability

PubMed Central

Chen, Junjian; Zhou, Wenwen; Jia, Qiong; Chen, Junwei; Zhang, Shuang; Yao, Wenting; Wei, Fuxiang; Zhang, Yuejin; Yang, Fang; Huang, Wei; Zhang, Yao; Zhang, Huafeng; Zhang, Yi; Huang, Bo; Zhang, Zhihong; Jia, Haibo; Wang, Ning

2016-01-01

Cancer metastasis is the most deadly stage in cancer progression. Despite significant efforts over the past decades, it remains elusive why only a very small fraction of cancer cells is able to generate micrometastasis and metastatic colonization. Recently we have shown that tumor-repopulating cells (TRCs), a highly tumorigenic subpopulation of mouse melanoma cells, can be selected by being cultured and grown in 3D soft fibrin gels. Here we show that when injected into the yolk of a 2 day-post-fertilization (dpf) embryo of Tg (fli1:EGFP or kdrl:mCherry) zebrafish, TRCs are much more efficient in surviving and growing at various secondary sites to generate micrometastasis and metastatic colonization than control melanoma cells that are grown on rigid plastic. The metastasis of TRCs is dependent on the presence of Sox2, a self-renewal gene, and silencing Sox2 leads to the inhibition of TRC metastasis. High-resolution of 3D confocal images of the TRCs at the secondary sites show that extravasation and formation of micrometastases by TRCs are more efficient than by the control cells. Remarkably, efficient extravasation of TRCs in vivo and transmigration in vitro are determined by TRC deformability, as a result of low Cdc42 and high Sox2. Our findings suggest that tumor cell deformability is a key factor in controlling extravasation dynamics during metastasis. PMID:26787224

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

BAF180 regulates cellular senescence and hematopoietic stem cell homeostasis through p21

PubMed Central

Lee, Hyemin; Dai, Fangyan; Zhuang, Li; Xiao, Zhen-Dong; Kim, Jongchan; Zhang, Yilei; Ma, Li; You, M. James; Wang, Zhong; Gan, Boyi

2016-01-01

BAF180 (also called PBRM1), a subunit of the SWI/SNF complex, plays critical roles in the regulation of chromatin remodeling and gene transcription, and is frequently mutated in several human cancers. However, the role of mammalian BAF180 in tumor suppression and tissue maintenance in vivo remains largely unknown. Here, using a conditional somatic knockout approach, we explored the cellular and organismal functions of BAF180 in mouse. BAF180 deletion in primary mouse embryonic fibroblasts (MEFs) triggers profound cell cycle arrest, premature cellular senescence, without affecting DNA damage response or chromosomal integrity. While somatic deletion of BAF180 in adult mice does not provoke tumor development, BAF180 deficient mice exhibit defects in hematopoietic system characterized by progressive reduction of hematopoietic stem cells (HSCs), defective long-term repopulating potential, and hematopoietic lineage developmental aberrations. BAF180 deletion results in elevated p21 expression in both MEFs and HSCs. Mechanistically, we showed that BAF180 binds to p21 promoter, and BAF180 deletion enhances the binding of modified histones associated with transcriptional activation on p21 promoter. Deletion of p21 rescues cell cycle arrest and premature senescence in BAF180 deficient MEFs, and partially rescues hematopoietic defects in BAF180 deficient mice. Together, our study identifies BAF180 as a critical regulator of cellular senescence and HSC homeostasis, which is at least partially regulated through BAF180-mediated suppression of p21 expression. Our results also suggest that senescence triggered by BAF180 inactivation may serve as a failsafe mechanism to restrain BAF180 deficiency-associated tumor development, providing a conceptual framework to further understand BAF180 function in tumor biology. PMID:26992241

Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1.

PubMed

Schiroli, Giulia; Ferrari, Samuele; Conway, Anthony; Jacob, Aurelien; Capo, Valentina; Albano, Luisa; Plati, Tiziana; Castiello, Maria C; Sanvito, Francesca; Gennery, Andrew R; Bovolenta, Chiara; Palchaudhuri, Rahul; Scadden, David T; Holmes, Michael C; Villa, Anna; Sitia, Giovanni; Lombardo, Angelo; Genovese, Pietro; Naldini, Luigi

2017-10-11

Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all IL2RG (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the IL2RG -edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of IL2RG editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Kinetics of CD4+ T cell repopulation of lymphoid tissues after treatment of HIV-1 infection

PubMed Central

Zhang, Zhi-Qiang; Notermans, Daan W.; Sedgewick, Gerald; Cavert, Winston; Wietgrefe, Stephen; Zupancic, Mary; Gebhard, Kristin; Henry, Keith; Boies, Lawrence; Chen, Zongming; Jenkins, Marc; Mills, Roger; McDade, Hugh; Goodwin, Carolyn; Schuwirth, Caspar M.; Danner, Sven A.; Haase, Ashley T.

1998-01-01

Potent combinations of antiretroviral drugs diminish the turnover of CD4+ T lymphocytes productively infected with HIV-1 and reduce the large pool of virions deposited in lymphoid tissue (LT). To determine to what extent suppression of viral replication and reduction in viral antigens in LT might lead correspondingly to repopulation of the immune system, we characterized CD4+ T lymphocyte populations in LT in which we previously had quantitated viral load and turnover of infected cells before and after treatment. We directly measured by quantitative image analysis changes in total CD4+ T cell counts, the CD45RA+ subset, and fractions of proliferating or apoptotic CD4+ T cells. Compared with normal controls, we documented decreased numbers of CD4+ T cells and increased proliferation and apoptosis. After treatment, proliferation returned to normal levels, and total CD4+ T and CD45RA+ cells increased. We discuss the effects of HIV-1 on this subset based on the concept that renewal mechanisms in the adult are operating at full capacity before infection and cannot meet the additional demand imposed by the loss of productively infected cells. The slow increases in the CD45RA+ CD4+ T cells are consistent with the optimistic conclusions that (i) renewal mechanisms have not been damaged irreparably even at relatively advanced stages of infection and (ii) CD4+ T cell populations can be partially restored by control of active replication without eradication of HIV-1. PMID:9448301

Kinetics of CD4+ T cell repopulation of lymphoid tissues after treatment of HIV-1 infection.

PubMed

Zhang, Z Q; Notermans, D W; Sedgewick, G; Cavert, W; Wietgrefe, S; Zupancic, M; Gebhard, K; Henry, K; Boies, L; Chen, Z; Jenkins, M; Mills, R; McDade, H; Goodwin, C; Schuwirth, C M; Danner, S A; Haase, A T

1998-02-03

Potent combinations of antiretroviral drugs diminish the turnover of CD4+ T lymphocytes productively infected with HIV-1 and reduce the large pool of virions deposited in lymphoid tissue (LT). To determine to what extent suppression of viral replication and reduction in viral antigens in LT might lead correspondingly to repopulation of the immune system, we characterized CD4+ T lymphocyte populations in LT in which we previously had quantitated viral load and turnover of infected cells before and after treatment. We directly measured by quantitative image analysis changes in total CD4+ T cell counts, the CD45RA+ subset, and fractions of proliferating or apoptotic CD4+ T cells. Compared with normal controls, we documented decreased numbers of CD4+ T cells and increased proliferation and apoptosis. After treatment, proliferation returned to normal levels, and total CD4+ T and CD45RA+ cells increased. We discuss the effects of HIV-1 on this subset based on the concept that renewal mechanisms in the adult are operating at full capacity before infection and cannot meet the additional demand imposed by the loss of productively infected cells. The slow increases in the CD45RA+ CD4+ T cells are consistent with the optimistic conclusions that (i) renewal mechanisms have not been damaged irreparably even at relatively advanced stages of infection and (ii) CD4+ T cell populations can be partially restored by control of active replication without eradication of HIV-1.

Platelet recruitment promotes keratocyte repopulation following corneal epithelial abrasion in the mouse

USDA-ARS?s Scientific Manuscript database

Corneal abrasion not only damages the epithelium but also induces stromal keratocyte death at the site of injury. While a coordinated cascade of inflammatory cell recruitment facilitates epithelial restoration, it is unclear if this cascade is necessary for keratocyte recovery. Since platelet and ne...

A different regional response by mouse oligodendrocyte progenitor cells (OPCs) to high-dose X-irradiation has consequences for repopulating OPC-depleted normal tissue.

PubMed

Irvine, Karen-Amanda; Blakemore, William F

2007-01-01

This study was designed to investigate whether the residual, dysfunctional oligodendrocyte progenitor cells (OPCs) observed following X-irradiation of the mouse spinal cord [D. M. Chari et al. (2003) Exp. Neurol., 198, 145-153], the presence of which prevented the endogenous repopulation of these areas from normal tissue, reflects a general response of OPCs in the mouse central nervous system (CNS) to X-irradiation. The brains of adult mice were exposed to 40 Gy of X-irradiation and the effect of X-irradiation on the OPCs was assessed up to 4 weeks post-irradiation using anti-NG2 antibodies. X-irradiation resulted in almost complete depletion of OPCs within the telencephalon (cortex, corpus callosum and hippocampus) by 7 days post-irradiation, which was followed by progressive repopulation of OPCs from non-irradiated areas of the cortex. By contrast, within the lower brain centres (the diencephalon and mesencephalon) OPC loss occurred much more slowly so that 26% of the OPCs still remained 4 weeks after X-irradiation. The consequence of this heterogeneous response to X-irradiation was that whereas transplanted and endogenous OPCs rapidly established themselves in the OPC-depleted telencephalon this did not occur in the areas where there was incomplete depletion of endogenous OPCs. Our findings confirm not only the requirement for almost complete OPC depletion in order to establish transplanted OPCs in normal tissue but also highlight a heterogeneity of progenitor populations in different areas of the mouse CNS.

Fractionated External Beam Radiotherapy as a Suitable Preparative Regimen for Hepatocyte Transplantation After Partial Hepatectomy

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

Krause, Petra; Wolff, Hendrik A.; Rave-Frank, Margret

2011-07-15

Purpose: Hepatocyte transplantation is strongly considered to be a promising option to correct chronic liver failure through repopulation of the diseased organ. We already reported on extensive liver repopulation by hepatocytes transplanted into rats preconditioned with 25-Gy single dose selective external beam irradiation (IR). Herein, we tested lower radiation doses and fractionated protocols, which would be applicable in clinical use. Methods and Material: Livers of dipeptidylpeptidase IV (DPPIV)-deficient rats were preconditioned with partial liver external beam single dose IR at 25 Gy, 8 Gy, or 5 Gy, or fractionated IR at 5 x 5 Gy or 5 x 2 Gy.more » Four days after completion of IR, a partial hepatectomy (PH) was performed to resect the untreated liver section. Subsequently, 12 million wild-type (DPPIV{sup +}) hepatocytes were transplanted via the spleen into the recipient livers. The degree of donor cell integration and liver repopulation was studied 16 weeks after transplantation by means of immunofluorescence and DPPIV-luminescence assay. Results: Donor hepatocyte integration and liver repopulation were more effective in the irradiated livers following pretreatment with the IR doses 1 x 25 Gy and 5 x 5 Gy (formation of large DPPIV-positive cell clusters) than single-dose irradiation at 8 Gy or 5 Gy (DPPIV-positive clusters noticeably smaller and less frequent). Quantitative analysis of extracted DPPIV revealed signals exceeding the control level in all transplanted animals treated with IR and PH. Compared with the standard treatment of 1 x 25 Gy, fractionation with 5 x 5 Gy was equally efficacious, the Mann-Whitney U test disclosing no statistically significant difference (p = 0.146). The lower doses of 1 x 5 Gy, 1 x 8 Gy, and 5 x 2 Gy were significantly less effective with p < 0.05. Conclusion: This study suggests that fractionated radiotherapy in combination with PH is a conceivable pretreatment approach to prime the host liver for hepatocyte transplantation, thus bringing the experimental model a step closer to clinical application.« less

Comparison of HIV- and EIAV-based vectors on their efficiency in transducing murine and human hematopoietic repopulating cells.

PubMed

Siapati, Elena K; Bigger, Brian W; Miskin, James; Chipchase, Daniel; Parsley, Kathryn L; Mitrophanous, Kyriacos; Themis, Mike; Thrasher, Adrian J; Bonnet, Dominique

2005-09-01

The use of lentiviral vectors for gene transfer into hematopoietic stem cells has raised considerable interest as these vectors can permanently integrate their genome into quiescent cells. Vectors based on alternative lentiviruses would theoretically be safer than HIV-1-based vectors and could also be used in HIV-positive patients, minimizing the risk of generating replication-competent virus. Here we report the use of third-generation equine infectious anemia virus (EIAV)- and HIV-1-based vectors with minimal viral sequences and absence of accessory proteins. We have compared their efficiency in transducing mouse and human hematopoietic stem cells both in vitro and in vivo to that of a previously documented second-generation HIV-1 vector. The third-generation EIAV- and HIV-based vectors gave comparable levels of transduction and transgene expression in both mouse and human NOD/SCID repopulating cells but were less efficient than the second-generation HIV-1 vector in human HSCs. For the EIAV vector this is possibly a reflection of the lower protein expression levels achieved in human cells, as vector copy number analysis revealed that this vector exhibited a trend to integrate equally efficiently compared to the third-generation HIV-1 vector in both mouse and human HSCs. Interestingly, the presence or absence of Tat in viral preparations did not influence the transduction efficiency of HIV-1 vectors in human HSCs.

Proteasome expression and activity in cancer and cancer stem cells.

PubMed

Voutsadakis, Ioannis A

2017-03-01

Proteasome is a multi-protein organelle that participates in cellular proteostasis by destroying damaged or short-lived proteins in an organized manner guided by the ubiquitination signal. By being in a central place in the cellular protein complement homeostasis, proteasome is involved in virtually all cell processes including decisions on cell survival or death, cell cycle, and differentiation. These processes are important also in cancer, and thus, the proteasome is an important regulator of carcinogenesis. Cancers include a variety of cells which, according to the cancer stem cell theory, descend from a small percentage of cancer stem cells, alternatively termed tumor-initiating cells. These cells constitute the subsets that have the ability to propagate the whole variety of cancer and repopulate tumors after cytostatic therapies. Proteasome plays a role in cellular processes in cancer stem cells, but it has been found to have a decreased function in them compared to the rest of cancer cells. This article will discuss the transcriptional regulation of proteasome sub-unit proteins in cancer and in particular cancer stem cells and the relationship of the proteasome with the pluripotency that is the defining characteristic of stem cells. Therapeutic opportunities that present from the understanding of the proteasome role will also be discussed.

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.

Dysfunctional oligodendrocyte progenitor cell (OPC) populations may inhibit repopulation of OPC depleted tissue.

PubMed

Chari, D M; Huang, W L; Blakemore, W F

2003-09-15

We have attempted to extend a previously described rat model of focal oligodendrocyte progenitor cell (OPC) depletion, using 40 Gy X-irradiation (Chari and Blakemore [2002] Glia 37:307-313), to the adult mouse spinal cord, to examine the ability of OPCs present in adjacent normal areas to colonise areas of progenitor depletion. In contrast to rat, OPCs in the mouse spinal cord appeared to be a comparatively radiation-resistant population, as 30-35% of OPCs survived in X-irradiated tissue (whereas <1% of OPCs survive in X-irradiated rat spinal cord). The numbers of surviving OPCs remained constant with time indicating that this population was incapable of regenerating itself in response to OPC loss. Additionally, these OPCs did not contribute to remyelination of axons when demyelinating lesions were placed in X-irradiated tissue, suggesting that the surviving cells are functionally impaired. Importantly, the length of the OPC-depleted area did not diminish with time, as would be expected if progressive repopulation of OPC-depleted areas by OPCs from normal areas was occurring. Our findings therefore raise the possibility that the presence of a residual dysfunctional OPC population may inhibit colonisation of such areas by normal OPCs. Copyright 2003 Wiley-Liss, Inc.

Protection from radiation-induced damage to spermatogenesis by hormone treatment

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

Kurdoglu, B.; Wilson, G.; Parchuri, N.

1994-07-01

Infertility caused by killing of the spermatogonial stem cells occurs frequently in men treated for cancer with radiotherapy and chemotherapy. We investigated whether pretreatment of rats with testosterone plus estradiol, which reversibly inhibits the completion of spermatogenesis and protects spermatogonial stem cells from procarbazine-induced damage, would also protect these cells from radiation. Adult male LBNF rats were implanted for 6 weeks with capsules containing testosterone and estradiol and then irradiated with doses from 2.5-7.0 Gy. Controls were irradiated with 1.8-3.5 Gy. Implants were removed 1 day after irradiation, and all animals were killed 10 weeks later for assessment of stemmore » cell survival by counting repopulating tubules in histological sections and by sperm head counts. At doses of 2.5 and 3.5 Gy the repopulation indices and sperm head counts were significantly higher (P < 0.001) in the rats treated with testosterone and estradiol than in the controls. Protection factors calculated from the dose-response curves were in the range of 1.5-2.2. Elucidation of the mechanism of protection is essential to apply it to clinical situations. The fact that the spermatogonia are protected against radiation as well as procarbazine indicates that the mechanism does not involve drug delivery or metabolism. 32 refs., 3 figs.« less

Regenerative medicine in kidney disease: where we stand and where to go.

PubMed

Borges, Fernanda T; Schor, Nestor

2017-07-22

The kidney is a complex organ with more than 20 types of specialized cells that play an important role in maintaining the body's homeostasis. The epithelial tubular cell is formed during embryonic development and has little proliferative capacity under physiological conditions, but after acute injury the kidney does have regenerative capacity. However, after repetitive or severe lesions, it may undergo a maladaptation process that predisposes it to chronic kidney injury. Regenerative medicine includes various repair and regeneration techniques, and these have gained increasing attention in the scientific literature. In the future, not only will these techniques contribute to the repair and regeneration of the human kidney, but probably also to the construction of an entire organ. New mechanisms studied for kidney regeneration and repair include circulating stem cells as mesenchymal stromal/stem cells and their paracrine mechanisms of action; renal progenitor stem cells; the leading role of tubular epithelial cells in the tubular repair process; the study of zebrafish larvae to understand the process of nephron development, kidney scaffold and its repopulation; and, finally, the development of organoids. This review elucidates where we are in terms of current scientific knowledge regarding these mechanisms and the promises of future scientific perspectives.

Combined short-term immunotherapy for experimental autoimmune myasthenia gravis

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

Pestronk, A.; Drachman, D.B.; Teoh, R.

1983-08-01

A therapeutic strategy was designed to eliminate the humoral immune response to acetylcholine receptor (AChR) in ongoing experimental autoimmune myasthenia gravis (EAMG). Rats with EAMG were treated with a protocol consisting of three components: (1) A single high dose of cyclophosphamide (200 mg/kg) was used to produce a rapid and sustained fall in the anti-AChR antibody levels by preferential destruction of antibody-producing B-lymphocytes. ''Memory'' lymphocytes were not eliminated by cyclophosphamide. (2) Irradiation (600 rads) was used to eliminate the ''memory'' cells. It eliminated the anamnestic response to a challenge with the antigen AChR. (3) Bone marrow transplantation was used tomore » repopulate the hematopoietic system after the otherwise lethal dose of cyclophosphamide. We used bone marrow from syngeneic rats with active EAMG to simulate an autologous transplant. Rats with EAMG treated with this combined protocol showed a prompt and sustained fall in the anti-AChR antibody levels and had no anamnestic response to a challenge with AChR. Thus, an affected animal's own marrow could be stored and used later for repopulation after cyclophosphamide-irradiation treatment. This treatment eliminates the animal's ongoing immune responses and reconstitutes the immune system in its original state. The success of this approach suggests that, if their safety could be established, similar ''curative'' strategies might be developed for the treatment of patients with severe antibody-mediated autoimmune disorders, such as myasthenia gravis.« less

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

Occurrence of Salmonella serotype Typhimurium DT104 on a commercial swine farm before, during, and after depopulation and repopulation.

PubMed

Erdman, Matthew M; Harris, Isabel T; Torremorell, Montserrat; Wilt, Vincil M; Harris, D L Hank

2005-08-01

To determine whether depopulation-repopulation could be used to eradicate Salmonella serotype Typhimurium DT104 from a commercial swine farm in the midwestern United States. Observational study A commercial swine farm undergoing depopulation-repopulation to eliminate porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae. Pooled fecal samples, tissue samples, and serum samples were collected from pigs on the farm before and after depopulation-repopulation. When there were no pigs on the farm, environmental swab specimens were collected for bacterial culture. Serum was analyzed for anti-Salmonella antibodies with an indirect ELISA. Salmonella isolates obtained by bacterial culture of fecal, tissue, and environmental samples were characterized by means of serotyping, phage typing, pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing. 167 Salmonella isolates representing 9 serotypes were recovered from the farm. Results of PFGE and antimicrobial susceptibility testing suggested that S. Typhimurium DT104 strain was not eradicated from the farm. However, seroprevalence of anti-Salmonella antibodies and the percentage of pooled fecal samples positive for Salmonella spp were significantly decreased following repopulation. Results suggested that depopulation-repopulation in conjunction with stringent cleaning and disinfection, attention to biosecurity procedures, control of other diseases, and changes in feed management may reduce the occurrence of, but likely will not eliminate, Salmonella spp in commercial swine herds.

Periodontal Wound Healing Responses to Varying Oxygen Concentrations and Atmospheric Pressures.

DTIC Science & Technology

1986-05-01

Presumably, epithelial and gingival connective tissue exclusion allowed periodontal ligament cells to repopulate the wound and to regenerate a new...However, it seems clear that the periodontal ligament cells provide a major source of connective tissue attachment and regeneration (Nyman et al., 1982a...Connective Tissue Regeneration to Periodontally Diseased Teeth. J. Perio. Res. 15:1. Davis, J. C., Dunn, J. M., Gates, G. A. and Heimbach, R. D. 1979

Decellularized aortic conduits: could their cryopreservation affect post-implantation outcomes? A morpho-functional study on porcine homografts.

PubMed

Gallo, Michele; Bonetti, Antonella; Poser, Helen; Naso, Filippo; Bottio, Tomaso; Bianco, Roberto; Paolin, Adolfo; Franci, Paolo; Busetto, Roberto; Frigo, Anna Chiara; Buratto, Edward; Spina, Michele; Marchini, Maurizio; Ortolani, Fulvia; Iop, Laura; Gerosa, Gino

2016-11-01

Decellularized porcine aortic valve conduits (AVCs) implanted in a Vietnamese Pig (VP) experimental animal model were matched against decellularized and then cryopreserved AVCs to assess the effect of cryopreservation on graft hemodynamic performance and propensity to in vivo repopulation by host's cells. VPs (n = 12) underwent right ventricular outflow tract substitution using AVC allografts and were studied for 15-month follow-up. VPs were randomized into two groups, receiving AVCs treated with decellularization alone (D; n = 6) or decellularization/cryopreservation (DC; n = 6), respectively. Serial echocardiography was carried out to follow up hemodynamic function. All explanted AVCs were processed for light and electron microscopy. No signs of dilatation, progressive stenosis, regurgitation, and macroscopic calcification were echocardiographically observed in both D and DC groups. Explanted D grafts exhibited near-normal features, whereas the presence of calcification, inflammatory infiltrates, and disarray of elastic lamellae occurred in some DC grafts. In the unaltered regions of AVCs from both groups, almost complete re-endothelialization was observed for both valve cusps and aorta walls. In addition, side-by-side repopulation by recipient's fibroblasts, myofibroblasts, and smooth muscle cells was paralleled by ongoing tissue remodeling, as revealed by the ultrastructural identification of typical canals of collagen fibrillogenesis and elastogenesis-related features. Incipient neo-vascularization and re-innervation of medial and adventitial tunicae of grafted aortic walls were also detected for both D and DC groups. Cryopreservation did not affect post-implantation AVC hemodynamic behavior and was topically propensive to cell repopulation and tissue renewal, although graft deterioration including calcification was present in several areas. Thus, these preliminary data provide essential information on feasibility of decellularization and cryopreservation coupling in the perspective of treatment optimization and subsequent clinical trials using similarly treated human allografts as innovative heart valve substitutes.

Mechanisms of radiation-induced normal tissue toxicity and implications for future clinical trials

PubMed Central

Jenrow, Kenneth A.; Brown, Stephen L.

2014-01-01

To summarize current knowledge regarding mechanisms of radiation-induced normal tissue injury and medical countermeasures available to reduce its severity. Advances in radiation delivery using megavoltage and intensity-modulated radiation therapy have permitted delivery of higher doses of radiation to well-defined tumor target tissues. Injury to critical normal tissues and organs, however, poses substantial risks in the curative treatment of cancers, especially when radiation is administered in combination with chemotherapy. The principal pathogenesis is initiated by depletion of tissue stem cells and progenitor cells and damage to vascular endothelial microvessels. Emerging concepts of radiation-induced normal tissue toxicity suggest that the recovery and repopulation of stromal stem cells remain chronically impaired by long-lived free radicals, reactive oxygen species, and pro-inflammatory cytokines/chemokines resulting in progressive damage after radiation exposure. Better understanding the mechanisms mediating interactions among excessive generation of reactive oxygen species, production of pro-inflammatory cytokines and activated macrophages, and role of bone marrow-derived progenitor and stem cells may provide novel insight on the pathogenesis of radiation-induced injury of tissues. Further understanding the molecular signaling pathways of cytokines and chemokines would reveal novel targets for protecting or mitigating radiation injury of tissues and organs. PMID:25324981

Evidence for naive T-cell repopulation despite thymus irradiation after autologous transplantation in adults with multiple myeloma: role of ex vivo CD34+ selection and age.

PubMed

Malphettes, Marion; Carcelain, Guislaine; Saint-Mezard, Pierre; Leblond, Véronique; Altes, Hester Korthals; Marolleau, Jean-Pierre; Debré, Patrice; Brouet, Jean-Claude; Fermand, Jean-Paul; Autran, Brigitte

2003-03-01

Immunodeficiency following autologous CD34+-purified peripheral blood stem cell (PBSC) transplantation could be related to T-cell depletion of the graft or impaired T-cell reconstitution due to thymus irradiation. Aiming to assess the role of irradiated thymus in T-cell repopulation, we studied 32 adults with multiple myeloma, randomly assigned to receive high-dose therapy including total body irradiation (TBI) followed by autologous transplantation with either unselected or CD34+-selected PBSCs. The median number of reinfused CD3+ cells was lower in the selected group (0.03 versus 14 x 10(6)/kg; P =.002). Lymphocyte subset counts were evaluated from month 3 to 24 after grafting. Naive CD4+ T cells were characterized both by phenotype and by quantification of T-cell receptor rearrangement excision circles (TRECs). The reconstitution of CD3+ and CD4+ T cells was significantly delayed in the CD34+-selected group, but eventually led to counts similar to those found in the unselected group after month 12. Mechanism of reconstitution differed, however, between both groups. Indeed, a marked increase in the naive CD62L+CD45RA+CD4+ subset was observed in the selected group, but not in the unselected group in which half of the CD45RA+CD4+ T cells appear to be CD62L-. Age was identified as an independent adverse factor for CD4+ and CD62L+CD45RA+CD4+ T-cell reconstitution. Our results provide evidence that infusing PBSCs depleted of T cells after TBI in adults delays T-cell reconstitution but accelerates thymic regeneration.

Repopulation of calibrations with samples from the target site: effect of the size of the calibration.

NASA Astrophysics Data System (ADS)

Guerrero, C.; Zornoza, R.; Gómez, I.; Mataix-Solera, J.; Navarro-Pedreño, J.; Mataix-Beneyto, J.; García-Orenes, F.

2009-04-01

Near infrared (NIR) reflectance spectroscopy offers important advantages because is a non-destructive technique, the pre-treatments needed in samples are minimal, and the spectrum of the sample is obtained in less than 1 minute without the needs of chemical reagents. For these reasons, NIR is a fast and cost-effective method. Moreover, NIR allows the analysis of several constituents or parameters simultaneously from the same spectrum once it is obtained. For this, a needed steep is the development of soil spectral libraries (set of samples analysed and scanned) and calibrations (using multivariate techniques). The calibrations should contain the variability of the target site soils in which the calibration is to be used. Many times this premise is not easy to fulfil, especially in libraries recently developed. A classical way to solve this problem is through the repopulation of libraries and the subsequent recalibration of the models. In this work we studied the changes in the accuracy of the predictions as a consequence of the successive addition of samples to repopulation. In general, calibrations with high number of samples and high diversity are desired. But we hypothesized that calibrations with lower quantities of samples (lower size) will absorb more easily the spectral characteristics of the target site. Thus, we suspect that the size of the calibration (model) that will be repopulated could be important. For this reason we also studied this effect in the accuracy of predictions of the repopulated models. In this study we used those spectra of our library which contained data of soil Kjeldahl Nitrogen (NKj) content (near to 1500 samples). First, those spectra from the target site were removed from the spectral library. Then, different quantities of samples of the library were selected (representing the 5, 10, 25, 50, 75 and 100% of the total library). These samples were used to develop calibrations with different sizes (%) of samples. We used partial least squares regression, and leave-one-out cross validation as methods of calibration. Two methods were used to select the different quantities (size of models) of samples: (1) Based on Characteristics of Spectra (BCS), and (2) Based on NKj Values of Samples (BVS). Both methods tried to select representative samples. Each of the calibrations (containing the 5, 10, 25, 50, 75 or 100% of the total samples of the library) was repopulated with samples from the target site and then recalibrated (by leave-one-out cross validation). This procedure was sequential. In each step, 2 samples from the target site were added to the models, and then recalibrated. This process was repeated successively 10 times, being 20 the total number of samples added. A local model was also created with the 20 samples used for repopulation. The repopulated, non-repopulated and local calibrations were used to predict the NKj content in those samples from the target site not included in repopulations. For the measurement of the accuracy of the predictions, the r2, RMSEP and slopes were calculated comparing predicted with analysed NKj values. This scheme was repeated for each of the four target sites studied. In general, scarce differences can be found between results obtained with BCS and BVS models. We observed that the repopulation of models increased the r2 of the predictions in sites 1 and 3. The repopulation caused scarce changes of the r2 of the predictions in sites 2 and 4, maybe due to the high initial values (using non-repopulated models r2 >0.90). As consequence of repopulation, the RMSEP decreased in all the sites except in site 2, where a very low RMESP was obtained before the repopulation (0.4 g×kg-1). The slopes trended to approximate to 1, but this value was reached only in site 4 and after the repopulation with 20 samples. In sites 3 and 4, accurate predictions were obtained using the local models. Predictions obtained with models using similar size of samples (similar %) were averaged with the aim to describe the main patterns. The r2 of predictions obtained with models of higher size were not more accurate than those obtained with models of lower size. After repopulation, the RMSEP of predictions using models with lower sizes (5, 10 and 25% of samples of the library) were lower than RMSEP obtained with higher sizes (75 and 100%), indicating that small models can easily integrate the variability of the soils from the target site. The results suggest that calibrations of small size could be repopulated and "converted" in local calibrations. According to this, we can focus most of the efforts in the obtainment of highly accurate analytical values in a reduced set of samples (including some samples from the target sites). The patterns observed here are in opposition with the idea of global models. These results could encourage the expansion of this technique, because very large data based seems not to be needed. Future studies with very different samples will help to confirm the robustness of the patterns observed. Authors acknowledge to "Bancaja-UMH" for the financial support of the project "NIRPROS".

Stem Cell Genetic Therapy for Fanconi Anemia - A New Hope.

PubMed

Hanenberg, Helmut; Roellecke, Katharina; Wiek, Constanze

2017-01-01

Fanconi anemia (FA) is a rare inherited DNA disorder clinically characterized by congenital malformations, progressive bone marrow failure, and cancer susceptibility. Due to a strong survival advantage of spontaneously corrected 'normal' hematopoietic stem cells (HSCs) in a few patients, FA is considered a model disorder for genetic correction of autologous stem cells, where genetically corrected stem cells and their progeny have a strong in vivo selective advantage, ultimately leading to normal hematopoiesis. Despite these apparently ideal circumstances, three HSC gene therapy trials with gammaretroviral vectors (stage I) designed to cure the hematological manifestation of FA completely failed to provide long-term clinical benefits for patients, predominantly due to the combination of insufficient gene transfer technologies and incompletely understood FA HSC pathobiology. Currently, FA gene therapy is in stage II where, based on an improved understanding of the cellular defects in FA HSCs, consequently adapted transduction protocols are being used in two phase I/II trials for in vitro genetic correction of FANCA-deficient hematopoietic stem cells. These results are eagerly awaited. Independent from the outcome of these studies, technologies are already available that seem highly attractive for testing in FA. In stage III, this would ultimately include targeted in vivo correction of autologous HSCs by overexpression of nonintegrating lentiviral vectors with scaffold/matrix attachment region elements using specific envelopes as pseudotypes. Although currently still challenging, in a few years in vivo genome editing approaches will be readily available in stage IV, in which the delivery of the editing machinery/ complex is targeted to the autologous FA HSCs by the nonintegrating lentiviral vectors established in stage III. Even low levels of corrected stem cells will then quickly repopulate the entire hematopoiesis of the patient. We therefore are sanguine that in the future, genetic therapy can be used clinically for the correction of FA HSCs in the standard care of FA patients. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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.

The Network of Epithelial-mesenchymal transition: potential new targets for tumor resistance

PubMed Central

Nantajit, Danupon; Lin, Dong; Li, Jian Jian

2014-01-01

Purpose In multiple cell metazoans, the ability of polarized epithelial cells to convert to motile mesenchymal cells in order to relocate to another location is governed by a unique process termed epithelial-mesenchymal transition (EMT). While being an essential process of cellular plasticity for normal tissue and organ developments, EMT is found to be involved in an array of malignant phenotypes of tumor cells including proliferation and invasion, angiogenesis, stemness of cancer cells and resistance to chemo-radiotherapy. Although EMT is being extensively studied and demonstrated to play a key role in tumor metastasis and in sustaining tumor hallmarks, there is a lack of clear picture of the overall EMT signaling network, wavering the potential clinical trials targeting EMT. Methods In this review, we highlight the potential key therapeutic targets of EMT linked with tumor aggressiveness, hypoxia, angiogenesis and cancer stem cells, emphasizing on an emerging EMT-associated NF-κB/HER2/STAT3 pathway in radioresistance of breast cancer stem cells. Results Further definition of cancer stem cell repopulation due to EMT-controlled tumor microenvironment will help to understand how tumors exploit the EMT mechanisms for their survival and expansion advantages. Conclusions The knowledge of EMT will offer more effective targets in clinical trials to treat therapy-resistant metastatic lesions. PMID:25270087

Eradication of Murine Norovirus from a Mouse Barrier Facility

PubMed Central

Kastenmayer, Robin J; Perdue, Kathy A; Elkins, William R

2008-01-01

Murine norovirus (MNV) is a common viral infection of mice in many research facilities. MNV infects hematopoietic cells and alters their cellular morphology. Because of MNV's probable effects on the systemic immune response of infected mice the decision was made to eradicate the virus from 2 rooms containing infected animals in our vivarium. Two different eradication methods were selected. One room, in which most of the indirectly exposed sentinels had antibodies to MNV, was depopulated and thoroughly cleaned prior to repopulation. In the other room, in which only 13% of the sentinels had positive MNV titers, selective testing was used, and MNV-positive animals were removed. Data from surveillance of the sentinel mice exposed to dirty bedding indicate that the test-and-removal method was ineffective in eliminating MNV from the room, whereas sentinel mice in the room that underwent depopulation and cleaning prior to repopulation have not shown any evidence of MNV since December 2006. PMID:18210995

Upcyte® Microvascular Endothelial Cells Repopulate Decellularized Scaffold

PubMed Central

Dally, Iris; Hartmann, Nadja; Münst, Bernhard; Braspenning, Joris; Walles, Heike

2013-01-01

A general problem in tissue engineering is the poor and insufficient blood supply to guarantee tissue cell survival as well as physiological tissue function. To address this limitation, we have developed an in vitro vascularization model in which a decellularized porcine small bowl segment, representing a capillary network within a collagen matrix (biological vascularized scaffold [BioVaSc]), is reseeded with microvascular endothelial cells (mvECs). However, since the supply of mvECs is limited, in general, and as these cells rapidly dedifferentiate, we have applied a novel technology, which allows the generation of large batches of quasi-primary cells with the ability to proliferate, whilst maintaining their differentiated functionality. These so called upcyte mvECs grew for an additional 15 population doublings (PDs) compared to primary cells. Upcyte mvECs retained endothelial characteristics, such as von Willebrandt Factor (vWF), CD31 and endothelial nitric oxide synthase (eNOS) expression, as well as positive Ulex europaeus agglutinin I staining. Upcyte mvECs also retained biological functionality such as tube formation, cell migration, and low density lipoprotein (LDL) uptake, which were still evident after PD27. Initial experiments using MTT and Live/Dead staining indicate that upcyte mvECs repopulate the BioVaSc Scaffold. As with conventional cultures, these cells also express key endothelial molecules (vWF, CD31, and eNOS) in a custom-made bioreactor system even after a prolonged period of 14 days. The combination of upcyte mvECs and the BioVaSc represents a novel and promising approach toward vascularizing bioreactor models which can better reflect organs, such as the liver. PMID:22799502

Effect of proinflammatory cytokines on PIGA- hematopoiesis.

PubMed

Kulkarni, Shashikant; Bessler, Monica

2003-09-01

Blood cells from patients with paroxysmal nocturnal hemoglobinuria lack glycosyl phosphatidylinositol (GPI)-linked proteins, due to a somatic mutation in the X-linked PIGA gene. It is believed that clonal expansion of PIGA- blood cells is due to a survival advantage in the hostile marrow environment of aplastic anemia. Here we investigated the effects of inhibitory cytokines in mice genetically engineered to have blood cells deficient in GPI-linked proteins. The effect of inhibitory cytokines (tumor necrosis factor-alpha [TNF-alpha], interferon-gamma [IFN-gamma], macrophage inflammatory protein-1 alpha [MIP-1alpha], and transforming growth factor-beta1 [TGF-beta1]) was investigated, using clonogenic assays, competitive repopulation, and in vivo induction of proinflammatory cytokines by double-stranded RNA. The expression of Fas on progenitor cells and its up-regulation by inhibitory cytokines were analyzed by flow cytometry. TNF-alpha, IFN-gamma, MIP-1alpha, and TGF-beta1 suppressed colony formation in a dose-dependent fashion that was similar for PIGA+ and PIGA- blood bone marrow cells. Competitive repopulation of bone marrow cells cultured in IFN-gamma and TNF-alpha resulted in a comparable ability of PIGA+ and PIGA- hematopoietic stem cells to reconstitute hematopoiesis. Fas expression was minimal on PIGA+ and PIGA- progenitor cells and was up-regulated to the same extent in response to IFN-gamma and TNF-alpha as assessed by Fas antibody-mediated apoptosis. Similarly, in vivo induction of proinflammatory cytokines by double-stranded RNA had no effect on the proportion of circulating PIGA- blood cells. These results indicate that PIGA+ and PIGA- hematopoietic progenitor cells respond similarly to inhibitory cytokines, suggesting that other factors are responsible for the clonal expansion of paroxysmal nocturnal hemoglobinuria cells.

Efficacy and safety of a clinically relevant foamy vector design in human hematopoietic repopulating cells.

PubMed

Everson, Elizabeth M; Hocum, Jonah D; Trobridge, Grant D

2018-06-23

Previous studies have shown that foamy viral (FV) vectors are a promising alternative to gammaretroviral and lentiviral vectors and insulators can improve FV vector safety. However, in a previous analysis of insulator effects on FV vector safety, strong viral promoters were used to elicit genotoxic events. Here we developed and analyzed the efficacy and safety of a high-titer, clinically relevant FV vector driven by the housekeeping promoter elongation factor-1α and insulated with an enhancer blocking A1 insulator (FV-EGW-A1). Human CD34 + cord blood cells were exposed to an enhanced green fluorescent protein expressing vector, FV-EGW-A1, at a multiplicity of infection of 10 and then maintained in vitro or transplanted into immunodeficient mice. Flow cytometry was used to measure engraftment and marking in vivo. FV vector integration sites were analyzed to assess safety. FV-EGW-A1 resulted in high-marking, multi-lineage engraftment of human repopulating cells with no evidence of silencing. Engraftment was highly polyclonal with no clonal dominance and a promising safety profile based on integration site analysis. An FV vector with an elongation factor-1α promoter and an A1 insulator is a promising vector design for use in the clinic. This article is protected by copyright. All rights reserved.

Telomerase deficiency in bone marrow-derived cells attenuates angiotensin II-induced abdominal aortic aneurysm formation.

PubMed

Findeisen, Hannes M; Gizard, Florence; Zhao, Yue; Cohn, Dianne; Heywood, Elizabeth B; Jones, Karrie L; Lovett, David H; Howatt, Deborah A; Daugherty, Alan; Bruemmer, Dennis

2011-02-01

Abdominal aortic aneurysms (AAA) are an age-related vascular disease and an important cause of morbidity and mortality. In this study, we sought to determine whether the catalytic component of telomerase, telomerase reverse transcriptase (TERT), modulates angiotensin (Ang) II-induced AAA formation. Low-density lipoprotein receptor-deficient (LDLr-/-) mice were lethally irradiated and reconstituted with bone marrow-derived cells from TERT-deficient (TERT-/-) mice or littermate wild-type mice. Mice were placed on a diet enriched in cholesterol, and AAA formation was quantified after 4 weeks of Ang II infusion. Repopulation of LDLr-/- mice with TERT-/- bone marrow-derived cells attenuated Ang II-induced AAA formation. TERT-deficient recipient mice revealed modest telomere attrition in circulating leukocytes at the study end point without any overt effect of the donor genotype on white blood cell counts. In mice repopulated with TERT-/- bone marrow, aortic matrix metalloproteinase-2 (MMP-2) activity was reduced, and TERT-/- macrophages exhibited decreased expression and activity of MMP-2 in response to stimulation with Ang II. Finally, we demonstrated in transient transfection studies that TERT overexpression activates the MMP-2 promoter in macrophages. TERT deficiency in bone marrow-derived macrophages attenuates Ang II-induced AAA formation in LDLr-/- mice and decreases MMP-2 expression. These results point to a previously unrecognized role of TERT in the pathogenesis of AAA.

The impact of left ventricular stretching in model cultivations with neonatal cardiomyocytes in a whole-heart bioreactor.

PubMed

Hülsmann, Jörn; Aubin, Hug; Wehrmann, Alexander; Lichtenberg, Artur; Akhyari, Payam

2017-05-01

Here, we investigate the impact of integrated three-dimensional (3D) left ventricular (LV) stretching on myocardial maturation in a whole-heart bioreactor setting. Therefore, decellularized rat hearts were selectively repopulated with rodent neonatal cardiomyocytes (5 · 10 6 cells per heart) and cultured over 5 days. Continuous medium perfusion was maintained through the coronary artery system in a customized whole-heart bioreactor system with or without integrated biomechanical stimulation of LV. 3D repopulation effectiveness and cellular vitality were evaluated by repetitive metabolic WST-1 assays and 3D confocal microscopy analysis through fluorescent staining, also assessing cellular organization. Moreover, specific myocardial vitality was verified by detecting spontaneous electrophysiological activity using a multielectrode assay. Western blot analysis of cardiac myosin heavychain (MHC) and quantitative RT-PCR for Connexin 43 was used to analyze cardiomyocyte maturation. Decellularized whole-heart constructs repopulated with neonatal cardiomyocytes (repopWHC) showed vital 3D cell populations throughout the repopulation sites within the LV with a significant increase in metabolic activity (326 ± 113% for stimulated constructs vs. 162 ± 32% for non-stimulated controls after 96 h of continuous cultivation as compared to their state 24 h after injection, directly prior to bioreactor cultivation). Further, bioreactor cultivation under integrated mechanical LV stimulation not only led to a higher degree of cellular organization and an increased MHC content, but also to a significant increase of Cx43 gene expression resulting in a regain of 60 ± 19% of native neonatal hearts expression level in contrast to 20 ± 9% for non-stimulated controls (P = 0.03). Therefore, our study suggests that the integration of LV stretching into whole-heart bioreactor cultivation may enhance cardiac maturation not only by promoting cellular organization but also through adaptive protein and gene expression with particular implications for the formation of the conductive apparatus. Further, this study emphasizes the importance of suitable bioprocessing strategies within sophisticated bioreactor systems as tools for customized stimulation and cultivation of tissue engineered tissues and organs. Biotechnol. Bioeng. 2017;114: 1107-1117. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

iRhom2 regulates CSF1R cell surface expression and non-steady state myelopoiesis in mice.

PubMed

Qing, Xiaoping; Rogers, Lindsay; Mortha, Arthur; Lavin, Yonit; Redecha, Patricia; Issuree, Priya D; Maretzky, Thorsten; Merad, Miriam; McIlwain, David; Mak, Tak W; Overall, Christopher M; Blobel, Carl P; Salmon, Jane E

2016-12-01

CSF1R (colony stimulating factor 1 receptor) is the main receptor for CSF1 and has crucial roles in regulating myelopoeisis. CSF1R can be proteolytically released from the cell surface by ADAM17 (A disintegrin and metalloprotease 17). Here, we identified CSF1R as a major substrate of ADAM17 in an unbiased degradomics screen. We explored the impact of CSF1R shedding by ADAM17 and its upstream regulator, inactive rhomboid protein 2 (iRhom2, gene name Rhbdf2), on homeostatic development of mouse myeloid cells. In iRhom2-/- mice, we found constitutive accumulation of membrane-bound CSF1R on myeloid cells at steady state, although cell numbers of these populations were not altered. However, in the context of mixed bone marrow (BM) chimera, under competitive pressure, iRhom2-/- BM progenitor-derived monocytes, tissue macrophages and lung DCs showed a repopulation advantage over those derived from wild-type (WT) BM progenitors, suggesting enhanced CSF1R signaling in the absence of iRhom2. In vitro experiments indicate that iRhom2-/- Lin - SCA-1 + c-Kit + (LSKs) cells, but not granulocyte-macrophage progenitors (GMPs), had faster growth rates than WT cells in response to CSF1. Our results shed light on an important role of iRhom2/ADAM17 pathway in regulation of CSF1R shedding and repopulation of monocytes, macrophages and DCs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

iRhom2 regulates cell surface expression of CSF1R and non-steady state myelopoiesis in mice

PubMed Central

Qing, Xiaoping; Lavin, Yonit; Redecha, Patricia; Issuree, Priya D.; Maretzky, Thorsten; Merad, Miriam; McIlwain, David; Mak, Tak W.; Overall, Christopher M.

2016-01-01

The colony stimulating factor 1 receptor (CSF1R) functions as the major receptor for macrophage colony stimulating factor (CSF1) with crucial roles in regulating myelopoeisis. CSF1R can be proteolytically released from the cell surface by A disintegrin and metalloprotease 17 (ADAM17). Here we identified CSF1R as a major substrate of ADAM17 in an unbiased degradomics screen. We explored the impact of CSF1R shedding by ADAM17 and its upstream regulator, inactive rhomboid protein 2 (iRhom2, gene name Rhbdf2), on homeostatic development of mouse myeloid cells. In iRhom2−/− mice, we found constitutive accumulation of membrane-bound CSF1R on myeloid cells at steady state, although cell numbers of these populations were not altered. However, in the context of mixed bone marrow (BM) chimera, under competitive pressure, iRhom2−/− BM progenitor-derived monocytes, tissue macrophages and lung DCs showed a repopulation advantage over those derived from wild type (WT) BM progenitors, suggesting enhanced CSF1R signaling in the absence of iRhom2. In vitro experiments indicate that iRhom2−/− Lin−SCA-1+c-Kit+ (LSKs) cells, but not granulocyte-macrophage progenitors (GMPs), had faster growth rates than WT cells in response to CSF1. Our results shed light on an important role of iRhom2/ADAM17 pathway in regulation of CSF1R shedding and repopulation of monocytes, macrophages and DCs. PMID:27601030

Esophageal tissue engineering: A new approach for esophageal replacement

PubMed Central

Totonelli, Giorgia; Maghsoudlou, Panagiotis; Fishman, Jonathan M; Orlando, Giuseppe; Ansari, Tahera; Sibbons, Paul; Birchall, Martin A; Pierro, Agostino; Eaton, Simon; De Coppi, Paolo

2012-01-01

A number of congenital and acquired disorders require esophageal tissue replacement. Various surgical techniques, such as gastric and colonic interposition, are standards of treatment, but frequently complicated by stenosis and other problems. Regenerative medicine approaches facilitate the use of biological constructs to replace or regenerate normal tissue function. We review the literature of esophageal tissue engineering, discuss its implications, compare the methodologies that have been employed and suggest possible directions for the future. Medline, Embase, the Cochrane Library, National Research Register and ClinicalTrials.gov databases were searched with the following search terms: stem cell and esophagus, esophageal replacement, esophageal tissue engineering, esophageal substitution. Reference lists of papers identified were also examined and experts in this field contacted for further information. All full-text articles in English of all potentially relevant abstracts were reviewed. Tissue engineering has involved acellular scaffolds that were either transplanted with the aim of being repopulated by host cells or seeded prior to transplantation. When acellular scaffolds were used to replace patch and short tubular defects they allowed epithelial and partial muscular migration whereas when employed for long tubular defects the results were poor leading to an increased rate of stenosis and mortality. Stenting has been shown as an effective means to reduce stenotic changes and promote cell migration, whilst omental wrapping to induce vascularization of the construct has an uncertain benefit. Decellularized matrices have been recently suggested as the optimal choice for scaffolds, but smart polymers that will incorporate signalling to promote cell-scaffold interaction may provide a more reproducible and available solution. Results in animal models that have used seeded scaffolds strongly sug- gest that seeding of both muscle and epithelial cells on scaffolds prior to implantation is a prerequisite for complete esophageal replacement. Novel approaches need to be designed to allow for peristalsis and vascularization in the engineered esophagus. Although esophageal tissue engineering potentially offers a real alternative to conventional treatments for severe esophageal disease, important barriers remain that need to be addressed. PMID:23322987

Esophageal tissue engineering: a new approach for esophageal replacement.

PubMed

Totonelli, Giorgia; Maghsoudlou, Panagiotis; Fishman, Jonathan M; Orlando, Giuseppe; Ansari, Tahera; Sibbons, Paul; Birchall, Martin A; Pierro, Agostino; Eaton, Simon; De Coppi, Paolo

2012-12-21

A number of congenital and acquired disorders require esophageal tissue replacement. Various surgical techniques, such as gastric and colonic interposition, are standards of treatment, but frequently complicated by stenosis and other problems. Regenerative medicine approaches facilitate the use of biological constructs to replace or regenerate normal tissue function. We review the literature of esophageal tissue engineering, discuss its implications, compare the methodologies that have been employed and suggest possible directions for the future. Medline, Embase, the Cochrane Library, National Research Register and ClinicalTrials.gov databases were searched with the following search terms: stem cell and esophagus, esophageal replacement, esophageal tissue engineering, esophageal substitution. Reference lists of papers identified were also examined and experts in this field contacted for further information. All full-text articles in English of all potentially relevant abstracts were reviewed. Tissue engineering has involved acellular scaffolds that were either transplanted with the aim of being repopulated by host cells or seeded prior to transplantation. When acellular scaffolds were used to replace patch and short tubular defects they allowed epithelial and partial muscular migration whereas when employed for long tubular defects the results were poor leading to an increased rate of stenosis and mortality. Stenting has been shown as an effective means to reduce stenotic changes and promote cell migration, whilst omental wrapping to induce vascularization of the construct has an uncertain benefit. Decellularized matrices have been recently suggested as the optimal choice for scaffolds, but smart polymers that will incorporate signalling to promote cell-scaffold interaction may provide a more reproducible and available solution. Results in animal models that have used seeded scaffolds strongly suggest that seeding of both muscle and epithelial cells on scaffolds prior to implantation is a prerequisite for complete esophageal replacement. Novel approaches need to be designed to allow for peristalsis and vascularization in the engineered esophagus. Although esophageal tissue engineering potentially offers a real alternative to conventional treatments for severe esophageal disease, important barriers remain that need to be addressed.

Pre-differentiation of human neural stem cells into GABAergic neurons prior to transplant results in greater repopulation of the damaged brain and accelerates functional recovery after transient ischemic stroke.

PubMed

Abeysinghe, Hima C S; Bokhari, Laita; Quigley, Anita; Choolani, Mahesh; Chan, Jerry; Dusting, Gregory J; Crook, Jeremy M; Kobayashi, Nao R; Roulston, Carli L

2015-09-29

Despite attempts to prevent brain injury during the hyperacute phase of stroke, most sufferers end up with significant neuronal loss and functional deficits. The use of cell-based therapies to recover the injured brain offers new hope. In the current study, we employed human neural stem cells (hNSCs) isolated from subventricular zone (SVZ), and directed their differentiation into GABAergic neurons followed by transplantation to ischemic brain. Pre-differentiated GABAergic neurons, undifferentiated SVZ-hNSCs or media alone were stereotaxically transplanted into the rat brain (n=7/group) 7 days after endothelin-1 induced stroke. Neurological outcome was assessed by neurological deficit scores and the cylinder test. Transplanted cell survival, cellular phenotype and maturation were assessed using immunohistochemistry and confocal microscopy. Behavioral assessments revealed accelerated improvements in motor function 7 days post-transplant in rats treated with pre-differentiated GABAergic cells in comparison to media alone and undifferentiated hNSC treated groups. Histopathology 28 days-post transplant indicated that pre-differentiated cells maintained their GABAergic neuronal phenotype, showed evidence of synaptogenesis and up-regulated expression of both GABA and calcium signaling proteins associated with neurotransmission. Rats treated with pre-differentiated cells also showed increased neurogenic activity within the SVZ at 28 days, suggesting an additional trophic role of these GABAergic cells. In contrast, undifferentiated SVZ-hNSCs predominantly differentiated into GFAP-positive astrocytes and appeared to be incorporated into the glial scar. Our study is the first to show enhanced exogenous repopulation of a neuronal phenotype after stroke using techniques aimed at GABAergic cell induction prior to delivery that resulted in accelerated and improved functional recovery.

INDUCTION OF IMMUNOLOGIC TOLERANCE IN OLDER NEW ZEALAND MICE REPOPULATED WITH YOUNG SPLEEN, BONE MARROW, OR THYMUS

PubMed Central

Staples, Parker J.; Steinberg, Alfred D.; Talal, Norman

1970-01-01

Newborn, 7–9 day, and 16–18 day old NZB and B/W mice were, unlike older New Zealand mice, rendered tolerant to single doses of 8–10 mg of soluble BGG. After challenge, this tolerance was of short duration and escape occurred rapidly. Age-matched and similarly treated C3H, Balb/c and C57Bl mice did not escape from tolerance. Partial tolerance could be maintained by repeated injections of BGG. Biofiltration ruled out hyperphagocytosis as an explanation for this resistance to tolerance. Tolerance could be induced in older B/W mice if they were thymectomized, irradiated, and repopulated with young (12–15 day), but not old (2–3 month), spleen or bone marrow cells. Old bone marrow cells gave a non-tolerant response even when combined with young thymic grafts. Young bone marrow gave a tolerant response which was followed by the expected rapid escape only if a young thymus graft was also present. Escape was retarded if old thymus, or old irradiated thymus, was combined with young bone marrow. These results are best explained by abnormalities of both lymphoid precursors and thymic regulation. PMID:4192570

Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization

PubMed Central

Uzarski, Joseph S.; Bijonowski, Brent M.; Wang, Bo; Ward, Heather H.; Wandinger-Ness, Angela

2015-01-01

Analysis of perfusion-based bioreactors for organ engineering and a detailed evaluation of physical and biochemical parameters that measure dynamic changes within maturing cell-laden scaffolds are critical components of ex vivo tissue development that remain understudied topics in the tissue and organ engineering literature. Intricately designed bioreactors that house developing tissue are critical to properly recapitulate the in vivo environment, deliver nutrients within perfused media, and monitor physiological parameters of tissue development. Herein, we provide an in-depth description and analysis of two dual-purpose perfusion bioreactors that improve upon current bioreactor designs and enable comparative analyses of ex vivo scaffold recellularization strategies and cell growth performance during long-term maintenance culture of engineered kidney or liver tissues. Both bioreactors are effective at maximizing cell seeding of small-animal organ scaffolds and maintaining cell survival in extended culture. We further demonstrate noninvasive monitoring capabilities for tracking dynamic changes within scaffolds as the native cellular component is removed during decellularization and model human cells are introduced into the scaffold during recellularization and proliferate in maintenance culture. We found that hydrodynamic pressure drop (ΔP) across the retained scaffold vasculature is a noninvasive measurement of scaffold integrity. We further show that ΔP, and thus resistance to fluid flow through the scaffold, decreases with cell loss during decellularization and correspondingly increases to near normal values for whole organs following recellularization of the kidney or liver scaffolds. Perfused media may be further sampled in real time to measure soluble biomarkers (e.g., resazurin, albumin, or kidney injury molecule-1) that indicate degree of cellular metabolic activity, synthetic function, or engraftment into the scaffold. Cell growth within bioreactors is validated for primary and immortalized cells, and the design of each bioreactor is scalable to accommodate any three-dimensional scaffold (e.g., synthetic or naturally derived matrix) that contains conduits for nutrient perfusion to deliver media to growing cells and monitor noninvasive parameters during scaffold repopulation, broadening the applicability of these bioreactor systems. PMID:25929317

Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization.

PubMed

Uzarski, Joseph S; Bijonowski, Brent M; Wang, Bo; Ward, Heather H; Wandinger-Ness, Angela; Miller, William M; Wertheim, Jason A

2015-10-01

Analysis of perfusion-based bioreactors for organ engineering and a detailed evaluation of physical and biochemical parameters that measure dynamic changes within maturing cell-laden scaffolds are critical components of ex vivo tissue development that remain understudied topics in the tissue and organ engineering literature. Intricately designed bioreactors that house developing tissue are critical to properly recapitulate the in vivo environment, deliver nutrients within perfused media, and monitor physiological parameters of tissue development. Herein, we provide an in-depth description and analysis of two dual-purpose perfusion bioreactors that improve upon current bioreactor designs and enable comparative analyses of ex vivo scaffold recellularization strategies and cell growth performance during long-term maintenance culture of engineered kidney or liver tissues. Both bioreactors are effective at maximizing cell seeding of small-animal organ scaffolds and maintaining cell survival in extended culture. We further demonstrate noninvasive monitoring capabilities for tracking dynamic changes within scaffolds as the native cellular component is removed during decellularization and model human cells are introduced into the scaffold during recellularization and proliferate in maintenance culture. We found that hydrodynamic pressure drop (ΔP) across the retained scaffold vasculature is a noninvasive measurement of scaffold integrity. We further show that ΔP, and thus resistance to fluid flow through the scaffold, decreases with cell loss during decellularization and correspondingly increases to near normal values for whole organs following recellularization of the kidney or liver scaffolds. Perfused media may be further sampled in real time to measure soluble biomarkers (e.g., resazurin, albumin, or kidney injury molecule-1) that indicate degree of cellular metabolic activity, synthetic function, or engraftment into the scaffold. Cell growth within bioreactors is validated for primary and immortalized cells, and the design of each bioreactor is scalable to accommodate any three-dimensional scaffold (e.g., synthetic or naturally derived matrix) that contains conduits for nutrient perfusion to deliver media to growing cells and monitor noninvasive parameters during scaffold repopulation, broadening the applicability of these bioreactor systems.

Pluripotent stem cells for cardiac regeneration: Overview of recent advances & emerging trends

PubMed Central

Pawani, Harsha; Bhartiya, Deepa

2013-01-01

Cell based regenerative therapy has emerged as one of the most promising options of treatment for patients suffering from heart failure. Various adult stem cells types have undergone extensive clinical trials with limited success which is believed to be more of a cytokine effect rather than cell therapy. Pluripotent human embryonic stem cells (hESCs) have emerged as an attractive candidate stem cell source for obtaining cardiomyocytes (CMs) because of their tremendous capacity for expansion and unquestioned potential to differentiate into CMs. Studies carried out in animal models indicate that ES-derived CMs can partially remuscularize infarcted hearts and improve contractile function; however, the effect was not sustained over long follow up periods due to their limited capacity of cell division in vivo. Thus, the concept of transplanting multipotent cardiovascular progenitors derived from ES cells has emerged since the progenitors retain robust proliferative ability and multipotent nature enabling repopulation of other myocardial elements also in addition to CMs. Transplantation of CMs (progenitors) seeded in biodegradable scaffold and gel based engineered constructs has met with modest success due to issues like cell penetration, nutrient and oxygen availability and inflammation triggered during scaffold degradation inversely affecting the seeded cells. Recently cell sheet based tissue engineering involving culturing cells on ‘intelligent’ polymers has been evolved. Generation of a 3-D pulsatile myocardial tissue has been achieved. However, these advances have to be looked at with cautious optimism as many challenges need to be overcome before using these in clinical practice. PMID:23563370

Hematopoietic stem cell loss and hematopoietic failure in severe aplastic anemia is driven by macrophages and aberrant podoplanin expression.

PubMed

McCabe, Amanda; Smith, Julianne N P; Costello, Angelica; Maloney, Jackson; Katikaneni, Divya; MacNamara, Katherine C

2018-05-17

Severe aplastic anemia results from profound hematopoietic stem cell loss. T cells and interferon gamma have long been associated with severe aplastic anemia, yet the underlying mechanisms driving hematopoietic stem cell loss remain unknown. Using a mouse model of severe aplastic anemia, we demonstrate that interferon gamma-dependent hematopoietic stem cell loss required macrophages. Interferon gamma was necessary for bone marrow macrophage persistence, despite loss of other myeloid cells and hematopoietic stem cells. Depleting macrophages or abrogating interferon gamma signaling specifically in macrophages did not impair T cell activation or interferon gamma production in the bone marrow but rescued hematopoietic stem cells and reduced mortality. Thus, macrophages are not required for induction of interferon gamma in severe aplastic anemia and rather act as sensors of interferon gamma. Macrophage depletion rescued thrombocytopenia, increased bone marrow megakaryocytes, preserved platelet-primed stem cells, and increased the platelet-repopulating capacity of transplanted hematopoietic stem cells. In addition to the hematopoietic effects, severe aplastic anemia induced loss of non-hematopoietic stromal populations, including podoplanin-positive stromal cells. However, a subset of podoplanin-positive macrophages was increased during disease, and blockade of podoplanin in mice was sufficient to rescue disease. Our data further our understanding of disease pathogenesis demonstrating a novel role for macrophages as sensors of interferon gamma, thus illustrating an important role for the microenvironment in pathogenesis of severe aplastic anemia. Copyright © 2018, Ferrata Storti Foundation.

Improvement of the in vivo cellular repopulation of decellularized cardiovascular tissues by a detergent-free, non-proteolytic, actin-disassembling regimen.

PubMed

Assmann, Alexander; Struß, Marc; Schiffer, Franziska; Heidelberg, Friederike; Munakata, Hiroshi; Timchenko, Elena V; Timchenko, Pavel E; Kaufmann, Tim; Huynh, Khon; Sugimura, Yukiharu; Leidl, Quentin; Pinto, Antonio; Stoldt, Volker R; Lichtenberg, Artur; Akhyari, Payam

2017-12-01

Low immunogenicity and high repopulation capacity are crucial determinants for the functional and structural performance of acellular cardiovascular implants. The present study evaluates a detergent-free, non-proteolytic, actin-disassembling regimen (BIO) for decellularization of heart valve and vessel grafts, particularly focusing on their bio-functionality. Rat aortic conduits (rAoC; n = 89) and porcine aortic valve samples (n = 106) are decellularized using detergents (group DET) or the BIO regimen. BIO decellularization results in effective elimination of cellular proteins and significantly improves removal of DNA as compared with group DET, while the extracellular matrix (ECM) structure as well as mechanical properties are preserved. The architecture of rAoC in group BIO allows for improved bio-functionalization with fibronectin (FN) in a standardized rat implantation model: BIO treatment significantly increases speed and amount of autologous medial cellular repopulation in vivo (p < 0.001) and decreases the formation of hyperplastic intima (p < 0.001) as compared with FN-coated DET-decellularized grafts. Moreover, there are no signs of infiltration with inflammatory cells. The present biological, detergent-free, non-proteolytic regimen balances effective decellularization and ECM preservation in cardiovascular grafts, and provides optimized bio-functionality. Additionally, this study implies that the actin-disassembling regimen may be a promising approach for bioengineering of acellular scaffolds from other muscular tissues, as for example myocardium or intestine. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Presentation, diagnosis and management of limbal stem cell deficiency.

PubMed

Sejpal, Kunjal; Bakhtiari, Pejman; Deng, Sophie X

2013-01-01

The human corneal surface epithelium is continuously repopulated by the limbal stem cells (LSCs). Limbal Stem Cell Deficiency (LSCD) can lead to corneal opacity and vascularization, with consequent visual impairment or blindness. Many acquired and congenital diseases can lead to LCSD by direct injury to the LSCs, destruction of LSC niche, or both. Based on the severity of the disease, LSCD can present with various symptoms and signs. Although LSCD can be detected clinically, laboratory tests are necessary to confirm the diagnosis and monitor the disease progression. This article concisely reviews the clinical presentation, techniques for diagnosis and management of LSCD.

Identification and Characterization of a Dendritic Cell Precursor in Parenchymal Lung Tissue.

PubMed

von Garnier, Christophe; Blank, Fabian; Rothen-Rutishauser, Barbara; Goethert, Joachim R; Holt, Patrick G; Stumbles, Philip A; Strickland, Deborah H

2017-03-01

The pulmonary parenchymal and mucosal microenvironments are constantly exposed to the external environment and thus require continuous surveillance to maintain steady-state immunological homeostasis. This is achieved by a mobile network of pulmonary dendritic cells (DC) and macrophages (mø) that constantly sample and process microenvironmental antigens into signals that can initiate or dampen inflammation, either locally or after onward migration to draining lymph nodes. The constant steady-state turnover of pulmonary DC and mø requires replenishment from bone marrow precursors; however, the nature of the pulmonary precursor cell (PC) remains unclear, although recent studies suggest that subsets of pulmonary DC may derive from circulating monocytic precursors. In the current study, we describe a population of cells in steady-state mouse lung tissue that has the surface phenotypic and ultrastructural characteristics of a common DC progenitor. Irradiation and reconstitution studies confirmed the bone marrow origins of this PC and showed that it had rapid depletion and reconstitution kinetics that were similar to those of DC, with a 50% repopulation by donor-derived cells by Days 7-9 after reconstitution. This was significantly faster than the rates observed for mø, which showed 50% repopulation by donor-derived cells beyond Days 16-21 after reconstitution. Purified PC gained antigen-presenting function and a cell surface phenotype similar to that of pulmonary DC after maturation in vitro, with light and electron microscopy confirming a myeloid DC morphology. To the best of our knowledge, this is the first study to describe a PC for DC in lung tissue; the findings have implications for the restoration of pulmonary immunological homeostasis after bone marrow transplant.

Vps26B-retromer negatively regulates plasma membrane resensitization of PAR-2.

PubMed

Bugarcic, Andrea; Vetter, Irina; Chalmers, Silke; Kinna, Genevieve; Collins, Brett M; Teasdale, Rohan D

2015-11-01

Retromer is a trimeric complex composed of Vps26, Vps29, and Vps35 and has been shown to be involved in trafficking and sorting of transmembrane proteins within the endosome. The Vps26 paralog, Vps26B, defines a distinct retromer complex (Vps26B-retromer) in vivo and in vitro. Although endosomally associated, Vps26B-retromer does not bind the established retromer transmembrane cargo protein, cation-independent mannose 6-phosphate receptor (CI-M6PR), indicating it has a distinct role to retromer containing the Vps26A paralog. In the present study we use the previously established Vps26B-expressing HEK293 cell model to address the role of Vps26B-retromer in trafficking of the protease activated G-protein coupled receptor PAR-2 to the plasma membrane. In these cells there is no apparent defect in the initial activation of the receptor, as evidenced by release of intracellular calcium, ERK1/2 signaling and endocytosis of activated receptor PAR-2 into degradative organelles. However, we observe a significant delay in plasma membrane repopulation of the protease activated G protein-coupled receptor PAR-2 following stimulation, resulting in a defect in PAR-2 activation after resensitization. Here we propose that PAR-2 plasma membrane repopulation is regulated by Vps26B-retromer, describing a potential novel role for this complex. © 2015 International Federation for Cell Biology.

Primordial germ cell-mediated chimera technology produces viable pure-line Houbara bustard offspring: potential for repopulating an endangered species.

PubMed

Wernery, Ulrich; Liu, Chunhai; Baskar, Vijay; Guerineche, Zhor; Khazanehdari, Kamal A; Saleem, Shazia; Kinne, Jörg; Wernery, Renate; Griffin, Darren K; Chang, Il-Kuk

2010-12-29

The Houbara bustard (Chlamydotis undulata) is a wild seasonal breeding bird populating arid sandy semi-desert habitats in North Africa and the Middle East. Its population has declined drastically during the last two decades and it is classified as vulnerable. Captive breeding programmes have, hitherto, been unsuccessful in reviving population numbers and thus radical technological solutions are essential for the long term survival of this species. The purpose of this study was to investigate the use of primordial germ cell-mediated chimera technology to produce viable Houbara bustard offspring. Embryonic gonadal tissue was dissected from Houbara bustard embryos at eight days post-incubation. Subsequently, Houbara tissue containing gonadal primordial germ cells (gPGCs) was injected into White Leghorn chicken (Gallus gallus domesticus) embryos, producing 83/138 surviving male chimeric embryos, of which 35 chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara gPGCs in chimeric chicken testis were assessed by PCR with Houbara-specific primers and 31.3% (5/16) gonads collected from the injected chicken embryos showed the presence of donor Houbara cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germline chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara; the other was female but died before hatching. Genotyping confirmed that the male chick was a pure-line Houbara derived from a chimeric rooster. This study demonstrates for the first time that Houbara gPGCs can migrate, differentiate and eventually give rise to functional sperm in the chimeric chicken testis. This approach may provide a promising tool for propagation and conservation of endangered avian species that cannot breed in captivity.

Physiological Srsf2 P95H expression causes impaired hematopoietic stem cell functions and aberrant RNA splicing in mice.

PubMed

Kon, Ayana; Yamazaki, Satoshi; Nannya, Yasuhito; Kataoka, Keisuke; Ota, Yasunori; Nakagawa, Masahiro Marshall; Yoshida, Kenichi; Shiozawa, Yusuke; Morita, Maiko; Yoshizato, Tetsuichi; Sanada, Masashi; Nakayama, Manabu; Koseki, Haruhiko; Nakauchi, Hiromitsu; Ogawa, Seishi

2018-02-08

Splicing factor mutations are characteristic of myelodysplastic syndromes (MDS) and related myeloid neoplasms and implicated in their pathogenesis, but their roles in the development of MDS have not been fully elucidated. In the present study, we investigated the consequence of mutant Srsf2 expression using newly generated Vav1-Cre -mediated conditional knockin mice. Mice carrying a heterozygous Srsf2 P95H mutation showed significantly reduced numbers of hematopoietic stem and progenitor cells (HSPCs) and differentiation defects both in the steady-state condition and transplantation settings. Srsf2 -mutated hematopoietic stem cells (HSCs) showed impaired long-term reconstitution compared with control mice in competitive repopulation assays. Although the Srsf2 mutant mice did not develop MDS under the steady-state condition, when their stem cells were transplanted into lethally irradiated mice, the recipients developed anemia, leukopenia, and erythroid dysplasia, which suggests the role of replicative stress in the development of an MDS-like phenotype in Srsf2 -mutated mice. RNA sequencing of the Srsf2 -mutated HSPCs revealed a number of abnormal splicing events and differentially expressed genes, including several potential targets implicated in the pathogenesis of hematopoietic malignancies, such as Csf3r , Fyn , Gnas , Nsd1 , Hnrnpa2b1 , and Trp53bp1 Among the mutant Srsf2 -associated splicing events, most commonly observed were the enhanced inclusion and/or exclusion of cassette exons, which were caused by the altered consensus motifs for the recognition of exonic splicing enhancers. Our findings suggest that the mutant Srsf2 leads to a compromised HSC function by causing abnormal RNA splicing and expression, contributing to the deregulated hematopoiesis that recapitulates the MDS phenotypes, possibly as a result of additional genetic and/or environmental insults. © 2018 by The American Society of Hematology.

Primordial Germ Cell-Mediated Chimera Technology Produces Viable Pure-Line Houbara Bustard Offspring: Potential for Repopulating an Endangered Species

PubMed Central

Wernery, Ulrich; Liu, Chunhai; Baskar, Vijay; Guerineche, Zhor; Khazanehdari, Kamal A.; Saleem, Shazia; Kinne, Jörg; Wernery, Renate

2010-01-01

Background The Houbara bustard (Chlamydotis undulata) is a wild seasonal breeding bird populating arid sandy semi-desert habitats in North Africa and the Middle East. Its population has declined drastically during the last two decades and it is classified as vulnerable. Captive breeding programmes have, hitherto, been unsuccessful in reviving population numbers and thus radical technological solutions are essential for the long term survival of this species. The purpose of this study was to investigate the use of primordial germ cell-mediated chimera technology to produce viable Houbara bustard offspring. Methodology/Principal Findings Embryonic gonadal tissue was dissected from Houbara bustard embryos at eight days post-incubation. Subsequently, Houbara tissue containing gonadal primordial germ cells (gPGCs) was injected into White Leghorn chicken (Gallus gallus domesticus) embryos, producing 83/138 surviving male chimeric embryos, of which 35 chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara gPGCs in chimeric chicken testis were assessed by PCR with Houbara-specific primers and 31.3% (5/16) gonads collected from the injected chicken embryos showed the presence of donor Houbara cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germline chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara; the other was female but died before hatching. Genotyping confirmed that the male chick was a pure-line Houbara derived from a chimeric rooster. Conclusion This study demonstrates for the first time that Houbara gPGCs can migrate, differentiate and eventually give rise to functional sperm in the chimeric chicken testis. This approach may provide a promising tool for propagation and conservation of endangered avian species that cannot breed in captivity. PMID:21209914

Enhancement of cancer stem-like and epithelial−mesenchymal transdifferentiation property in oral epithelial cells with long-term nicotine exposure: Reversal by targeting SNAIL

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

Yu, Cheng-Chia; School of Dentistry, Chung Shan Medical University, Taichung, Taiwan; Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwan

Cigarette smoking is one of the major risk factors in the development and further progression of tumorigenesis, including oral squamous cell carcinoma (OSCC). Recent studies suggest that interplay cancer stem-like cells (CSCs) and epithelial−mesenchymal transdifferentiation (EMT) properties are responsible for the tumor maintenance and metastasis in OSCC. The aim of the present study was to investigate the effects of long-term exposure with nicotine, a major component in cigarette, on CSCs and EMT characteristics. The possible reversal regulators were further explored in nicotine-induced CSCs and EMT properties in human oral epithelial (OE) cells. Long-term exposure with nicotine was demonstrated to up-regulatemore » ALDH1 population in normal gingival and primary OSCC OE cells dose-dependently. Moreover, long-term nicotine treatment was found to enhance the self-renewal sphere-forming ability and stemness gene signatures expression and EMT regulators in OE cells. The migration/cell invasiveness/anchorage independent growth and in vivo tumor growth by nude mice xenotransplantation assay was enhanced in long-term nicotine-stimulated OE cells. Knockdown of Snail in long-term nicotine-treated OE cells was found to reduce their CSCs properties. Therapeutic delivery of Si-Snail significantly blocked the xenograft tumorigenesis of long-term nicotine-treated OSCC cells and largely significantly improved the recipient survival. The present study demonstrated that the enrichment of CSCs coupled EMT property in oral epithelial cells induced by nicotine is critical for the development of OSCC tumorigenesis. Targeting Snail might offer a new strategy for the treatment of OSCC patients with smoking habit. -- Highlights: ► Sustained nicotine treatment induced CSCs properties of oral epithelial cells. ► Long-term nicotine treatment enhance EMT properties of oral epithelial cells. ► Long-term nicotine exposure increased tumorigenicity of oral epithelial cells. ► Si-Snail blocked xenograft tumorigenesis of long-term nicotine-treated OSCC cells.« less

Depletion of CD52-positive cells inhibits the development of central nervous system autoimmune disease, but deletes an immune-tolerance promoting CD8 T-cell population. Implications for secondary autoimmunity of alemtuzumab in multiple sclerosis.

PubMed

von Kutzleben, Stephanie; Pryce, Gareth; Giovannoni, Gavin; Baker, David

2017-04-01

The objective was to determine whether CD52 lymphocyte depletion can act to promote immunological tolerance induction by way of intravenous antigen administration such that it could be used to either improve efficiency of multiple sclerosis (MS) inhibition or inhibit secondary autoimmunities that may occur following alemtuzumab use in MS. Relapsing experimental autoimmune encephalomyelitis was induced in ABH mice and immune cell depletion was therapeutically applied using mouse CD52 or CD4 (in conjunction with CD8 or CD20) depleting monoclonal antibodies. Immunological unresponsiveness was then subsequently induced using intravenous central nervous system antigens and responses were assessed clinically. A dose-response of CD4 monoclonal antibody depletion indicated that the 60-70% functional CD4 T-cell depletion achieved in perceived failed trials in MS was perhaps too low to even stop disease in animals. However, more marked (~75-90%) physical depletion of CD4 T cells by CD4 and CD52 depleting antibodies inhibited relapsing disease. Surprisingly, in contrast to CD4 depletion, CD52 depletion blocked robust immunological unresponsiveness through a mechanism involving CD8 T cells. Although efficacy was related to the level of CD4 T-cell depletion, the observations that CD52 depletion of CD19 B cells was less marked in lymphoid organs than in the blood provides a rationale for the rapid B-cell hyper-repopulation that occurs following alemtuzumab administration in MS. That B cells repopulate in the relative absence of T-cell regulatory mechanisms that promote immune tolerance may account for the secondary B-cell autoimmunities, which occur following alemtuzumab treatment of MS. © 2016 The Authors. Immunology Published by John Wiley & Sons Ltd.

Fetal liver-derived mesenchymal stromal cells augment engraftment of transplanted hepatocytes

PubMed Central

Joshi, Meghnad; Patil, Pradeep B.; He, Zhong; Holgersson, Jan; Olausson, Michael; Sumitran-Holgersson, Suchitra

2012-01-01

Background aims One important problem commonly encountered after hepatocyte transplantation is the low numbers of transplanted cells found in the graft. If hepatocyte transplantation is to be a viable therapeutic approach, significant liver parenchyma repopulation is required. Mesenchymal stromal cells (MSC) produce high levels of various growth factors, cytokines and metalloproteinases, and have immunomodulatory effects. We therefore hypothesized that co-transplantation of MSC with human fetal hepatocytes (hFH) could augment in vivo expansion after transplantation. We investigated the ability of human fetal liver MSC (hFLMSC) to augment expansion of phenotypically and functionally well-characterized hFH. Methods Two million hFH (passage 6) were either transplanted alone or together (1:1 ratio) with green fluorescence protein-expressing hFLMSC into the spleen of C57BL/6 nude mice with retrorsine-induced liver injury. Results After 4 weeks, engraftment of cells was detected by fluorescence in situ hybridization using a human-specific DNA probe. Significantly higher numbers of cells expressing human cytokeratin (CK)8, CK18, CK19, Cysteine-rich MNNG HOS Transforming gene (c-Met), alpha-fetoprotein (AFP), human nuclear antigen, mitochondrial antigen, hepatocyte-specific antigen and albumin (ALB) were present in the livers of recipient animals co-transplanted with hFLMSC compared with those without. Furthermore, expression of human hepatocyte nuclear factor (HNF)-4α and HNF-1β, and cytochrome P450 (CYP) 3A7 mRNA was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) in these animals. In addition, significantly increased amounts of human ALB were detected. Importantly, hFLMSC did not transdifferentiate into hepatocytes. Conclusions Our study reports the use of a novel strategy for enhanced liver repopulation and thereby advances this experimental procedure closer to clinical liver cell therapy. PMID:22424216

Optimization and critical evaluation of decellularization strategies to develop renal extracellular matrix scaffolds as biological templates for organ engineering and transplantation.

PubMed

Caralt, M; Uzarski, J S; Iacob, S; Obergfell, K P; Berg, N; Bijonowski, B M; Kiefer, K M; Ward, H H; Wandinger-Ness, A; Miller, W M; Zhang, Z J; Abecassis, M M; Wertheim, J A

2015-01-01

The ability to generate patient-specific cells through induced pluripotent stem cell (iPSC) technology has encouraged development of three-dimensional extracellular matrix (ECM) scaffolds as bioactive substrates for cell differentiation with the long-range goal of bioengineering organs for transplantation. Perfusion decellularization uses the vasculature to remove resident cells, leaving an intact ECM template wherein new cells grow; however, a rigorous evaluative framework assessing ECM structural and biochemical quality is lacking. To address this, we developed histologic scoring systems to quantify fundamental characteristics of decellularized rodent kidneys: ECM structure (tubules, vessels, glomeruli) and cell removal. We also assessed growth factor retention--indicating matrix biofunctionality. These scoring systems evaluated three strategies developed to decellularize kidneys (1% Triton X-100, 1% Triton X-100/0.1% sodium dodecyl sulfate (SDS) and 0.02% Trypsin-0.05% EGTA/1% Triton X-100). Triton and Triton/SDS preserved renal microarchitecture and retained matrix-bound basic fibroblast growth factor and vascular endothelial growth factor. Trypsin caused structural deterioration and growth factor loss. Triton/SDS-decellularized scaffolds maintained 3 h of leak-free blood flow in a rodent transplantation model and supported repopulation with human iPSC-derived endothelial cells and tubular epithelial cells ex vivo. Taken together, we identify an optimal Triton/SDS-based decellularization strategy that produces a biomatrix that may ultimately serve as a rodent model for kidney bioengineering. © Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

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

Neodymium: yttrium-aluminum-garnet long impulse laser for the elimination of superfluous hair: experiences and considerations from 3 years of activity.

PubMed

Ferraro, G A; Perrotta, A; Rossano, F; D'Andrea, F

2004-01-01

This study examined the results obtained with a modern apparatus for laser hair removal (neodymium: yttrium-aluminum-garnet [Nd:YAG] laser at long impulses with a wave-length of 1,064 nm; Q-switched laser) over a follow-up period of 3 years. A large heterogeneous group of 480 patients was taken into consideration. These patients were treated according to a standard protocol with monthly checkups and a personalized protocol at deferred appointments. The results, discovered by means of the most objective procedure possible, were retrieved and put into a graph showing two different curves for the repopulation of hair. In their clinical travels, the authors observed an average variable regrowth of 40% to 65%, allowing them to affirm that laser hair removal using Nd:YAG at long impulses is decisively efficient in obtaining long-term results. The use of a protocol (denominated "prolonged monthly checkup") with laser sessions at ever-decreasing periods permits, among other things, more outstanding and advantageous results for the patient. Thanks to more efficiently synchronized phases of the biologic hair cycle, this shortens and moves the telegenic phases closer and also renders the anagenic phases (those in which the selective photoermolysis on the pilipheric follicle proves to be efficient) more efficient. Personalization of the treatment relative to the monthly health checkup sessions is of fundamental importance to the scope of obtaining the best results in terms of cost-benefit rate, provided submassimal fluxes are (i.e., those well-tolerated by the patient) used. All this allows hair removal that is not definitive, but which becomes progressively permanent (i.e., characterized by ever-growing periods of lack of hair sustained by sporadic maintenance laser sessions based on the individual's necessity).

In Vivo Selection of Transplanted Hepatocytes by Pharmacological Inhibition of Fumarylacetoacetate Hydrolase in Wild-type Mice

PubMed Central

Paulk, Nicole K; Wursthorn, Karsten; Haft, Annelise; Pelz, Carl; Clarke, Gregory; Newell, Amy H; Olson, Susan B; Harding, Cary O; Finegold, Milton J; Bateman, Raymond L; Witte, John F; McClard, Ronald; Grompe, Markus

2012-01-01

Genetic fumarylacetoacetate hydrolase (Fah) deficiency is unique in that healthy gene-corrected hepatocytes have a strong growth advantage and can repopulate the diseased liver. Unfortunately, similar positive selection of gene-corrected cells is absent in most inborn errors of liver metabolism and it is difficult to reach the cell replacement index required for therapeutic benefit. Therefore, methods to transiently create a growth advantage for genetically modified hepatocytes in any genetic background would be advantageous. To mimic the selective pressure of Fah deficiency in normal animals, an efficient in vivo small molecule inhibitor of FAH, 4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate (CEHPOBA) was developed. Microarray analysis demonstrated that pharmacological inhibition of FAH produced highly similar gene expression changes to genetic deficiency. As proof of principle, hepatocytes lacking homogentisic acid dioxygenase (Hgd) and hence resistant to FAH inhibition were transplanted into sex-mismatched wild-type recipients. Time course analyses of 4–6 weeks of CEHPOBA administration after transplantation showed a linear relationship between treatment length and replacement index. Compared to controls, recipients treated with the FAH-inhibitor had 20–100-fold increases in liver repopulation. We conclude that pharmacological inhibition of FAH is a promising approach to in vivo selection of hepatocytes. PMID:22871666

Long-term Culture of Human iPS Cell-derived Telencephalic Neuron Aggregates on Collagen Gel.

PubMed

Oyama, Hiroshi; Takahashi, Koji; Tanaka, Yoshikazu; Takemoto, Hiroshi; Haga, Hisashi

2018-01-01

It takes several months to form the 3-dimensional morphology of the human embryonic brain. Therefore, establishing a long-term culture method for neuronal tissues derived from human induced pluripotent stem (iPS) cells is very important for studying human brain development. However, it is difficult to keep primary neurons alive for more than 3 weeks in culture. Moreover, long-term adherent culture to maintain the morphology of telencephalic neuron aggregates induced from human iPS cells is also difficult. Although collagen gel has been widely used to support long-term culture of cells, it is not clear whether human iPS cell-derived neuron aggregates can be cultured for long periods on this substrate. In the present study, we differentiated human iPS cells to telencephalic neuron aggregates and examined long-term culture of these aggregates on collagen gel. The results indicated that these aggregates could be cultured for over 3 months by adhering tightly onto collagen gel. Furthermore, telencephalic neuronal precursors within these aggregates matured over time and formed layered structures. Thus, long-term culture of telencephalic neuron aggregates derived from human iPS cells on collagen gel would be useful for studying human cerebral cortex development.Key words: Induced pluripotent stem cell, forebrain neuron, collagen gel, long-term culture.

Thrombopoietin: biology and clinical potentials.

PubMed

Miyazaki, H; Kato, T

1999-12-01

Thrombopoietin (TPO) is the principal physiologic regulator of platelet production. In vitro, TPO induces the growth of colony-forming units-megakaryocyte (CFU-MK) and the generation of mature polyploid megakaryocytes, which subsequently form extended cytoplasmic processes, termed proplatelets. On more differentiated CFU-MK, but not on megakaryocytes, TPO is critical for enhancing proplatelet formation. TPO has multilineage effects in hematopoiesis, not only stimulating megakaryocytopoiesis but also acting in synergy with other cytokines to enhance proliferation and survival of committed erythroid progenitors and primitive hematopoietic stem cells. Surface c-MPL, the receptor for TPO, defines a phenotype of hematopoietic stem cells with long-term repopulating ability. Treatment with various cytokine combinations, including TPO, results in an extensive ex vivo expansion of hematopoietic stem cells and blood cell precursors. In normal animals, pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) or glycosylated TPO increases the number of bone marrow megakaryocytes and their progenitors and greatly enhance the production of morphologically and functionally normal platelets. In contrast, they have only minimal effects on peripheral white blood cell and red blood cell counts. PEG-rHuMGDF used alone markedly expands circulating levels of multiple types of hematopoietic progenitors, and its effect is enhanced in combination with granulocyte colony-stimulating factor (G-CSF). Although PEG-rHuMGDF augments platelet aggregation induced by agonists in vitro, it has no influence in an animal model of thrombus formation. PEG-rHuMGDF or glycosylated TPO has a profound effect in a variety of animal models of thrombocytopenia, including myelosuppressive therapy. PEG-rHuMGDF treatment accelerates multilineage hematopoietic recovery, effectively improving thrombocytopenia, and, in most models, neutropenia and anemia. The concurrent administration of PEG-rHuMGDF and G-CSF does not interfere with the in vivo activity of cytokines but rather has synergistic effects. To further accelerate hematopoietic recovery, PEG-rHuMGDF administration should start at the earliest time following myelosuppressive treatment; this time sensitivity may result from the presence of a greater number of residual hematopoietic progenitors in the bone marrow soon after treatment. Moreover, if a relatively large dose of PEG-rHuMGDF is administered, a single intravenous injection is fully effective in improving impaired hematopoiesis. This effectiveness appears to be related to the persistence of PEG-rHuMGDF in the circulation. The safety and efficacy of two forms of the recombinant hormone, PEG-rHuMDGF and glycosylated human full-length TPO produced in mammalian cells, are currently under clinical investigation.

Elucidating the identity and behavior of spermatogenic stem cells in the mouse testis.

PubMed

Yoshida, Shosei

2012-09-01

Spermatogenesis in mice and other mammalians is supported by a robust stem cell system. Stem cells maintain themselves and continue to produce progeny that will differentiate into sperm over a long period. The pioneering studies conducted from the 1950s to the 1970s, which were based largely on extensive morphological analyses, have established the fundamentals of mammalian spermatogenesis and its stem cells. The prevailing so-called A(single) (A(s)) model, which was originally established in 1971, proposes that singly isolated A(s) spermatogonia are in fact the stem cells. In 1994, the first functional stem cell assay was established based on the formation of repopulating colonies after transplantation in germ cell-depleted host testes, which substantially accelerated the understanding of spermatogenic stem cells. However, because testicular tissues are dissociated into single-cell suspension before transplantation, it was impossible to evaluate the A(s) and other classical models solely by this technique. From 2007 onwards, functional assessment of stem cells without destroying the tissue architecture has become feasible by means of pulse-labeling and live-imaging strategies. Results obtained from these experiments have been challenging the classical thought of stem cells, in which stem cells are a limited number of specialized cells undergoing asymmetric division to produce one self-renewing and one differentiating daughter cells. In contrast, the emerging data suggest that an extended and heterogeneous population of cells exhibiting different degrees of self-renewing and differentiating probabilities forms a reversible, flexible, and stochastic stem cell system as a population. These features may lead to establishment of a more universal principle on stem cells that is shared by other systems.

Epidermal Homeostasis and Radiation Responses in a Multiscale Tissue Modeling Framework

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Cucinotta, Francis A.

2013-01-01

The surface of skin is lined with several thin layers of epithelial cells that are maintained throughout life time by a small population of stem cells. High dose radiation exposures could injure and deplete the underlying proliferative cells and induce cutaneous radiation syndrome. In this work we propose a multiscale computational model for skin epidermal dynamics that links phenomena occurring at the subcellular, cellular, and tissue levels of organization, to simulate the experimental data of the radiation response of swine epidermis, which is closely similar to human epidermis. Incorporating experimentally measured histological and cell kinetic parameters, we obtain results of population kinetics and proliferation indexes comparable to observations in unirradiated and acutely irradiated swine experiments. At the sub-cellular level, several recently published Wnt signaling controlled cell-cycle models are applied and the roles of key components and parameters are analyzed. Based on our simulation results, we demonstrate that a moderate increase of proliferation rate for the survival proliferative cells is sufficient to fully repopulate the area denuded by high dose radiation, as long as the integrity of underlying basement membrane is maintained. Our work highlights the importance of considering proliferation kinetics as well as the spatial organization of tissues when conducting in vivo investigations of radiation responses. This integrated model allow us to test the validity of several basic biological rules at the cellular level and sub-cellular mechanisms by qualitatively comparing simulation results with published research, and enhance our understanding of the pathophysiological effects of ionizing radiation on skin.

Orthotopic Transplantation of Achilles Tendon Allograft in Rats: With or without Incorporation of Autologous Mesenchymal Stem Cells.

PubMed

Aynardi, Michael; Zahoor, Talal; Mitchell, Reed; Loube, Jeffrey; Feltham, Tyler; Manandhar, Lumanti; Paudel, Sharada; Schon, Lew; Zhang, Zijun

2018-02-01

The biology and function of orthotopic transplantation of Achilles tendon allograft are unknown. Particularly, the revitalization of Achilles allograft is a clinical concern. Achilles allografts were harvested from donor rats and stored at -80 °C. Subcutaneous adipose tissue was harvested from the would-be allograft recipient rats for isolation of mesenchymal stem cells (MSCs). MSCs were cultured with growth differentiation factor-5 (GDF-5) and applied onto Achilles allografts on the day of transplantation. After the native Achilles tendon was resected from the left hind limb of the rats, Achilles allograft, with or without autologous MSCs, was implanted and sutured with calf muscles proximally and calcaneus distally. Animal gait was recorded presurgery and postsurgery weekly. The animals were sacrificed at week 4, and the transplanted Achilles allografts were collected for biomechanical testing and histology. The operated limbs had altered gait. By week 4, the paw print intensity, stance time, and duty cycle (percentage of the stance phase in a step cycle) of the reconstructed limbs were mostly recovered to the baselines recorded before surgery. Maximum load of failure was not different between Achilles allografts, with or without MSCs, and the native tendons. The Achilles allograft supplemented with MSCs had higher cellularity than the Achilles allograft without MSCs. Deposition of fine collagen (type III) fibers was active in Achilles allograft, with or without MSCs, but it was more evenly distributed in the allografts that were incubated with MSCs. In conclusion, orthotopically transplanted Achilles allograft healed with host tissues, regained strength, and largely restored Achilles function in 4 wk in rats. It is therefore a viable option for the reconstruction of a large Achilles tendon defect. Supplementation of MSCs improved repopulation of Achilles allograft, but large animal models, with long-term follow up and cell tracking, may be required to fully appreciate the functional benefits of MSCs.

Protective Properties of Radio-Chemoresistant Glioblastoma Stem Cell Clones Are Associated with Metabolic Adaptation to Reduced Glucose Dependence

PubMed Central

Yamada, Kazunari; Tso, Jonathan L.; Menjivar, Jimmy C.; Tian, Jane Y.; Yong, William H.; Schaue, Dörthe; Mischel, Paul S.; Cloughesy, Timothy F.; Nelson, Stanley F.; Liau, Linda M.; McBride, William; Tso, Cho-Lea

2013-01-01

Glioblastoma stem cells (GSC) are a significant cell model for explaining brain tumor recurrence. However, mechanisms underlying their radiochemoresistance remain obscure. Here we show that most clonogenic cells in GSC cultures are sensitive to radiation treatment (RT) with or without temozolomide (TMZ). Only a few single cells survive treatment and regain their self-repopulating capacity. Cells re-populated from treatment-resistant GSC clones contain more clonogenic cells compared to those grown from treatment-sensitive GSC clones, and repeated treatment cycles rapidly enriched clonogenic survival. When compared to sensitive clones, resistant clones exhibited slower tumor development in animals. Upregulated genes identified in resistant clones via comparative expression microarray analysis characterized cells under metabolic stress, including blocked glucose uptake, impaired insulin/Akt signaling, enhanced lipid catabolism and oxidative stress, and suppressed growth and inflammation. Moreover, many upregulated genes highlighted maintenance and repair activities, including detoxifying lipid peroxidation products, activating lysosomal autophagy/ubiquitin-proteasome pathways, and enhancing telomere maintenance and DNA repair, closely resembling the anti-aging effects of caloric/glucose restriction (CR/GR), a nutritional intervention that is known to increase lifespan and stress resistance in model organisms. Although treatment–introduced genetic mutations were detected in resistant clones, all resistant and sensitive clones were subclassified to either proneural (PN) or mesenchymal (MES) glioblastoma subtype based on their expression profiles. Functional assays demonstrated the association of treatment resistance with energy stress, including reduced glucose uptake, fatty acid oxidation (FAO)-dependent ATP maintenance, elevated reactive oxygen species (ROS) production and autophagic activity, and increased AMPK activity and NAD+ levels accompanied by upregulated mRNA levels of SIRT1/PGC-1α axis and DNA repair genes. These data support the view that treatment resistance may arise from quiescent GSC exhibiting a GR-like phenotype, and suggest that targeting stress response pathways of resistant GSC may provide a novel strategy in combination with standard treatment for glioblastoma. PMID:24260384

Morphological changes in human melanoma cells following irradiation with thermal neutrons.

PubMed

Barkla, D H; Allen, B J; Brown, J K; Mountford, M; Mishima, Y; Ichihashi, M

1989-01-01

Morphological changes in two human melanoma cell lines, MM96 and MM418, following irradiation with thermal neutrons, were studied using light and electron microscopy. The results show that the response of human malignant melanoma cells to neutron irradiation is both cell line dependent and dose dependent, and that in any given cell line, some cells are more resistant to irradiation than others, thus demonstrating heterogeneity in respect to radiosensitivity. Cells repopulating MM96 flasks after irradiation were morphologically similar to the cells of origin whereas in MM418 flasks cells differentiated into five morphologically distinct subgroups and showed increased melanization. The results also show that radiation causes distinctive morphological patterns of damage although ultrastructural changes unique to the high LET particles released from boron 10 neutron capture are yet to be identified.

Gap junction turnover is achieved by the internalization of small endocytic double-membrane vesicles.

PubMed

Falk, Matthias M; Baker, Susan M; Gumpert, Anna M; Segretain, Dominique; Buckheit, Robert W

2009-07-01

Double-membrane-spanning gap junction (GJ) channels cluster into two-dimensional arrays, termed plaques, to provide direct cell-to-cell communication. GJ plaques often contain circular, channel-free domains ( approximately 0.05-0.5 mum in diameter) identified >30 y ago and termed nonjunctional membrane (NM) domains. We show, by expressing the GJ protein connexin43 (Cx43) tagged with green fluorescent protein, or the novel photoconvertible fluorescent protein Dendra2, that NM domains appear to be remnants generated by the internalization of small GJ channel clusters that bud over time from central plaque areas. Channel clusters internalized within seconds forming endocytic double-membrane GJ vesicles ( approximately 0.18-0.27 mum in diameter) that were degraded by lysosomal pathways. Surprisingly, NM domains were not repopulated by surrounding channels and instead remained mobile, fused with each other, and were expelled at plaque edges. Quantification of internalized, photoconverted Cx43-Dendra2 vesicles indicated a GJ half-life of 2.6 h that falls within the estimated half-life of 1-5 h reported for GJs. Together with previous publications that revealed continuous accrual of newly synthesized channels along plaque edges and simultaneous removal of channels from plaque centers, our data suggest how the known dynamic channel replenishment of functional GJ plaques can be achieved. Our observations may have implications for the process of endocytic vesicle budding in general.

Long-term three-dimensional perfusion culture of human adult bone marrow mononuclear cells in bioreactors.

PubMed

Schmelzer, Eva; Finoli, Anthony; Nettleship, Ian; Gerlach, Jörg C

2015-04-01

The construction and long-term maintenance of three-dimensional in vitro bone marrow models is of great interest but still quite challenging. Here we describe the use of a multi-compartment hollow-fiber membrane based three-dimensional perfusion bioreactor for long-term culture of whole human bone marrow mononuclear cells. We also investigated bioreactors with incorporated open-porous foamed hydroxyapatite scaffolds, mimicking the in vivo bone matrix. Cells in bioreactors with and without scaffolds were cultured to 6 weeks and compared to Petri dish controls. Cells were analyzed for gene expression, surface markers by flow cytometry, metabolic activity, hematopoietic potential, viability, and attachment by immunocytochemistry. Cells in bioreactors were metabolic active during long-term culture. The percentages of hematopoietic stem cell and mature endothelial cell fractions were maintained in bioreactors. The expression of most of the analyzed genes stabilized and increased after long-term culture of 6 weeks. Compared to Petri dish culture controls, bioreactor perfusion culture improved in both the short and long-term, the colony formation unit capacity of hematopoietic progenitors. Cells attached to the ample surface area provided by hydroxyapatite scaffolds. The implementation of a hydroxyapatite scaffold did not influence colony formation capacity, percentages of cell type specific fractions, gene expression, cell viability or metabolic turnover when compared to control cells cultured in bioreactors without scaffolds. In conclusion, three-dimensional perfusion bioreactor culture enables long-term maintenance of primary human bone marrow cells, with hydroxyapatite scaffolds providing an in vivo-like scaffold for three-dimensional culture. © 2015 Wiley Periodicals, Inc.

Mice with chimeric livers are an improved model for human lipoprotein metabolism.

PubMed

Ellis, Ewa C S; Naugler, Willscott Edward; Nauglers, Scott; Parini, Paolo; Mörk, Lisa-Mari; Jorns, Carl; Zemack, Helen; Sandblom, Anita Lövgren; Björkhem, Ingemar; Ericzon, Bo-Göran; Wilson, Elizabeth M; Strom, Stephen C; Grompe, Markus

2013-01-01

Rodents are poor model for human hyperlipidemias because total cholesterol and low density lipoprotein levels are very low on a normal diet. Lipoprotein metabolism is primarily regulated by hepatocytes and we therefore assessed whether chimeric mice extensively repopulated with human cells can model human lipid and bile acid metabolism. FRG [ F ah(-/-) R ag2(-/-)Il2r g (-/-)]) mice were repopulated with primary human hepatocytes. Serum lipoprotein lipid composition and distribution (VLDL, LDL, and HDL) was analyzed by size exclusion chromatography. Bile was analyzed by LC-MS or by GC-MS. RNA expression levels were measured by quantitative RT-PCR. Chimeric mice displayed increased LDL and VLDL fractions and a lower HDL fraction compared to wild type, thus significantly shifting the ratio of LDL/HDL towards a human profile. Bile acid analysis revealed a human-like pattern with high amounts of cholic acid and deoxycholic acid (DCA). Control mice had only taurine-conjugated bile acids as expcted, but highly repopulated mice had glycine-conjugated cholic acid as found in human bile. RNA levels of human genes involved in bile acid synthesis including CYP7A1, and CYP27A1 were significantly upregulated as compared to human control liver. However, administration of recombinant hFGF19 restored human CYP7A1 levels to normal. Humanized-liver mice showed a typical human lipoprotein profile with LDL as the predominant lipoprotein fraction even on a normal diet. The bile acid profile confirmed presence of an intact enterohepatic circulation. Although bile acid synthesis was deregulated in this model, this could be fully normalized by FGF19 administration. Taken together these data indicate that chimeric FRG-mice are a useful new model for human lipoprotein and bile-acid metabolism.

Successful Establishment of Plasmids R1 and pMV158 in a New Host Requires the Relief of the Transcriptional Repression of Their Essential rep Genes

PubMed Central

Ruiz-Masó, José Á.; Luengo, Luis M.; Moreno-Córdoba, Inmaculada; Díaz-Orejas, Ramón; del Solar, Gloria

2017-01-01

Although differing in size, encoded traits, host range, and replication mechanism, both narrow-host-range theta-type conjugative enterobacterial plasmid R1 and promiscuous rolling-circle-type mobilizable streptococcal plasmid pMV158 encode a transcriptional repressor protein, namely CopB in R1 and CopG in pMV158, involved in replication control. The gene encoding CopB or CopG is cotranscribed with a downstream gene that encodes the replication initiator Rep protein of the corresponding plasmid. However, whereas CopG is an auto-repressor that inhibits transcription of the entire copG-repB operon, CopB is expressed constitutively and represses a second, downstream promoter that directs transcription of repA. As a consequence of the distinct regulatory pathways implied by CopB and CopG, these repressor proteins play a different role in control of plasmid replication during the steady state: while CopB has an auxiliary role by keeping repressed the regulated promoter whenever the plasmid copy number is above a low threshold, CopG plays a primary role by acting coordinately with RNAII. Here, we have studied the role of the regulatory circuit mediated by these transcriptional repressors during the establishment of these two plasmids in a new host cell, and found that excess Cop repressor molecules in the recipient cell result in a severe decrease in the frequency and/or the velocity of appearance of transformant colonies for the cognate plasmid but not for unrelated plasmids. Using the pMV158 replicon as a model system, together with highly sensitive real-time qPCR and inverse PCR methods, we have also analyzed the effect of CopG on the kinetics of repopulation of the plasmid in Streptococcus pneumoniae. We show that, whereas in the absence of CopG pMV158 repopulation occurs mainly during the first 45 min following plasmid transfer, the presence of the transcriptional repressor in the recipient cell severely impairs the replicon repopulation and makes the plasmid replicate at approximately the same rate as the chromosome at any time after transformation, which results in maximal plasmid loss rate in the absence of selection. Overall, these findings indicate that unrepressed activity of the Cop-regulated promoter is crucial for the successful colonization of the recipient bacterial cells by the plasmid. PMID:29250051

Embryonic attenuated Wnt/β-catenin signaling defines niche location and long-term stem cell fate in hair follicle

PubMed Central

Xu, Zijian; Wang, Wenjie; Jiang, Kaiju; Yu, Zhou; Huang, Huanwei; Wang, Fengchao; Zhou, Bin; Chen, Ting

2015-01-01

Long-term adult stem cells sustain tissue regeneration throughout the lifetime of an organism. They were hypothesized to originate from embryonic progenitor cells that acquire long-term self-renewal ability and multipotency at the end of organogenesis. The process through which this is achieved often remains unclear. Here, we discovered that long-term hair follicle stem cells arise from embryonic progenitor cells occupying a niche location that is defined by attenuated Wnt/β-catenin signaling. Hair follicle initiation is marked by placode formation, which depends on the activation of Wnt/β-catenin signaling. Soon afterwards, a region with attenuated Wnt/β-catenin signaling emerges in the upper follicle. Embryonic progenitor cells residing in this region gain expression of adult stem cell markers and become definitive long-term hair follicle stem cells at the end of organogenesis. Attenuation of Wnt/β-catenin signaling is a prerequisite for hair follicle stem cell specification because it suppresses Sox9, which is required for stem cell formation. DOI: http://dx.doi.org/10.7554/eLife.10567.001 PMID:26653852

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

Can Stem Cells be Used to Generate New Lungs? Ex Vivo Lung Bioengineering with Decellularized Whole Lung Scaffolds

PubMed Central

Wagner, Darcy E.; Bonvillain, Ryan W.; Jensen, Todd J.; Girard, Eric D.; Bunnell, Bruce A.; Finck, Christine M.; Hoffman, Andrew M.; Weiss, Daniel J.

2013-01-01

For patients with end-stage lung diseases, lung transplantation is the only available therapeutic option. However, the number of suitable donor lungs is insufficient and lung transplants are complicated by significant graft failure and complications of immunosuppressive regimens. An alternative to classic organ replacement is desperately needed. Engineering of bioartificial organs using either natural or synthetic scaffolds is an exciting new potential option for generation of functional pulmonary tissue for human clinical application. Natural organ scaffolds can be generated by decellularization of native tissues; these acellular scaffolds retain the native organ ultrastructure and can be seeded with autologous cells toward the goal of regenerating functional tissues. Several decellularization strategies have been employed for lung, however, there is no consensus on the optimal approach. A variety of cell types have been investigated as potential candidates for effective recellularization of acellular lung scaffolds. Candidate cells that might be best utilized are those which can be easily and reproducibly isolated, expanded in vitro, seeded onto decellularized matrices, induced to differentiate into pulmonary lineage cells, and which survive to functional maturity. Whole lung cell suspensions, endogenous progenitor cells, embryonic and adult stem cells, and induced pluripotent stem (iPS) cells have been investigated for their applicability to repopulate acellular lung matrices. Ideally, patient-derived autologous cells would be used for lung recellularization as they have the potential to reduce the need for post-transplant immunosuppression. Several studies have performed transplantation of rudimentary bioengineered lung scaffolds in animal models with limited, short-term functionality but much further study is needed. PMID:23614471

α6β1- and αV-integrins are required for long-term self-renewal of murine embryonic stem cells in the absence of LIF.

PubMed

Cattavarayane, Sandhanakrishnan; Palovuori, Riitta; Tanjore Ramanathan, Jayendrakishore; Manninen, Aki

2015-02-27

The growth properties and self-renewal capacity of embryonic stem (ES) cells are regulated by their immediate microenvironment such as the extracellular matrix (ECM). Integrins, a central family of cellular ECM receptors, have been implicated in these processes but their specific role in ES cell self-renewal remains unclear. Here we have studied the effects of different ECM substrates and integrins in mouse ES cells in the absence of Leukemia Inhibitory Factor (LIF) using short-term assays as well as long-term cultures. Removal of LIF from ES cell culture medium induced morphological differentiation of ES cells into polarized epistem cell-like cells. These cells maintained epithelial morphology and expression of key stemness markers for at least 10 passages in the absence of LIF when cultured on laminin, fibronectin or collagen IV substrates. The specific functional roles of α6-, αV- and β1-integrin subunits were dissected using stable lentivirus-mediated RNAi methodology. β1-integrins were required for ES cell survival in long-term cultures and for the maintenance of stem cell marker expression. Inhibition of α6-integrin expression compromised self-renewal on collagen while αV-integrins were required for robust ES cell adhesion on laminin. Analysis of the stemness marker expression revealed subtle differences between α6- and αV-depleted ES cells but the expression of both was required for optimal self-renewal in long-term ES cell cultures. In the absence of LIF, long-term ES cell cultures adapt an epistem cell-like epithelial phenotype and retain the expression of multiple stem cell markers. Long-term maintenance of such self-renewing cultures depends on the expression of β1-, α6- and αV-integrins.

Loss of the Wnt receptor frizzled 7 in the mouse gastric epithelium is deleterious and triggers rapid repopulation in vivo.

PubMed

Flanagan, Dustin J; Barker, Nick; Nowell, Cameron; Clevers, Hans; Ernst, Matthias; Phesse, Toby J; Vincan, Elizabeth

2017-08-01

The gastric epithelium consists of tubular glandular units, each containing several differentiated cell types, and populations of stem cells, which enable the stomach to secrete the acid, mucus and various digestive enzymes required for its function. Very little is known about which cell signalling pathways are required for homeostasis of the gastric epithelium. Many diseases, such as cancer, arise as a result of deregulation of signalling pathways that regulate homeostasis of the diseased organ. Therefore, it is important to understand the biology of how normal conditions are maintained in a tissue to help inform the mechanisms driving disease in that same tissue, and to identify potential points of therapeutic intervention. Wnt signalling regulates several cell functions, including proliferation, differentiation and migration, and plays a crucial role during homeostasis of several tissues, including the intestinal epithelium. Wnt3a is required in the culture medium of gastric organoids, suggesting it is also important for the homeostasis of the gastric epithelium, but this has not been investigated in vivo Here, we show that the Wnt receptor frizzled 7 (Fzd7), which is required for the homeostasis of the intestine, is expressed in the gastric epithelium and is required for gastric organoid growth. Gastric-specific loss of Fzd7 in the adult gastric epithelium of mice is deleterious and triggers rapid epithelial repopulation, which we believe is the first observation of this novel function for this tissue. Taken together, these data provide functional evidence of a crucial role for Wnt signalling, via the Fzd7 receptor, during homeostasis of the gastric epithelium. © 2017. Published by The Company of Biologists Ltd.

Loss of the Wnt receptor frizzled 7 in the mouse gastric epithelium is deleterious and triggers rapid repopulation in vivo

PubMed Central

2017-01-01

ABSTRACT The gastric epithelium consists of tubular glandular units, each containing several differentiated cell types, and populations of stem cells, which enable the stomach to secrete the acid, mucus and various digestive enzymes required for its function. Very little is known about which cell signalling pathways are required for homeostasis of the gastric epithelium. Many diseases, such as cancer, arise as a result of deregulation of signalling pathways that regulate homeostasis of the diseased organ. Therefore, it is important to understand the biology of how normal conditions are maintained in a tissue to help inform the mechanisms driving disease in that same tissue, and to identify potential points of therapeutic intervention. Wnt signalling regulates several cell functions, including proliferation, differentiation and migration, and plays a crucial role during homeostasis of several tissues, including the intestinal epithelium. Wnt3a is required in the culture medium of gastric organoids, suggesting it is also important for the homeostasis of the gastric epithelium, but this has not been investigated in vivo. Here, we show that the Wnt receptor frizzled 7 (Fzd7), which is required for the homeostasis of the intestine, is expressed in the gastric epithelium and is required for gastric organoid growth. Gastric-specific loss of Fzd7 in the adult gastric epithelium of mice is deleterious and triggers rapid epithelial repopulation, which we believe is the first observation of this novel function for this tissue. Taken together, these data provide functional evidence of a crucial role for Wnt signalling, via the Fzd7 receptor, during homeostasis of the gastric epithelium. PMID:28600348

Stem cells--clinical application and perspectives.

PubMed

Brehm, Michael; Zeus, Tobias; Strauer, Bodo Eckehard

2002-11-01

Augmentation of myocardial performance in experimental models of therapeutic infarction and heart failure has been achieved by transplantation of exogenous cells into damaged myocardium. The quest for suitable donor cells has prompted research into the use of both embryonic stem cells and adult somatic stem cells. Recently, there has been a growing body of evidence that multipotent somatic stem cells in adult bone marrow exhibit tremendous functional plasticity and can reprogram in a new environmental tissue niche to give rise to cell lineages specific for new organ site. This phenomenon has made huge impact on myocardial biology, while multipotent adult bone marrow hematopoeitic stem cells and mesechymal stem cells can repopulate infarcted rodent myocardium and differentiate into both cardiomyocytes and new blood vessels. These data, coupled with the identification of a putative primitive cardiac stem cell population in the adult human heart, may open the way for novel therapeutic modalities for enhancing myocardial performance and treating heart failure.

Mammary Stem Cells and Breast Cancer Stem Cells: Molecular Connections and Clinical Implications.

PubMed

Celià-Terrassa, Toni

2018-05-04

Cancer arises from subpopulations of transformed cells with high tumor initiation and repopulation ability, known as cancer stem cells (CSCs), which share many similarities with their normal counterparts. In the mammary gland, several studies have shown common molecular regulators between adult mammary stem cells (MaSCs) and breast cancer stem cells (bCSCs). Cell plasticity and self-renewal are essential abilities for MaSCs to maintain tissue homeostasis and regenerate the gland after pregnancy. Intriguingly, these properties are similarly executed in breast cancer stem cells to drive tumor initiation, tumor heterogeneity and recurrence after chemotherapy. In addition, both stem cell phenotypes are strongly influenced by external signals from the microenvironment, immune cells and supportive specific niches. This review focuses on the intrinsic and extrinsic connections of MaSC and bCSCs with clinical implications for breast cancer progression and their possible therapeutic applications.

The impact of MIS-3 climate events at the transition from Neanderthals to modern humans in Europe

NASA Astrophysics Data System (ADS)

Staubwasser, M.; Dragusin, V.; Assonov, S.; Ersek, V.; Hoffmann, D.; Veres, D.; Onac, B. P.

2017-12-01

We report on last glacial stable C and O isotope records from two U-Th dated speleothems from Romania. The southerly record (Ascunsa Cave, South Carpathians) from the Danube region matches the pacing and relative change in amplitude of the Greenland ice temperature record at 30-50 ka BP as well as the abundance of coastal winter sea ice in the Black Sea. The northerly record (Tausoare Cave, East Carpathians) in parts shares the pacing of events with the Greenland or the southern Romanian record, but best matches northern Black Sea summer season temperature change. Heinrich events do not stand out in either record, but the temperature amplitudes of Greenland stadials and Black Sea records are generally reproduced. Based on similarity with the Black Sea we interpret the combined two speleothem records in terms of seasonal temperature change in central Eastern Europe. A climatic influence on the transition from Neanderthals to modern humans has long been suspected. However, the diachronous and spatially complex archaeologic succession across the Middle-Upper Paleolithic (MUPL) in Europe ( 38 - 48 ka) is difficult to reconcile with the millennial-scale pacing of northern hemisphere paleoclimate. Two extreme cold events at 44.0-43.3 recorded and 40.7-39.8 ka in the speleothems bracket the dates of the first known appearance of modern humans - the Aurignacian complex - and the disappearance of Neanderthals from most of Europe. These cold events are coeval with Greenland Stadials GS-12 and GS-10. The speleothem records generally match the paleosol/loess succession from central Europe across the MUPL. The combined record suggests that permafrost advance may have made central Europe uninhabitable at least during winter. The combined paleoclimate and archaeologic records suggest that depopulation-repopulation cycles may have occurred during and after each cold event. Repopulation of central Europe geographically favored the modern human Aurignacians from SE Europe.

CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

PubMed Central

Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

2010-01-01

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

Photostable fluorescent organic dots with aggregation-induced emission (AIE dots) for noninvasive long-term cell tracing

NASA Astrophysics Data System (ADS)

Li, Kai; Qin, Wei; Ding, Dan; Tomczak, Nikodem; Geng, Junlong; Liu, Rongrong; Liu, Jianzhao; Zhang, Xinhai; Liu, Hongwei; Liu, Bin; Tang, Ben Zhong

2013-01-01

Long-term noninvasive cell tracing by fluorescent probes is of great importance to life science and biomedical engineering. For example, understanding genesis, development, invasion and metastasis of cancerous cells and monitoring tissue regeneration after stem cell transplantation require continual tracing of the biological processes by cytocompatible fluorescent probes over a long period of time. In this work, we successfully developed organic far-red/near-infrared dots with aggregation-induced emission (AIE dots) and demonstrated their utilities as long-term cell trackers. The high emission efficiency, large absorptivity, excellent biocompatibility, and strong photobleaching resistance of the AIE dots functionalized by cell penetrating peptides derived from transactivator of transcription proteins ensured outstanding long-term noninvasive in vitro and in vivo cell tracing. The organic AIE dots outperform their counterparts of inorganic quantum dots, opening a new avenue in the development of fluorescent probes for following biological processes such as carcinogenesis.

Long-Term Dynamics of Autonomous Fractional Differential Equations

NASA Astrophysics Data System (ADS)

Liu, Tao; Xu, Wei; Xu, Yong; Han, Qun

This paper aims to investigate long-term dynamic behaviors of autonomous fractional differential equations with effective numerical method. The long-term dynamic behaviors predict where systems are heading after long-term evolution. We make some modification and transplant cell mapping methods to autonomous fractional differential equations. The mapping time duration of cell mapping is enlarged to deal with the long memory effect. Three illustrative examples, i.e. fractional Lotka-Volterra equation, fractional van der Pol oscillator and fractional Duffing equation, are studied with our revised generalized cell mapping method. We obtain long-term dynamics, such as attractors, basins of attraction, and saddles. Compared with some existing stability and numerical results, the validity of our method is verified. Furthermore, we find that the fractional order has its effect on the long-term dynamics of autonomous fractional differential equations.

Analysis of the immune system of multiple myeloma patients achieving long-term disease control by multidimensional flow cytometry

PubMed Central

Pessoa de Magalhães, Roberto J.; Vidriales, María-Belén; Paiva, Bruno; Fernandez-Gimenez, Carlos; García-Sanz, Ramón; Mateos, Maria-Victoria; Gutierrez, Norma C.; Lecrevisse, Quentin; Blanco, Juan F; Hernández, Jose; de las Heras, Natalia; Martinez-Lopez, Joaquin; Roig, Monica; Costa, Elaine Sobral; Ocio, Enrique M.; Perez-Andres, Martin; Maiolino, Angelo; Nucci, Marcio; De La Rubia, Javier; Lahuerta, Juan-Jose; San-Miguel, Jesús F.; Orfao, Alberto

2013-01-01

Multiple myeloma remains largely incurable. However, a few patients experience more than 10 years of relapse-free survival and can be considered as operationally cured. Interestingly, long-term disease control in multiple myeloma is not restricted to patients with a complete response, since some patients revert to having a profile of monoclonal gammopathy of undetermined significance. We compared the distribution of multiple compartments of lymphocytes and dendritic cells in the bone marrow and peripheral blood of multiple myeloma patients with long-term disease control (n=28), patients with newly diagnosed monoclonal gammopathy of undetermined significance (n=23), patients with symptomatic multiple myeloma (n=23), and age-matched healthy adults (n=10). Similarly to the patients with monoclonal gammopathy of undetermined significance and symptomatic multiple myeloma, patients with long-term disease control showed an expansion of cytotoxic CD8+ T cells and natural killer cells. However, the numbers of bone marrow T-regulatory cells were lower in patients with long-term disease control than in those with symptomatic multiple myeloma. It is noteworthy that B cells were depleted in patients with monoclonal gammopathy of undetermined significance and in those with symptomatic multiple myeloma, but recovered in both the bone marrow and peripheral blood of patients with long-term disease control, due to an increase in normal bone marrow B-cell precursors and plasma cells, as well as pre-germinal center peripheral blood B cells. The number of bone marrow dendritic cells and tissue macrophages differed significantly between patients with long-term disease control and those with symptomatic multiple myeloma, with a trend to cell count recovering in the former group of patients towards levels similar to those found in healthy adults. In summary, our results indicate that multiple myeloma patients with long-term disease control have a constellation of unique immune changes favoring both immune cytotoxicity and recovery of B-cell production and homing, suggesting improved immune surveillance. PMID:22773604

Method and Apparatus for a Miniature Bioreactor System for Long-Term Cell Culture

NASA Technical Reports Server (NTRS)

Kleis, Stanley J. (Inventor); Geffert, Sandra K. (Inventor); Gonda, Steve R. (Inventor)

2015-01-01

A bioreactor and method that permits continuous and simultaneous short, moderate, or long term cell culturing of one or more cell types or tissue in a laminar flow configuration is disclosed, where the bioreactor supports at least two laminar flow zones, which are isolated by laminar flow without the need for physical barriers between the zones. The bioreactors of this invention are ideally suited for studying short, moderate and long term studies of cell cultures and the response of cell cultures to one or more stressors such as pharmaceuticals, hypoxia, pathogens, or any other stressor. The bioreactors of this invention are also ideally suited for short, moderate or long term cell culturing with periodic cell harvesting and/or medium processing for secreted cellular components.

Implications of delayed initiation of radiotherapy: accelerated repopulation after induction chemotherapy for stage III non-small cell lung cancer.

PubMed

Chen, Chien P; Weinberg, Vivian K; Jahan, Thierry M; Jablons, David M; Yom, Sue S

2011-11-01

For patients with stage III non-small cell lung cancer treated with induction chemotherapy (ICT), delayed initiation of subsequent radiotherapy (RT) may allow for repopulation in the interval between treatment modalities and during the early phase of RT. We quantified the impact of postinduction RT timing by evaluating the pace of tumor regrowth. Institutionally approved retrospective review identified 21 analyzable patients with stage III non-small cell lung cancer who had platinum-based ICT followed by RT+/- chemotherapy from 2002 to 2009. Radiographic response was determined by RECIST criteria and the volume of the single largest tumor mass on the pre-ICT, post-ICT, and RT-planning computed tomography scans. After ICT, the median percent volume change from pre-ICT baseline was -41% (range -86 to +86%). By the RT-planning computed tomography scan, the median percent volume change from the post-ICT timepoint was +40% (range -11 to +311%) and the median volume change was +20 ml (range -4 to 102 ml); these changes were significant (p = 0.0002). Similar results were seen for tumor diameter. A correlation was observed between the amount of delay and degree of regrowth for percent volume (p = 0.0006) and percent diameter change (p = 0.003). A delay greater than 21 days produced greater increases in percent volume change (p = 0.002) and percent diameter (p = 0.055) than lesser delays. After ICT, tumor regrowth can occur within a few weeks. Radiation treatment planning should begin as soon as possible after the administration of ICT to maximize the benefits of cytoreduction.

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.

rhEPO Enhances Cellular Anti-oxidant Capacity to Protect Long-Term Cultured Aging Primary Nerve Cells.

PubMed

Wang, Huqing; Fan, Jiaxin; Chen, Mengyi; Yao, Qingling; Gao, Zhen; Zhang, Guilian; Wu, Haiqin; Yu, Xiaorui

2017-08-01

Erythropoietin (EPO) may protect the nervous system of animals against aging damage, making it a potential anti-aging drug for the nervous system. However, experimental evidence from natural aging nerve cell models is lacking, and the efficacy of EPO and underlying mechanism of this effect warrant further study. Thus, the present study used long-term cultured primary nerve cells to successfully mimic the natural aging process of nerve cells. Starting on the 11th day of culture, cells were treated with different concentrations of recombinant human erythropoietin (rhEPO). Using double immunofluorescence labeling, we found that rhEPO significantly improved the morphology of long-term cultured primary nerve cells and increased the total number of long-term cultured primary cells. However, rhEPO did not improve the ratio of nerve cells. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure nerve cell activity and showed that rhEPO significantly improved the activity of long-term cultured primary nerve cells. Moreover, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double immunofluorescence labeling flow cytometry revealed that rhEPO reduced the apoptotic rate of long-term cultured primary nerve cells. Senescence-associated β-galactosidase (SA-β-gal) immunohistochemistry staining showed that rhEPO significantly reduced the aging rate of long-term cultured primary nerve cells. Immunochemistry revealed that rhEPO enhanced intracellular superoxide dismutase (SOD) activity and glutathione (GSH) abundance and reduced the intracellular malondialdehyde (MDA) level. In addition, this effect depended on the dose, was maximized at a dose of 100 U/ml and was more pronounced than that of vitamin E. In summary, this study finds that rhEPO protects long-term cultured primary nerve cells from aging in a dose-dependent manner. The mechanism of this effect may be associated with the enhancement of the intracellular anti-oxidant capacity. These findings provide a theoretical basis to further the anti-aging mechanism of EPO in the nervous system, and they provide experimental evidence at the cellular level for the clinical application of EPO to protect the nervous system from aging.

Long-Term Live Cell Imaging of Cell Migration: Effects of Pathogenic Fungi on Human Epithelial Cell Migration.

PubMed

Wöllert, Torsten; Langford, George M

2016-01-01

Long-term live cell imaging was used in this study to determine the responses of human epithelial cells to pathogenic biofilms formed by Candida albicans. Epithelial cells of the skin represent the front line of defense against invasive pathogens such as C. albicans but under certain circumstances, especially when the host's immune system is compromised, the skin barrier is breached. The mechanisms by which the fungal pathogen penetrates the skin and invade the deeper layers are not fully understood. In this study we used keratinocytes grown in culture as an in vitro model system to determine changes in host cell migration and the actin cytoskeleton in response to virulence factors produced by biofilms of pathogenic C. albicans. It is clear that changes in epithelial cell migration are part of the response to virulence factors secreted by biofilms of C. albicans and the actin cytoskeleton is the downstream effector that mediates cell migration. Our goal is to understand the mechanism by which virulence factors hijack the signaling pathways of the actin cytoskeleton to alter cell migration and thereby invade host tissues. To understand the dynamic changes of the actin cytoskeleton during infection, we used long-term live cell imaging to obtain spatial and temporal information of actin filament dynamics and to identify signal transduction pathways that regulate the actin cytoskeleton and its associated proteins. Long-term live cell imaging was achieved using a high resolution, multi-mode epifluorescence microscope equipped with specialized light sources, high-speed cameras with high sensitivity detectors, and specific biocompatible fluorescent markers. In addition to the multi-mode epifluorescence microscope, a spinning disk confocal long-term live cell imaging system (Olympus CV1000) equipped with a stage incubator to create a stable in vitro environment for long-term real-time and time-lapse microscopy was used. Detailed descriptions of these two long-term live cell imaging systems are provided.

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 potential approach to clinical application for HPC peripheral mobilization.

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.

Modeling the Treatment of Glioblastoma Multiforme and Cancer Stem Cells with Ordinary Differential Equations.

PubMed

Abernathy, Kristen; Burke, Jeremy

2016-01-01

Despite improvements in cancer therapy and treatments, tumor recurrence is a common event in cancer patients. One explanation of recurrence is that cancer therapy focuses on treatment of tumor cells and does not eradicate cancer stem cells (CSCs). CSCs are postulated to behave similar to normal stem cells in that their role is to maintain homeostasis. That is, when the population of tumor cells is reduced or depleted by treatment, CSCs will repopulate the tumor, causing recurrence. In this paper, we study the application of the CSC Hypothesis to the treatment of glioblastoma multiforme by immunotherapy. We extend the work of Kogan et al. (2008) to incorporate the dynamics of CSCs, prove the existence of a recurrence state, and provide an analysis of possible cancerous states and their dependence on treatment levels.

Late stage infection in sleeping sickness.

PubMed

Wolburg, Hartwig; Mogk, Stefan; Acker, Sven; Frey, Claudia; Meinert, Monika; Schönfeld, Caroline; Lazarus, Michael; Urade, Yoshihiro; Kubata, Bruno Kilunga; Duszenko, Michael

2012-01-01

At the turn of the 19(th) century, trypanosomes were identified as the causative agent of sleeping sickness and their presence within the cerebrospinal fluid of late stage sleeping sickness patients was described. However, no definitive proof of how the parasites reach the brain has been presented so far. Analyzing electron micrographs prepared from rodent brains more than 20 days after infection, we present here conclusive evidence that the parasites first enter the brain via the choroid plexus from where they penetrate the epithelial cell layer to reach the ventricular system. Adversely, no trypanosomes were observed within the parenchyma outside blood vessels. We also show that brain infection depends on the formation of long slender trypanosomes and that the cerebrospinal fluid as well as the stroma of the choroid plexus is a hostile environment for the survival of trypanosomes, which enter the pial space including the Virchow-Robin space via the subarachnoid space to escape degradation. Our data suggest that trypanosomes do not intend to colonize the brain but reside near or within the glia limitans, from where they can re-populate blood vessels and disrupt the sleep wake cycles.

TH-E-BRF-01: Exploiting Tumor Shrinkage in Split-Course Radiotherapy

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

Unkelbach, J; Craft, D; Hong, T

2014-06-15

Purpose: In split-course radiotherapy, a patient is treated in several stages separated by weeks or months. This regimen has been motivated by radiobiological considerations. However, using modern image-guidance, it also provides an approach to reduce normal tissue dose by exploiting tumor shrinkage. In this work, we consider the optimal design of split-course treatments, motivated by the clinical management of large liver tumors for which normal liver dose constraints prohibit the administration of an ablative radiation dose in a single treatment. Methods: We introduce a dynamic tumor model that incorporates three factors: radiation induced cell kill, tumor shrinkage, and tumor cellmore » repopulation. The design of splitcourse radiotherapy is formulated as a mathematical optimization problem in which the total dose to the liver is minimized, subject to delivering the prescribed dose to the tumor. Based on the model, we gain insight into the optimal administration of radiation over time, i.e. the optimal treatment gaps and dose levels. Results: We analyze treatments consisting of two stages in detail. The analysis confirms the intuition that the second stage should be delivered just before the tumor size reaches a minimum and repopulation overcompensates shrinking. Furthermore, it was found that, for a large range of model parameters, approximately one third of the dose should be delivered in the first stage. The projected benefit of split-course treatments in terms of liver sparing depends on model assumptions. However, the model predicts large liver dose reductions by more than a factor of two for plausible model parameters. Conclusion: The analysis of the tumor model suggests that substantial reduction in normal tissue dose can be achieved by exploiting tumor shrinkage via an optimal design of multi-stage treatments. This suggests taking a fresh look at split-course radiotherapy for selected disease sites where substantial tumor regression translates into reduced target volumes.« less

Micro patterned surfaces allow long-term digital holographic microscopy live cell imaging

NASA Astrophysics Data System (ADS)

Mues, Sarah; Lilge, Inga; Schönherr, Holger; Kemper, Björn; Schnekenburger, Jürgen

2017-07-01

During long-term imaging, cells move out of the field of view. We have generated functionalized substrates containing rectangular areas, which were capable in keeping cells over the whole observation period.

Ageing and long-term CD4 cell count trends in HIV-positive patients with 5 years or more combination antiretroviral therapy experience.

PubMed

Wright, S T; Petoumenos, K; Boyd, M; Carr, A; Downing, S; O'Connor, C C; Grotowski, M; Law, M G

2013-04-01

The aim of this study was to describe the long-term changes in CD4 cell counts beyond 5 years of combination antiretroviral therapy (cART). If natural ageing leads to a long-term decline in the immune system via low-grade chronic immune activation/inflammation, then one might expect to see a greater or earlier decline in CD4 counts in older HIV-positive patients with increasing duration of cART. Retrospective and prospective data were examined from long-term virologically stable HIV-positive adults from the Australian HIV Observational Database. We estimated mean CD4 cell count changes following the completion of 5 years of cART using linear mixed models. A total of 37 916 CD4 measurements were observed for 892 patients over a combined total of 9753 patient-years. Older patients (> 50 years old) at cART initiation had estimated mean (95% confidence interval) changes in CD4 counts by year-5 CD4 count strata (< 500, 500-750 and > 750 cells/μL) of 14 (7 to 21), 3 (-5 to 11) and -6 (-17 to 4) cells/μL/year. Of the CD4 cell count rates of change estimated, none were indicative of long-term declines in CD4 cell counts. Our results suggest that duration of cART and increasing age do not result in decreasing mean changes in CD4 cell counts for long-term virologically suppressed patients, indicating that the level of immune recovery achieved during the first 5 years of treatment is sustained through long-term cART. © 2012 British HIV Association.

Ageing & long-term CD4 cell count trends in HIV-positive patients with 5 years or more combination antiretroviral therapy experience

PubMed Central

WRIGHT, ST; PETOUMENOS, K; BOYD, M; CARR, A; DOWNING, S; O’CONNOR, CC; GROTOWSKI, M; LAW, MG

2012-01-01

Background The aim of this analysis is to describe the long-term changes in CD4 cell counts beyond 5 years of combination antiretroviral therapy (cART). If natural ageing leads to a long-term decline in the immune system via low-grade chronic immune activation/inflammation, then one might expect to see a greater or earlier decline in CD4 counts in older HIV-positive patients with increasing duration of cART. Methods Retrospective and prospective data were examined from long-term virologically stable HIV-positive adults from the Australian HIV Observational Database. We estimated mean CD4 cell counts changes following the completion of 5 years of cART using linear mixed models. Results A total of 37,916 CD4 measurements were observed for 892 patients over a combined total of 9,753 patient years. Older patients (>50 years) at cART initiation had estimated mean(95% confidence interval) change in CD4 counts by Year-5 CD4 count strata (<500, 501–750 and >750 cells/μL) of 14(7 to 21), 3(−5 to 11) and −6(−17 to 4) cells/μL/year. Of the CD4 cell count rates of change estimated, none were indicative of long-term declines in CD4 cell counts. Conclusions Our results suggest that duration of cART and increasing age does not result in decreasing mean changes in CD4 cell counts for long-term virologically suppressed patients. Indicating that level of immune recovery achieved during the first 5 years of treatment are sustained through long-term cART. PMID:23036045

Ex vivo expanded cord blood cells provide rapid engraftment in fetal sheep but lack long-term engrafting potential.

PubMed

McNiece, Ian K; Almeida-Porada, Graça; Shpall, Elizabeth J; Zanjani, Esmail

2002-06-01

Cord blood (CB) products are becoming routinely used in unrelated allogeneic transplantation for smaller pediatric patients. Because of the low numbers of cells in CB compared to bone marrow or peripheral blood progenitor cells, their use is more limited in larger adults. Therefore, we developed ex vivo expansion conditions for CB and currently are transplanting ex vivo expanded CB products to patients receiving high-dose chemotherapy. As there is concern that ex vivo expansion may exhaust long-term engrafting cells, the current clinical protocols consist of both an expanded fraction and an unexpanded fraction. To determine the effect of expansion culture on long-term engrafting cells, we evaluated the short- and long-term engrafting potential of ex vivo expanded CB using a fetal sheep xenogeneic transplant model. CD 34(+) cells were selected from CB products and cultured in a two-step procedure in the presence of stem cell factor, megakaryocyte growth and differentiation factor, and granulocyte colony-stimulating factor for 14 days. Starting cells (CD34(+) cells), and cultured cells (day 7 and day 14 cells) were transplanted in 60-day-old fetal sheep and evaluated at various time points post transplant for the presence of human cells. Long-term engrafting cells were assessed by serial passage into secondary and tertiary recipients. Day 14 expanded CB cells provided more rapid engraftment than either the day 7 expanded cells or the day 0 cells; however, this engraftment was transient, and no human cells were detectable at 16 months post transplant in the animals that received the day 14 expanded cells. Day 0 cells had engrafted animals at 2 months post transplant and both the day 0 and day 7 cells persisted to 16 months or longer. In the secondary animals, the day 0 and day 7 cells engrafted equivalently at 3 months post transplant; however, no secondary engraftment resulted from the day 14 cells. The levels of engraftment in secondary animals receiving day 7 cells decreased with time to barely detectable levels at 12 months post transplant. Ex vivo expansion of CB CD34(+) cells under the conditions described results in the generation of increased mature cells and progenitors that are capable of more rapid engraftment in fetal sheep compared to unexpanded CB CD34(+) cells. The expanded cells engrafted primary sheep but lacked secondary and tertiary engrafting potential. These studies demonstrate that although ex vivo expanded cells may be able to provide rapid short-term engraftment, the long-term potential of expanded grafts may be compromised. Therefore, clinical protocols may require transplantation of two fractions of cells, an expanded CB graft to provide rapid short-term engraftment and an unmanipulated fraction of CB graft to provide stem cells for long-term engraftment.

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

Meijne, E.I.; van der Winden-van Groenewegen, R.J.; Ploemacher, R.E.

The sensitivity for x-irradiation of a series of hematopoietic stem cell populations has been determined. The most primitive cells identified, cells with marrow-repopulating ability (MRA), showed the highest degree of radioresistance. These MRA cells which generate many secondary day-twelve spleen colony-forming units (MRA(CFU-S-12)) or colony-forming units in culture (MRA(CFU-C)) in the marrow of primary recipients had Do values equal to 1.18 and 1.13 Gy, respectively. The more mature CFU-S-12 had intermediate radiosensitivity (Do = 0.94 Gy), whereas the less primitive CFU-S-7 were the most radiosensitive (Do = 0.71 Gy). The in vitro colony-forming precursor cells (CFU-C) showed low radiosensitivity. Thesemore » data clearly show that the most primitive hematopoietic stem cell measured is less sensitive to ionizing radiation than generally has been assumed on the basis of measurements on CFU-S-7 or CFU-S-12.« less

Osteoclasts control reactivation of dormant myeloma cells by remodelling the endosteal niche

PubMed Central

Lawson, Michelle A.; McDonald, Michelle M.; Kovacic, Natasa; Hua Khoo, Weng; Terry, Rachael L.; Down, Jenny; Kaplan, Warren; Paton-Hough, Julia; Fellows, Clair; Pettitt, Jessica A.; Neil Dear, T.; Van Valckenborgh, Els; Baldock, Paul A.; Rogers, Michael J.; Eaton, Colby L.; Vanderkerken, Karin; Pettit, Allison R.; Quinn, Julian M. W.; Zannettino, Andrew C. W.; Phan, Tri Giang; Croucher, Peter I.

2015-01-01

Multiple myeloma is largely incurable, despite development of therapies that target myeloma cell-intrinsic pathways. Disease relapse is thought to originate from dormant myeloma cells, localized in specialized niches, which resist therapy and repopulate the tumour. However, little is known about the niche, and how it exerts cell-extrinsic control over myeloma cell dormancy and reactivation. In this study, we track individual myeloma cells by intravital imaging as they colonize the endosteal niche, enter a dormant state and subsequently become activated to form colonies. We demonstrate that dormancy is a reversible state that is switched ‘on' by engagement with bone-lining cells or osteoblasts, and switched ‘off' by osteoclasts remodelling the endosteal niche. Dormant myeloma cells are resistant to chemotherapy that targets dividing cells. The demonstration that the endosteal niche is pivotal in controlling myeloma cell dormancy highlights the potential for targeting cell-extrinsic mechanisms to overcome cell-intrinsic drug resistance and prevent disease relapse. PMID:26632274

The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.

PubMed

van Galen, Peter; Kreso, Antonija; Mbong, Nathan; Kent, David G; Fitzmaurice, Timothy; Chambers, Joseph E; Xie, Stephanie; Laurenti, Elisa; Hermans, Karin; Eppert, Kolja; Marciniak, Stefan J; Goodall, Jane C; Green, Anthony R; Wouters, Bradly G; Wienholds, Erno; Dick, John E

2014-06-12

The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage. Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis. Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis. Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the co-chaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells.

Fibronectin regulates Wnt7a signaling and satellite cell expansion

PubMed Central

Bentzinger, C. Florian; Wang, Yu Xin; von Maltzahn, Julia; Soleimani, Vahab D.; Yin, Hang; Rudnicki, Michael A.

2012-01-01

SUMMARY The influence of the extracellular matrix (ECM) within the stem cell niche remains poorly understood. We found that Syndecan-4 (Sdc4) and Frizzled-7 (Fzd7) form a co-receptor complex in satellite cells and that binding of the ECM glycoprotein Fibronectin (FN) to Sdc4 stimulates the ability of Wnt7a to induce the symmetric expansion of satellite stem cells. Newly activated satellite cells dynamically remodel their niche by transient high-level expression of FN. Knockdown of FN in prospectively isolated satellite cells severely impaired their ability to repopulate the satellite cell niche. Conversely, in vivo over-expression of FN with Wnt7a dramatically stimulated the expansion of satellite stem cells in regenerating muscle. Therefore, activating satellite cells remodel their niche through autologous expression of FN that provides feedback to stimulate Wnt7a signaling through the Fzd7/Sdc4 co-receptor complex. Thus, FN and Wnt7a together regulate the homeostatic levels of satellite stem cells and satellite myogenic cells during regenerative myogenesis. PMID:23290138

Cell-to-cell movement of plastids in plants.

PubMed

Thyssen, Gregory; Svab, Zora; Maliga, Pal

2012-02-14

Our objective was to test whether or not plastids and mitochondria, the two DNA-containing organelles, move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new cell-to-cell contacts, creating an opportunity to detect cell-to-cell organelle movement between the genetically distinct plants. We initiated tissue culture from sliced graft junctions and selected for clonal lines in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids. Here, we present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. We also found that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. Our finding supports the universality of intercellular organelle trafficking and may enable development of future biotechnological applications.

Distinct Molecular Signature of Murine Fetal Liver and Adult Hematopoietic Stem Cells Identify Novel Regulators of Hematopoietic Stem Cell Function

PubMed Central

Manesia, Javed K.; Franch, Monica; Tabas-Madrid, Daniel; Nogales-Cadenas, Ruben; Vanwelden, Thomas; Van Den Bosch, Elisa; Xu, Zhuofei; Pascual-Montano, Alberto; Khurana, Satish; Verfaillie, Catherine M.

2018-01-01

During ontogeny, fetal liver (FL) acts as a major site for hematopoietic stem cell (HSC) maturation and expansion, whereas HSCs in the adult bone marrow (ABM) are largely quiescent. HSCs in the FL possess faster repopulation capacity as compared with ABM HSCs. However, the molecular mechanism regulating the greater self-renewal potential of FL HSCs has not yet extensively been assessed. Recently, we published RNA sequencing-based gene expression analysis on FL HSCs from 14.5-day mouse embryo (E14.5) in comparison to the ABM HSCs. We reanalyzed these data to identify key transcriptional regulators that play important roles in the expansion of HSCs during development. The comparison of FL E14.5 with ABM HSCs identified more than 1,400 differentially expressed genes. More than 200 genes were shortlisted based on the gene ontology (GO) annotation term “transcription.” By morpholino-based knockdown studies in zebrafish, we assessed the function of 18 of these regulators, previously not associated with HSC proliferation. Our studies identified a previously unknown role for tdg, uhrf1, uchl5, and ncoa1 in the emergence of definitive hematopoiesis in zebrafish. In conclusion, we demonstrate that identification of genes involved in transcriptional regulation differentially expressed between expanding FL HSCs and quiescent ABM HSCs, uncovers novel regulators of HSC function. PMID:27958775

Orthotopic Transplantation of Achilles Tendon Allograft in Rats

PubMed Central

Aynardi, Michael; Zahoor, Talal; Mitchell, Reed; Loube, Jeffrey; Feltham, Tyler; Manandhar, Lumanti; Paudel, Sharada; Schon, Lew; Zhang, Zijun

2018-01-01

The biology and function of orthotopic transplantation of Achilles tendon allograft are unknown. Particularly, the revitalization of Achilles allograft is a clinical concern. Achilles allografts were harvested from donor rats and stored at −80 °C. Subcutaneous adipose tissue was harvested from the would-be allograft recipient rats for isolation of mesenchymal stem cells (MSCs). MSCs were cultured with growth differentiation factor-5 (GDF-5) and applied onto Achilles allografts on the day of transplantation. After the native Achilles tendon was resected from the left hind limb of the rats, Achilles allograft, with or without autologous MSCs, was implanted and sutured with calf muscles proximally and calcaneus distally. Animal gait was recorded presurgery and postsurgery weekly. The animals were sacrificed at week 4, and the transplanted Achilles allografts were collected for biomechanical testing and histology. The operated limbs had altered gait. By week 4, the paw print intensity, stance time, and duty cycle (percentage of the stance phase in a step cycle) of the reconstructed limbs were mostly recovered to the baselines recorded before surgery. Maximum load of failure was not different between Achilles allografts, with or without MSCs, and the native tendons. The Achilles allograft supplemented with MSCs had higher cellularity than the Achilles allograft without MSCs. Deposition of fine collagen (type III) fibers was active in Achilles allograft, with or without MSCs, but it was more evenly distributed in the allografts that were incubated with MSCs. In conclusion, orthotopically transplanted Achilles allograft healed with host tissues, regained strength, and largely restored Achilles function in 4 wk in rats. It is therefore a viable option for the reconstruction of a large Achilles tendon defect. Supplementation of MSCs improved repopulation of Achilles allograft, but large animal models, with long-term follow up and cell tracking, may be required to fully appreciate the functional benefits of MSCs. PMID:29637821

Promoting long-term survival of insulin-producing cell grafts that differentiate from adipose tissue-derived stem cells to cure type 1 diabetes.

PubMed

Zhang, Shuzi; Dai, Hehua; Wan, Ni; Moore, Yolonda; Dai, Zhenhua

2011-01-01

Insulin-producing cell clusters (IPCCs) have recently been generated in vitro from adipose tissue-derived stem cells (ASCs) to circumvent islet shortage. However, it is unknown how long they can survive upon transplantation, whether they are eventually rejected by recipients, and how their long-term survival can be induced to permanently cure type 1 diabetes. IPCC graft survival is critical for their clinical application and this issue must be systematically addressed prior to their in-depth clinical trials. Here we found that IPCC grafts that differentiated from murine ASCs in vitro, unlike their freshly isolated islet counterparts, did not survive long-term in syngeneic mice, suggesting that ASC-derived IPCCs have intrinsic survival disadvantage over freshly isolated islets. Indeed, β cells retrieved from IPCC syngrafts underwent faster apoptosis than their islet counterparts. However, blocking both Fas and TNF receptor death pathways inhibited their apoptosis and restored their long-term survival in syngeneic recipients. Furthermore, blocking CD40-CD154 costimulation and Fas/TNF signaling induced long-term IPCC allograft survival in overwhelming majority of recipients. Importantly, Fas-deficient IPCC allografts exhibited certain immune privilege and enjoyed long-term survival in diabetic NOD mice in the presence of CD28/CD40 joint blockade while their islet counterparts failed to do so. Long-term survival of ASC-derived IPCC syngeneic grafts requires blocking Fas and TNF death pathways, whereas blocking both death pathways and CD28/CD40 costimulation is needed for long-term IPCC allograft survival in diabetic NOD mice. Our studies have important clinical implications for treating type 1 diabetes via ASC-derived IPCC transplantation. © 2011 Zhang et al.

Promoting Long-Term Survival of Insulin-Producing Cell Grafts That Differentiate from Adipose Tissue-Derived Stem Cells to Cure Type 1 Diabetes

PubMed Central

Zhang, Shuzi; Dai, Hehua; Wan, Ni; Moore, Yolonda; Dai, Zhenhua

2011-01-01

Background Insulin-producing cell clusters (IPCCs) have recently been generated in vitro from adipose tissue-derived stem cells (ASCs) to circumvent islet shortage. However, it is unknown how long they can survive upon transplantation, whether they are eventually rejected by recipients, and how their long-term survival can be induced to permanently cure type 1 diabetes. IPCC graft survival is critical for their clinical application and this issue must be systematically addressed prior to their in-depth clinical trials. Methodology/Principal Findings Here we found that IPCC grafts that differentiated from murine ASCs in vitro, unlike their freshly isolated islet counterparts, did not survive long-term in syngeneic mice, suggesting that ASC-derived IPCCs have intrinsic survival disadvantage over freshly isolated islets. Indeed, β cells retrieved from IPCC syngrafts underwent faster apoptosis than their islet counterparts. However, blocking both Fas and TNF receptor death pathways inhibited their apoptosis and restored their long-term survival in syngeneic recipients. Furthermore, blocking CD40-CD154 costimulation and Fas/TNF signaling induced long-term IPCC allograft survival in overwhelming majority of recipients. Importantly, Fas-deficient IPCC allografts exhibited certain immune privilege and enjoyed long-term survival in diabetic NOD mice in the presence of CD28/CD40 joint blockade while their islet counterparts failed to do so. Conclusions/Significance Long-term survival of ASC-derived IPCC syngeneic grafts requires blocking Fas and TNF death pathways, whereas blocking both death pathways and CD28/CD40 costimulation is needed for long-term IPCC allograft survival in diabetic NOD mice. Our studies have important clinical implications for treating type 1 diabetes via ASC-derived IPCC transplantation. PMID:22216347

In silico modelling of radiation effects towards personalised treatment in radiotherapy

NASA Astrophysics Data System (ADS)

Marcu, Loredana G.; Marcu, David

2017-12-01

In silico models applied in medical physics are valuable tools to assist in treatment optimization and personalization, which represent the ultimate goal of today's radiotherapy. Next to several biological and biophysical factors that influence tumour response to ionizing radiation, hypoxia and cancer stem cells are critical parameters that dictate the final outcome. The current work presents the results of an in silico model of tumour growth and response to radiation developed using Monte Carlo techniques. We are presenting the impact of partial oxygen tension and repopulation via cancer stem cells on tumour control after photon irradiation, highlighting some of the gaps that clinical research needs to fill for better customized treatment.

Allogenic fetal liver cells have a distinct competitive engraftment advantage over adult bone marrow cells when infused into fetal as compared with adult severe combined immunodeficient recipients.

PubMed

Taylor, Patricia A; McElmurry, Ronald T; Lees, Christopher J; Harrison, David E; Blazar, Bruce R

2002-03-01

In utero transplantation (IUT) is becoming a viable option for the treatment of various immune and metabolic disorders diagnosed early in gestation. In this study, donor fetal liver cells had a 10-fold competitive engraftment advantage relative to adult bone marrow in allogeneic fetal severe combined immunodeficient (SCID) recipients compared with adult recipients. In contrast, adult bone marrow cells engrafted slightly better than fetal liver cells in allogeneic adult SCID transplant recipients. By using different ratios of fetal and adult cell mixtures, fetal liver cells repopulated 8.2 times better than adult bone marrow cells in fetal recipients, but only 0.8 times as well in adult recipients. Fetal SCID recipients were more permissive to an allogeneic donor graft than adult recipients. These data indicate that the recipient microenvironment may regulate the engraftment efficiency of a given stem cell source and suggest that the use of cord blood should be tested in clinical IUT.

Cancer stem cells, cancer cell plasticity and radiation therapy.

PubMed

Vlashi, Erina; Pajonk, Frank

2015-04-01

Since the first prospective identification of cancer stem cells in solid cancers the cancer stem cell hypothesis has reemerged as a research topic of increasing interest. It postulates that solid cancers are organized hierarchically with a small number of cancer stem cells driving tumor growth, repopulation after injury and metastasis. They give rise to differentiated progeny, which lack these features. The model predicts that for any therapy to provide cure, all cancer stem cells have to be eliminated while the survival of differentiated progeny is less critical. In this review we discuss recent reports challenging the idea of a unidirectional differentiation of cancer cells. These reports provide evidence supporting the idea that non-stem cancer cells exhibit a remarkable degree of plasticity that allows them to re-acquire cancer stem cell traits, especially in the context of radiation therapy. We summarize conditions under which differentiation is reversed and discuss the current knowledge of the underlying mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Radiosensitivity of cancer-initiating cells and normal stem cells (or what the Heisenberg uncertainly principle has to do with biology).

PubMed

Woodward, Wendy Ann; Bristow, Robert Glen

2009-04-01

Mounting evidence suggests that parallels between normal stem cell biology and cancer biology may provide new targets for cancer therapy. Prospective identification and isolation of cancer-initiating cells from solid tumors has promoted the descriptive and functional identification of these cells allowing for characterization of their response to contemporary cancer therapies, including chemotherapy and radiation. In clinical radiation therapy, the failure to clinically eradicate all tumor cells (eg, a lack of response, partial response, or nonpermanent complete response by imaging) is considered a treatment failure. As such, biologists have explored the characteristics of the small population of clonogenic cancer cells that can survive and are capable of repopulating the tumor after subcurative therapy. Herein, we discuss the convergence of these clonogenic studies with contemporary radiosensitivity studies that use cell surface markers to identify cancer-initiating cells. Implications for and uncertainties regarding incorporation of these concepts into the practice of modern radiation oncology are discussed.

A novel intranuclear RNA vector system for long-term stem cell modification

PubMed Central

Ikeda, Yasuhiro; Makino, Akiko; Matchett, William E.; Holditch, Sara J.; Lu, Brian; Dietz, Allan B.; Tomonaga, Keizo

2015-01-01

Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, Borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression, or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells (iPSCs), while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction. PMID:26632671

Short-term and long-term clinostat and vibration-induced biochemical changes in dwarf Marigold stems

NASA Astrophysics Data System (ADS)

Siegel, S. M.; Siegel, B. Z.

Stems of 21-day dwarf Marigold plants cultivated on the clinostat were compared with plants cultivated on vertical axis rotators (``vibrational controls'') and stationary controls for long-term changes in cell wall composition. Stems of 21-day plants grown under stationary conditions and subsequently exposed to the clinostat for 24 hours were also analyzed. Among the long-term markers, calcium, lignin, and protein-bound hemicellulose (possibly cell wall glycoprotein) clearly differentiated the effects of vibration from those of the clinostat. Short-term differential responses included rate of ethylene production, nastic movement and peroxidase activity of the cell wall, but not of the protoplast.

Short-term and long-term clinostat and vibration-induced biochemical changes in dwarf marigold stems

NASA Technical Reports Server (NTRS)

Siegel, S. M.; Siegel, B. Z.

1983-01-01

Stems of 21-day dwarf marigold plants cultivated on the clinostat were compared with plants cultivated on vertical axis rotators ('vibrational controls') and stationary controls for long-term changes in cell wall composition. Stems of 21-day plants grown under stationary conditions and subsequently exposed to the clinostat for 24 hours were also analyzed. Among the long-term markers, calcium, lignin, and protein-bound hemicellulose (possibly cell wall glycoprotein) clearly differentiated the effects of vibration from those of the clinostat. Short-term differential responses included rate of ethylene production, nastic movement and peroxidase activity of the cell wall, but not of the protoplast.

Safe engineering of CAR T cells for adoptive cell therapy of cancer using long-term episomal gene transfer.

PubMed

Jin, Chuan; Fotaki, Grammatiki; Ramachandran, Mohanraj; Nilsson, Berith; Essand, Magnus; Yu, Di

2016-07-01

Chimeric antigen receptor (CAR) T-cell therapy is a new successful treatment for refractory B-cell leukemia. Successful therapeutic outcome depends on long-term expression of CAR transgene in T cells, which is achieved by delivering transgene using integrating gamma retrovirus (RV) or lentivirus (LV). However, uncontrolled RV/LV integration in host cell genomes has the potential risk of causing insertional mutagenesis. Herein, we describe a novel episomal long-term cell engineering method using non-integrating lentiviral (NILV) vector containing a scaffold/matrix attachment region (S/MAR) element, for either expression of transgenes or silencing of target genes. The insertional events of this vector into the genome of host cells are below detection level. CD19 CAR T cells engineered with a NILV-S/MAR vector have similar levels of CAR expression as T cells engineered with an integrating LV vector, even after numerous rounds of cell division. NILV-S/MAR-engineered CD19 CAR T cells exhibited similar cytotoxic capacity upon CD19(+) target cell recognition as LV-engineered T cells and are as effective in controlling tumor growth in vivo We propose that NILV-S/MAR vectors are superior to current options as they enable long-term transgene expression without the risk of insertional mutagenesis and genotoxicity. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Long- and short-term exposure to PM2.5 and mortality: using novel exposure models.

PubMed

Kloog, Itai; Ridgway, Bill; Koutrakis, Petros; Coull, Brent A; Schwartz, Joel D

2013-07-01

Many studies have reported associations between ambient particulate matter (PM) and adverse health effects, focused on either short-term (acute) or long-term (chronic) PM exposures. For chronic effects, the studied cohorts have rarely been representative of the population. We present a novel exposure model combining satellite aerosol optical depth and land-use data to investigate both the long- and short-term effects of PM2.5 exposures on population mortality in Massachusetts, United States, for the years 2000-2008. All deaths were geocoded. We performed two separate analyses: a time-series analysis (for short-term exposure) where counts in each geographic grid cell were regressed against cell-specific short-term PM2.5 exposure, temperature, socioeconomic data, lung cancer rates (as a surrogate for smoking), and a spline of time (to control for season and trends). In addition, for long-term exposure, we performed a relative incidence analysis using two long-term exposure metrics: regional 10 × 10 km PM2.5 predictions and local deviations from the cell average based on land use within 50 m of the residence. We tested whether these predicted the proportion of deaths from PM-related causes (cardiovascular and respiratory diseases). For short-term exposure, we found that for every 10-µg/m increase in PM 2.5 exposure there was a 2.8% increase in PM-related mortality (95% confidence interval [CI] = 2.0-3.5). For the long-term exposure at the grid cell level, we found an odds ratio (OR) for every 10-µg/m increase in long-term PM2.5 exposure of 1.6 (CI = 1.5-1.8) for particle-related diseases. Local PM2.5 had an OR of 1.4 (CI = 1.3-1.5), which was independent of and additive to the grid cell effect. We have developed a novel PM2.5 exposure model based on remote sensing data to assess both short- and long-term human exposures. Our approach allows us to gain spatial resolution in acute effects and an assessment of long-term effects in the entire population rather than a selective sample from urban locations.

A human bone marrow mesodermal-derived cell population with hemogenic potential.

PubMed

Mokhtari, Saloomeh; Colletti, Evan; Yin, Weihong; Sanada, Chad; Lamar, Zanetta; Simmons, Paul J; Walker, Steven; Bishop, Colin; Atala, Anthony; Zanjani, Esmail D; Porada, Christopher D; Almeida-Porada, Graça

2018-02-02

The presence, within the human bone marrow, of cells with both endothelial and hemogenic potential has been controversial. Herein, we identify, within the human fetal bone marrow, prior to establishment of hematopoiesis, a unique APLNR+, Stro-1+ cell population, co-expressing markers of early mesodermal precursors and/or hemogenic endothelium. In adult marrow, cells expressing similar markers are also found, but at very low frequency. These adult-derived cells can be extensively culture expanded in vitro without loss of potential, they preserve a biased hemogenic transcriptional profile, and, upon in vitro induction with OCT4, assume a hematopoietic phenotype. In vivo, these cells, upon transplantation into a fetal microenvironment, contribute to the vasculature, and generate hematopoietic cells that provide multilineage repopulation upon serial transplantation. The identification of this human somatic cell population provides novel insights into human ontogenetic hematovascular potential, which could lead to a better understanding of, and new target therapies for, malignant and nonmalignant hematologic disorders.

Development and function of human innate immune cells in a humanized mouse model.

PubMed

Rongvaux, Anthony; Willinger, Tim; Martinek, Jan; Strowig, Till; Gearty, Sofia V; Teichmann, Lino L; Saito, Yasuyuki; Marches, Florentina; Halene, Stephanie; Palucka, A Karolina; Manz, Markus G; Flavell, Richard A

2014-04-01

Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models cannot support development of human innate immune cells, including myeloid cells and natural killer (NK) cells. Here we describe two mouse strains called MITRG and MISTRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked into their respective mouse loci. The human cytokines support the development and function of monocytes, macrophages and NK cells derived from human fetal liver or adult CD34(+) progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MITRG and MISTRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

Development and function of human innate immune cells in a humanized mouse model

PubMed Central

Rongvaux, Anthony; Willinger, Tim; Martinek, Jan; Strowig, Till; Gearty, Sofia V.; Teichmann, Lino L.; Saito, Yasuyuki; Marches, Florentina; Halene, Stephanie; Palucka, A. Karolina; Manz, Markus G.; Flavell, Richard A.

2014-01-01

Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models are unable to support development of human innate immune cells, including myeloid cells and NK cells. Here we describe a mouse strain, called MI(S)TRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked in to their respective mouse loci. The human cytokines support the development and function of monocytes/macrophages and natural killer cells derived from human fetal liver or adult CD34+ progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MI(S)TRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology. PMID:24633240

Hepatic tissue environment in NEMO-deficient mice critically regulates positive selection of donor cells after hepatocyte transplantation.

PubMed

Kaldenbach, Michaela; Cubero, Francisco Javier; Erschfeld, Stephanie; Liedtke, Christian; Trautwein, Christian; Streetz, Konrad

2014-01-01

Hepatocyte transplantation (HT) is a promising alternative treatment strategy for end-stage liver diseases compared with orthotopic liver transplantation. A limitation for this approach is the low engraftment of donor cells. The deletion of the I-kappa B kinase-regulatory subunit IKKγ/NEMO in hepatocytes prevents nuclear factor (NF)-kB activation and triggers spontaneous liver apoptosis, chronic hepatitis and the development of liver fibrosis and hepatocellular carcinoma. We hypothesized that NEMOΔhepa mice may therefore serve as an experimental model to study HT. Pre-conditioned NEMOΔhepa mice were transplanted with donor-hepatocytes from wildtype (WT) and mice deficient for the pro-apoptotic mediator Caspase-8 (Casp8Δhepa). Transplantation of isolated WT-hepatocytes into pre-conditioned NEMOΔhepa mice resulted in a 6-7 fold increase of donor cells 12 weeks after HT, while WT-recipients showed no liver repopulation. The use of apoptosis-resistant Casp8Δhepa-derived donor cells further enhanced the selection 3-fold after 12-weeks and up to 10-fold increase after 52 weeks compared with WT donors. While analysis of NEMOΔhepa mice revealed strong liver injury, HT-recipient NEMOΔhepa mice showed improved liver morphology and decrease in serum transaminases. Concomitant with these findings, the histological examination elicited an improved liver tissue architecture associated with significantly lower levels of apoptosis, decreased proliferation and a lesser amount of liver fibrogenesis. Altogether, our data clearly support the therapeutic benefit of the HT procedure into NEMOΔhepa mice. This study demonstrates the feasibility of the NEMOΔhepa mouse as an in vivo tool to study liver repopulation after HT. The improvement of the characteristic phenotype of chronic liver injury in NEMOΔhepa mice after HT suggests the therapeutic potential of HT in liver diseases with a chronic inflammatory phenotype and opens a new door for the applicability of this technique to combat liver disease in the human clinic.

Hepatic Tissue Environment in NEMO-Deficient Mice Critically Regulates Positive Selection of Donor Cells after Hepatocyte Transplantation

PubMed Central

Kaldenbach, Michaela; Cubero, Francisco Javier; Erschfeld, Stephanie; Liedtke, Christian; Trautwein, Christian; Streetz, Konrad

2014-01-01

Background Hepatocyte transplantation (HT) is a promising alternative treatment strategy for end-stage liver diseases compared with orthotopic liver transplantation. A limitation for this approach is the low engraftment of donor cells. The deletion of the I-kappa B kinase-regulatory subunit IKKγ/NEMO in hepatocytes prevents nuclear factor (NF)-kB activation and triggers spontaneous liver apoptosis, chronic hepatitis and the development of liver fibrosis and hepatocellular carcinoma. We hypothesized that NEMOΔhepa mice may therefore serve as an experimental model to study HT. Methods Pre-conditioned NEMOΔhepa mice were transplanted with donor-hepatocytes from wildtype (WT) and mice deficient for the pro-apoptotic mediator Caspase-8 (Casp8Δhepa). Results Transplantation of isolated WT-hepatocytes into pre-conditioned NEMOΔhepa mice resulted in a 6-7 fold increase of donor cells 12 weeks after HT, while WT-recipients showed no liver repopulation. The use of apoptosis-resistant Casp8Δhepa-derived donor cells further enhanced the selection 3-fold after 12-weeks and up to 10-fold increase after 52 weeks compared with WT donors. While analysis of NEMOΔhepa mice revealed strong liver injury, HT-recipient NEMOΔhepa mice showed improved liver morphology and decrease in serum transaminases. Concomitant with these findings, the histological examination elicited an improved liver tissue architecture associated with significantly lower levels of apoptosis, decreased proliferation and a lesser amount of liver fibrogenesis. Altogether, our data clearly support the therapeutic benefit of the HT procedure into NEMOΔhepa mice. Conclusion This study demonstrates the feasibility of the NEMOΔhepa mouse as an in vivo tool to study liver repopulation after HT. The improvement of the characteristic phenotype of chronic liver injury in NEMOΔhepa mice after HT suggests the therapeutic potential of HT in liver diseases with a chronic inflammatory phenotype and opens a new door for the applicability of this technique to combat liver disease in the human clinic. PMID:24979756

Lipid-Antigen Presentation by CD1d+ B Cells Is Essential for the Maintenance of Invariant Natural Killer T Cells

PubMed Central

Bosma, Anneleen; Abdel-Gadir, Azza; Isenberg, David A.; Jury, Elizabeth C.; Mauri, Claudia

2012-01-01

Summary B cells perform many immunological functions, including presenting lipid antigen to CD1d-restricted invariant natural killer T (iNKT) cells, known to contribute to maintaining tolerance in autoimmunity. Patients with systemic lupus erythematous (SLE) display dysregulated B cell responses and reduced peripheral iNKT cell frequencies. The significance of these defects and how they relate to SLE pathogenesis remain elusive. We report that B cells are essential for iNKT cell expansion and activation in healthy donors but fail to exert a similar effect in SLE patients. Defective B cell-mediated stimulation of iNKT cells in SLE patients was associated with altered CD1d recycling, a defect recapitulated in B cells from healthy donors after stimulation with interferon-α (IFN-α) and anti-immunoglobulin (Ig). iNKT cell number and function were restored in SLE patients responding to anti-CD20 treatment upon normalization of CD1d expression exclusively in repopulated immature B cells. We propose that healthy B cells are pivotal for iNKT cell homeostasis. PMID:22406267

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.

Lgr4 regulates mammary gland development and stem cell activity through the pluripotency transcription factor Sox2.

PubMed

Wang, Ying; Dong, Jie; Li, Dali; Lai, Li; Siwko, Stefan; Li, Yi; Liu, Mingyao

2013-09-01

The key signaling networks regulating mammary stem cells are poorly defined. The leucine-rich repeat containing G protein-coupled receptor (Lgr) family has been implicated in intestinal, gastric, and epidermal stem cell functions. We investigated whether Lgr4 functions in mammary gland development and mammary stem cells. We found that Lgr4(-/-) mice had delayed ductal development, fewer terminal end buds, and decreased side-branching. Crucially, the mammary stem cell repopulation capacity was severely impaired. Mammospheres from Lgr4(-/-) mice showed decreased Wnt signaling. Wnt3a treatment prevented the adverse effects of Lgr4 loss on organoid formation. Chromatin immunoprecipitation analysis indicated that Sox2 expression was controlled by the Lgr4/Wnt/β-catenin/Lef1 pathway. Importantly, Sox2 overexpression restored the in vivo mammary regeneration potential of Lgr4(-/-) mammary stem cells. Therefore, Lgr4 activates Sox2 to regulate mammary development and stem cell functions via Wnt/β-catenin/Lef1. © AlphaMed Press.

A Longitudinal Study on Feeding Behaviour and Activity Patterns of Released Chimpanzees in Conkouati-Douli National Park, Republic of Congo

PubMed Central

Renaud, Amandine; Jamart, Aliette; Goossens, Benoit; Ross, Caroline

2013-01-01

Simple Summary Wild chimpanzee populations are dramatically declining due to anthropogenic pressure. One way of increasing wild population numbers and/or repopulating areas where local extinction has occurred is to release captive animals. HELP Congo was the first project to successfully release wild-born orphan chimpanzees in their natural environment. We studied the behaviour of eight released chimpanzees over eight years. Over time, they modified their behaviour, suggesting long-term behavioural and ecological adaptations. This suggests that successful release programmes may reinforce existing populations of endangered species. Abstract Wild chimpanzee populations are still declining due to logging, disease transmission and hunting. The bushmeat trade frequently leads to an increase in the number of orphaned primates. HELP Congo was the first project to successfully release wild-born orphan chimpanzees into an existing chimpanzee habitat. A collection of post monitoring data over 16 years now offers the unique opportunity to investigate possible behavioural adaptations in these chimpanzees. We investigated the feeding and activity patterns in eight individuals via focal observation techniques from 1997–1999 and 2001–2005. Our results revealed a decline in the number of fruit and insect species in the diet of released chimpanzees over the years, whereas within the same period of time, the number of consumed seed species increased. Furthermore, we found a decline in time spent travelling, but an increase in time spent on social activities, such as grooming, as individuals matured. In conclusion, the observed changes in feeding and activity patterns seem to reflect important long-term behavioural and ecological adaptations in wild-born orphan released chimpanzees, demonstrating that the release of chimpanzees can be successful, even if it takes time for full adaptation. PMID:26487416

Hazard assessment of three haloacetic acids, as byproducts of water disinfection, in human urothelial cells.

PubMed

Marsà, Alicia; Cortés, Constanza; Hernández, Alba; Marcos, Ricard

2018-05-15

Disinfection by-products (DBPs) are compounds produced in the raw water disinfection processes. Although increased cancer incidence has been associated with exposure to this complex mixture, the carcinogenic potential of individual DBPs remains not well known; thus, further studies are required. Haloacetic acids (HAAs) constitute an important group among DBPs. In this study, we have assessed the in vitro carcinogenic potential of three HAAs namely chloro-, bromo-, and iodoacetic acids. Using a long-term (8 weeks) and sub-toxic doses exposure scenario, different in vitro transformation markers were evaluated using a human urothelial cell line (T24). Our results indicate that long-term exposure to low doses of HAAs did not reproduce the genotoxic effects observed in acute treatments, where oxidative DNA damage was induced. No changes in the transformation endpoints analyzed were observed, as implied by the absence of significant morphological, cell growth rate and anchorage-independent cell growth pattern modifications. Interestingly, HAA-long-term exposed cells developed resistance to oxidative stress damage, what would explain the observed differences between acute and long-term exposure conditions. Accordingly, data obtained under long-term exposure to sub-toxic doses of HAAs could be more accurate, in terms of risk assessment, than under acute exposure scenarios. Copyright © 2018 Elsevier Inc. All rights reserved.

Induced neural stem cells achieve long-term survival and functional integration in the adult mouse brain.

PubMed

Hemmer, Kathrin; Zhang, Mingyue; van Wüllen, Thea; Sakalem, Marna; Tapia, Natalia; Baumuratov, Aidos; Kaltschmidt, Christian; Kaltschmidt, Barbara; Schöler, Hans R; Zhang, Weiqi; Schwamborn, Jens C

2014-09-09

Differentiated cells can be converted directly into multipotent neural stem cells (i.e., induced neural stem cells [iNSCs]). iNSCs offer an attractive alternative to induced pluripotent stem cell (iPSC) technology with regard to regenerative therapies. Here, we show an in vivo long-term analysis of transplanted iNSCs in the adult mouse brain. iNSCs showed sound in vivo long-term survival rates without graft overgrowths. The cells displayed a neural multilineage potential with a clear bias toward astrocytes and a permanent downregulation of progenitor and cell-cycle markers, indicating that iNSCs are not predisposed to tumor formation. Furthermore, the formation of synaptic connections as well as neuronal and glial electrophysiological properties demonstrated that differentiated iNSCs migrated, functionally integrated, and interacted with the existing neuronal circuitry. We conclude that iNSC long-term transplantation is a safe procedure; moreover, it might represent an interesting tool for future personalized regenerative applications. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Effector and memory T cell subsets in the response to bovine tuberculosis

USDA-ARS?s Scientific Manuscript database

Long-term (i.e., 14 days) cultured IFN-gamma ELISPOT assays of peripheral blood mononuclear cells (PBMC) are used to access T cell central memory (Tcm) responses in both cattle and humans. With bovine tuberculosis, vaccine-elicited long-term IFN-gamma ELISPOT response correlates with protection; how...

Stable multilineage xenogeneic replacement of definitive hematopoiesis in adult zebrafish.

PubMed

Hess, Isabell; Boehm, Thomas

2016-01-18

Bony fishes are the most numerous and phenotypically diverse group of vertebrates inhabiting our planet, making them an ideal target for identifying general principles of tissue development and function. However, lack of suitable experimental platforms prevents the exploitation of this rich source of natural phenotypic variation. Here, we use a zebrafish strain lacking definitive hematopoiesis for interspecific analysis of hematopoietic cell development. Without conditioning prior to transplantation, hematopoietic progenitor cells from goldfish stably engraft in adult zebrafish homozygous for the c-myb(I181N) mutation. However, in competitive repopulation experiments, zebrafish hematopoietic cells exhibit an advantage over their goldfish counterparts, possibly owing to subtle species-specific functional differences in hematopoietic microenvironments resulting from over 100 million years of independent evolution. Thus, our unique animal model provides an unprecedented opportunity to genetically and functionally disentangle universal and species-specific contributions of the microenvironment to hematopoietic progenitor cell maintenance and development.

Leptin deficiency shifts mast cells toward anti-inflammatory actions and protects mice from obesity and diabetes by polarizing M2 macrophages

PubMed Central

Zhou, Yi; Yu, Xueqing; Chen, Huimei; Sjöberg, Sara; Roux, Joséphine; Zhang, Lijun; Ivoulsou, Al-Habib; Bensaid, Farid; Liu, Conglin; Liu, Jian; Tordjman, Joan; Clement, Karine; Lee, Chih-Hao; Hotamisligil, Gokhan S.; Libby, Peter; Shi, Guo-Ping

2015-01-01

SUMMARY Mast cells (MCs) contribute to the pathogenesis of obesity and diabetes. This study demonstrates that leptin deficiency slants MCs toward anti-inflammatory functions. MCs in the white adipose tissues (WAT) of lean humans and mice express negligible leptin. Adoptive transfer of leptin-deficient MCs expanded ex vivo mitigates diet-induced and pre-established obesity and diabetes in mice. Mechanistic studies show that leptin-deficient MCs polarize macrophages from M1 to M2 functions because of impaired cell signaling and an altered balance between pro- and anti-inflammatory cytokines, but do not affect T-cell differentiation. Rampant body weight gain in ob/ob mice, a strain that lacks leptin, associates with reduced MC content in WAT. In ob/ob mice, genetic depletion of MCs exacerbates obesity and diabetes, and repopulation of ex vivo expanded ob/ob MCs ameliorates these diseases. PMID:26481668

Coarse-scale movement patterns of a small-bodied fish inhabiting a desert stream

USGS Publications Warehouse

Dzul, M.C.; Quist, M.C.; Dinsmore, S.J.; Gaines, D.B.; Bower, M.R.

2013-01-01

Located on the floor of Death Valley (CA, USA), Salt Creek harbors a single fish species, the Salt Creek pupfish, Cyprinodon salinus salinus, which has adapted to this extremely harsh environment. Salt Creek is fed by an underground spring and is comprised of numerous pools, runs, and marshes that exhibit substantial variability in temperature, salinity, and dissolved oxygen concentrations. In addition, the wetted area of Salt Creek is reduced throughout the summer months due to high rates of evaporation, with some reaches drying completely. Therefore, it seems logical that short- and long-term movement patterns may play an important role in Salt Creek pupfish population dynamics. The objective of this study was to describe coarse-scale movements of Salt Creek pupfish in Salt Creek during their breeding season from March to May. Sex ratios and length–frequency distributions varied spatially within Salt Creek, suggesting population segregation during the breeding season. Long-distance movements were generally rare, although two fish moved more than 1.2 km. Movement in upstream reaches was rare or absent, in contrast to the greater movement observed in downstream reaches (29% of recaptures). Temporal trends and demographic patterns in movement were not observed. Because the two most downstream habitats dry up in the summer, our results indicate that coarse-scale movements that re-populate downstream reaches likely occur during other times of year. Consequently, the importance of small- and large-scale movements is influenced by season. Further assessment of Salt Creek movement patterns conducted during other times of year may better illuminate long-distance movement patterns and source-sink dynamics.

Enrichment of human bone marrow aspirates for low-density mononuclear cells using a haemonetics discontinuous blood cell separator.

PubMed

Raijmakers, R; de Witte, T; Koekman, E; Wessels, J; Haanen, C

1986-01-01

Isopycnic density floatation centrifugation has been proven to be a suitable technique to enrich bone marrow aspirates for clonogenic cells on a small scale. We have tested a Haemonetics semicontinuous blood cell separator in order to process large volumes of bone marrow with minimal bone marrow manipulation. The efficacy of isopycnic density floatation was tested in a one and a two-step procedure. Both procedures showed a recovery of about 20% of the nucleated cells and 1-2% of the erythrocytes. The enrichment of clonogenic cells in the one-step procedure appeared superior to the two-step enrichment, first separating buffy coat cells. The recovery of clonogenic cells was 70 and 50%, respectively. Repopulation capacity of the low-density cell fraction containing the clonogenic cells was excellent after autologous reinfusion (6 cases) and allogeneic bone marrow transplantation (3 cases). Fast enrichment of large volumes of bone marrow aspirates with low-density cells containing the clonogenic cells by isopycnic density floatation centrifugation can be done safely using a Haemonetics blood cell separator.

Tree Line Structure and Dynamics at the Northern Limit of the Larch Forest: Anabar Plateau, Siberia, Russia

NASA Technical Reports Server (NTRS)

Kharuk, Viacheslav I.; Ranson, Kenneth J.; Im, Sergey T.; Oskorbin, Pavel A.; Dvinskaya, Maria L.; Ovchinnikov, Dmitriy V.

2013-01-01

The goal of the study was to provide an analysis of climate impact before, during, and after the Little Ice Age (LIA) on the larch (Larix gmelinii) tree line at the northern extreme of Siberian forests. Recent decadal climate change impacts on the tree line, regeneration abundance, and age structure were analyzed. The location of the study area was within the forest-tundra ecotone (elevation range 170-450 m) in the Anabar Plateau, northern Siberia. Field studies were conducted along elevational transects. Tree natality/mortality and radial increment were determined based on dendrochronology analyses. Tree morphology, number of living and subfossil trees, regeneration abundance, and age structure were studied. Locations of pre-LIA, LIA, and post-LIA tree lines and refugia boundaries were established. Long-term climate variables and drought index were included in the analysis. It was found that tree mortality from the 16th century through the beginning of the 19th century caused a downward tree line recession. Sparse larch stands experienced deforestation, transforming into tundra with isolated relict trees. The maximum tree mortality and radial growth decrease were observed to have occurred at the beginning of 18th century. Now larch, at its northern boundary in Siberia, is migrating into tundra areas. Upward tree migration was induced by warming in the middle of the 19th century. Refugia played an important role in repopulation of the forest-tundra ecotone by providing a seed source and shelter for recruitment of larch regeneration. Currently this ecotone is being repopulated mainly by tree cohorts that were established after the 1930s. The last two decades of warming did not result in an acceleration of regeneration recruitment because of increased drought conditions. The regeneration line reached (but did not exceed) the pre-LIA tree line location, although contemporary tree heights and stand densities are comparatively lower than in the pre-LIA period. The mean rate of tree line upward migration has been about 0.35 m/yr (with a range of 0.21-0.58), which translates to a tree line response to temperature of about 55 m/deg C.

Wound signaling: The missing link in plant regeneration.

PubMed

Chen, Lyuqin; Sun, Beibei; Xu, Lin; Liu, Wu

2016-10-02

Wounding is the first event that occurs in plant regeneration. However, wound signaling in plant regeneration is barely understood. Using a simple system of de novo root organogenesis from Arabidopsis thaliana leaf explants, we analyzed the genes downstream of wound signaling. Leaf explants may produce at least two kinds of wound signals to trigger short-term and long-term wound signaling. Short-term wound signaling is primarily involved in controlling auxin behavior and the fate transition of regeneration-competent cells, while long-term wound signaling mainly modulates the cellular environment at the wound site and maintains the auxin level in regeneration-competent cells. YUCCA (YUC) genes, which are involved in auxin biogenesis, are targets of short-term wound signaling in mesophyll cells and of long-term wound signaling in regeneration-competent cells. The expression patterns of YUCs provide important information about the molecular basis of wound signaling in plant regeneration.

A potential role for cell-based therapeutics in the treatment of intervertebral disc herniation.

PubMed

Ganey, Timothy M; Meisel, Hans Joerg

2002-10-01

Lower back pain and disc degeneration negatively affect quality of life and impose an enormous financial burden. An extensive body of scientific work has evolved that characterizes the disc, demonstrating spinal anatomy and morphology that contribute to risk and likely promote failure. Ultimately, matrix failure is responsible for mechanical failure, which in turn results in spinal compromise anatomically and subsequent pain. One intervening approach to breaking this sequence has been to repopulate the anatomy with autologous disc chondrocytes--cells capable of restoring the matrix and retaining the mechanical balance by which the disc functions. This strategy has been implemented both in patients and in animal models, and early results, although preliminary, support the premise as a positive approach.

Zscan4 restores the developmental potency of embryonic stem cells

PubMed Central

Amano, Tomokazu; Hirata, Tetsuya; Falco, Geppino; Monti, Manuela; Sharova, Lioudmila V.; Amano, Misa; Sheer, Sarah; Hoang, Hien G.; Piao, Yulan; Stagg, Carole A.; Yamamizu, Kohei; Akiyama, Tomohiko; Ko, Minoru S.H.

2013-01-01

The developmental potency of mouse embryonic stem (ES) cells, which is the ability to contribute to a whole embryo is known to deteriorate during long-term cell culture. Previously we have shown that ES cells oscillate between Zscan4- and Zscan4+ states, and the transient activation of Zscan4 is required for the maintenance of telomeres and genome stability of ES cells. Here we show that increasing the frequency of Zscan4 activation in mouse ES cells restores and maintains their developmental potency in long-term cell culture. Injection of a single ES cell with such increased potency into a tetraploid blastocyst gives rise to an entire embryo with a higher success rate. These results not only provide a means to rejuvenate ES cells by manipulating Zscan4 expression, but also indicate the active roles of Zscan4 in the long-term maintenance of ES cell potency. PMID:23739662

Implications of long-term culture for mesenchymal stem cells: genetic defects or epigenetic regulation?

PubMed Central

2012-01-01

Mesenchymal stem cells change dramatically during culture expansion. Long-term culture has been suspected to evoke oncogenic transformation: overall, the genome appears to be relatively stable throughout culture but transient clonal aneuploidies have been observed. Oncogenic transformation does not necessarily entail growth advantage in vitro and, therefore, the available methods - such as karyotypic analysis or genomic profiling - cannot exclude this risk. On the other hand, long-term culture is associated with specific senescence-associated DNA methylation (SA-DNAm) changes, particularly in developmental genes. SA-DNAm changes are highly reproducible and can be used to monitor the state of senescence for quality control. Notably, neither telomere attrition nor SA-DNAm changes occur in pluripotent stem cells, which can evade the 'Hayflick limit'. Long-term culture of mesenchymal stem cells seems to involve a tightly regulated epigenetic program. These epigenetic modifications may counteract dominant clones, which are more prone to transformation. PMID:23257053

Implications of long-term culture for mesenchymal stem cells: genetic defects or epigenetic regulation?

PubMed

Wagner, Wolfgang

2012-12-20

Mesenchymal stem cells change dramatically during culture expansion. Long-term culture has been suspected to evoke oncogenic transformation: overall, the genome appears to be relatively stable throughout culture but transient clonal aneuploidies have been observed. Oncogenic transformation does not necessarily entail growth advantage in vitro and, therefore, the available methods - such as karyotypic analysis or genomic profiling - cannot exclude this risk. On the other hand, long-term culture is associated with specific senescence-associated DNA methylation (SA-DNAm) changes, particularly in developmental genes. SA-DNAm changes are highly reproducible and can be used to monitor the state of senescence for quality control. Notably, neither telomere attrition nor SA-DNAm changes occur in pluripotent stem cells, which can evade the 'Hayflick limit'. Long-term culture of mesenchymal stem cells seems to involve a tightly regulated epigenetic program. These epigenetic modifications may counteract dominant clones, which are more prone to transformation.

Assessment of genetic and epigenetic variation during long-term Taxus cell culture.

PubMed

Fu, Chunhua; Li, Liqin; Wu, Wenjuan; Li, Maoteng; Yu, Xiaoqing; Yu, Longjiang

2012-07-01

Gradual loss of secondary metabolite production is a common obstacle in the development of a large-scale plant cell production system. In this study, cell morphology, paclitaxel (Taxol®) biosynthetic ability, and genetic and epigenetic variations in the long-term culture of Taxus media cv Hicksii cells were assessed over a 5-year period to evaluate the mechanisms of the loss of secondary metabolites biosynthesis capacity in Taxus cell. The results revealed that morphological variations, gradual loss of paclitaxel yield and decreased transcriptional level of paclitaxel biosynthesis key genes occurred during long-term subculture. Genetic and epigenetic variations in these cultures were also studied at different times during culture using amplified fragment-length polymorphism (AFLP), methylation-sensitive amplified polymorphism (MSAP), and high-performance liquid chromatography (HPLC) analyses. A total of 32 primer combinations were used in AFLP amplification, and none of the AFLP loci were found to be polymorphic, thus no major genetic rearrangements were detected in any of the tested samples. However, results from both MSAP and HPLC indicated that there was a higher level of DNA methylation in the low-paclitaxel yielding cell line after long-term culture. Based on these results, we proposed that accumulation of paclitaxel in Taxus cell cultures might be regulated by DNA methylation. To our knowledge, this is the first report of increased methylation with the prolongation of culture time in Taxus cell culture. It provides substantial clues for exploring the gradual loss of the taxol biosynthesis capacity of Taxus cell lines during long-term subculture. DNA methylation maybe involved in the regulation of paclitaxel biosynthesis in Taxus cell culture.

Long-term efficiency of mesenchymal stromal cell-mediated CD-MSC/5FC therapy in human melanoma xenograft model.

PubMed

Kucerova, L; Skolekova, S; Demkova, L; Bohovic, R; Matuskova, M

2014-10-01

Mesenchymal stromal cells (MSC) can be exploited as cellular delivery vehicles for the enzymes converting non-toxic prodrugs to toxic substances. Because of their inherent chemoresistance, they exert potent bystander and antitumor effect. Here we show that the human adipose tissue-derived MSC expressing fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CD-MSC) in combination with 5-fluorocytosine (5FC) mediated a long-term tumor-free survival in the 83.3% of tumor-bearing animals. CD-MSC/5FC treatment induced cytotoxicity against model human melanoma cells EGFP-A375. Only 4% of the therapeutic CD-MSC cells eliminated >98.5% of the tumor cells in vitro. Long-term tumor-free survival was confirmed in 15 out of the 18 animals. However, repeatedly used CD-MSC/5FC therapeutic regimen generated more aggressive and metastatic variant of the melanoma cells EGFP-A375/Rel3. These cells derived from the refractory xenotransplants exhibited increased resistance to the CD-MSC/5FC treatment, altered cell adhesion, migration, tumorigenic and metastatic properties. However, long-term curative effect was achieved by the augmentation of the CD-MSC/5FC regimen along with the inhibition of c-Met/hepatocyte growth factor signaling axis in this aggressive melanoma derivative. In summary, the CD-MSC/5FC regimen can be regarded as a very effective antitumor approach to achieve long-term tumor-free survival as demonstrated on a mouse model of aggressive human melanoma xenografts.

The Long-Term Performance of Small-Cell Batteries Without Cell-Balancing Electronics

NASA Technical Reports Server (NTRS)

Pearson, C.; Thwaite, C.; Curzon, D.; Rao, G.

2006-01-01

Tests approx.8 yrs ago showed Sony HC do not imbalance. AEA developed a theory (ESPC 2002): a) Self-discharge (SD) decreases with state-of-charge (SOC); b) Cells diverge to a state of dynamic equilibrium; c) Equilibrium spread depends on cell SD uniformity. Balancing model verified against test data. Short-term measures of SD difficult in Sony cells and very small values, depends on technique. Long-term evidence supports lower SD at low SD. Battery testing best proof of performance, typically mission specific tests.

Interplay between Short- and Long-Term Plasticity in Cell-Assembly Formation

PubMed Central

Hiratani, Naoki; Fukai, Tomoki

2014-01-01

Various hippocampal and neocortical synapses of mammalian brain show both short-term plasticity and long-term plasticity, which are considered to underlie learning and memory by the brain. According to Hebb’s postulate, synaptic plasticity encodes memory traces of past experiences into cell assemblies in cortical circuits. However, it remains unclear how the various forms of long-term and short-term synaptic plasticity cooperatively create and reorganize such cell assemblies. Here, we investigate the mechanism in which the three forms of synaptic plasticity known in cortical circuits, i.e., spike-timing-dependent plasticity (STDP), short-term depression (STD) and homeostatic plasticity, cooperatively generate, retain and reorganize cell assemblies in a recurrent neuronal network model. We show that multiple cell assemblies generated by external stimuli can survive noisy spontaneous network activity for an adequate range of the strength of STD. Furthermore, our model predicts that a symmetric temporal window of STDP, such as observed in dopaminergic modulations on hippocampal neurons, is crucial for the retention and integration of multiple cell assemblies. These results may have implications for the understanding of cortical memory processes. PMID:25007209

Development of a cell-based treatment for long-term neurotrophin expression and spiral ganglion neuron survival.

PubMed

Zanin, M P; Hellström, M; Shepherd, R K; Harvey, A R; Gillespie, L N

2014-09-26

Spiral ganglion neurons (SGNs), the target cells of the cochlear implant, undergo gradual degeneration following loss of the sensory epithelium in deafness. The preservation of a viable population of SGNs in deafness can be achieved in animal models with exogenous application of neurotrophins such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3. For translation into clinical application, a suitable delivery strategy that provides ongoing neurotrophic support and promotes long-term SGN survival is required. Cell-based neurotrophin treatment has the potential to meet the specific requirements for clinical application, and we have previously reported that Schwann cells genetically modified to express BDNF can support SGN survival in deafness for 4 weeks. This study aimed to investigate various parameters important for the development of a long-term cell-based neurotrophin treatment to support SGN survival. Specifically, we investigated different (i) cell types, (ii) gene transfer methods and (iii) neurotrophins, in order to determine which variables may provide long-term neurotrophin expression and which, therefore, may be the most effective for supporting long-term SGN survival in vivo. We found that fibroblasts that were nucleofected to express BDNF provided the most sustained neurotrophin expression, with ongoing BDNF expression for at least 30 weeks. In addition, the secreted neurotrophin was biologically active and elicited survival effects on SGNs in vitro. Nucleofected fibroblasts may therefore represent a method for safe, long-term delivery of neurotrophins to the deafened cochlea to support SGN survival in deafness. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Acute myeloid leukemia-targeted toxin activates both apoptotic and necroptotic death mechanisms.

PubMed

Horita, Henrick; Frankel, Arthur E; Thorburn, Andrew

2008-01-01

Acute myelogenous leukemia (AML) is the second most common leukemia with approximately 13,410 new cases and 8,990 deaths annually in the United States. A novel fusion toxin treatment, diphtheria toxin GM-CSF (DT-GMCSF) has been shown to selectively eliminate leukemic repopulating cells that are critical for the formation of AML. We previously showed that DT-GMCSF treatment of U937 cells, an AML cell line, causes activation of caspases and the induction of apoptosis. In this study we further investigate the mechanisms of cell death induced by DT-GMCSF and show that, in addition to the activation of caspase-dependent apoptosis, DT-GMCSF also kills AML cells by simultaneously activating caspase-independent necroptosis. These mechanisms depend on the ability of the targeted toxin to inhibit protein synthesis, and are not affected by the receptor that is targeted or the mechanism through which protein synthesis is blocked. We conclude that fusion toxin proteins may be effective for treating AML cells whether or not they are defective in apoptosis.

Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros.

PubMed

Ng, Elizabeth S; Azzola, Lisa; Bruveris, Freya F; Calvanese, Vincenzo; Phipson, Belinda; Vlahos, Katerina; Hirst, Claire; Jokubaitis, Vanta J; Yu, Qing C; Maksimovic, Jovana; Liebscher, Simone; Januar, Vania; Zhang, Zhen; Williams, Brenda; Conscience, Aude; Durnall, Jennifer; Jackson, Steven; Costa, Magdaline; Elliott, David; Haylock, David N; Nilsson, Susan K; Saffery, Richard; Schenke-Layland, Katja; Oshlack, Alicia; Mikkola, Hanna K A; Stanley, Edouard G; Elefanty, Andrew G

2016-11-01

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34 + cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34 + cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34 + hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17 + vessels from which RUNX1C + blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34 + hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

Bovine central memory T cells are highly proliferative in response to bovine tuberculosis infection

USDA-ARS?s Scientific Manuscript database

Long-term (i.e., 14 days) cultured IFN-gamma responses of peripheral blood mononuclear cells are used as a correlate of T cell central memory (Tcm) responses in both humans and cattle. With bovine tuberculosis, vaccine-elicited long-term IFN-gamma ELISPOT assays are a correlate of protection. Recent...

Long-term Blue Light Effects on the Histology of Lettuce and Soybean Leaves and Stems

NASA Technical Reports Server (NTRS)

Dougher, Tracy A. O.; Bugbee, Bruce

2004-01-01

Blue light (320 to 496 nm) alters hypocotyl and stem elongation and leaf expansion in short-term, cell-level experiments, but histological effects of blue light in long-term studies of whole plants have not been described. We measured cell size and number in stems of soybean (Glycine max L.) and leaves of soybean and lettuce (Lactuca sativa L.), at two blue light fractions. Short-term studies have shown that cell expansion in stems is rapidly inhibited when etiolated tissue is exposed to blue light. However, under long-term light exposure, an increase in the blue light fraction from less than 0.1% to 26% decreased internode length, specifically by inhibiting soybean cell division in stems. In contrast, an increase in blue light fraction from 6% to 26% reduced soybean leaf area by decreasing cell expansion. Surprisingly, lettuce leaf area increased with increasing blue light fraction (0% to 6%), which was attributed to a 3.1-fold increase in cell expansion and a 1.6-fold increase in cell division.

Micro patterned surfaces: an effective tool for long term digital holographic microscopy cell imaging

NASA Astrophysics Data System (ADS)

Mues, Sarah; Lilge, Inga; Schönherr, Holger; Kemper, Björn; Schnekenburger, Jürgen

2017-02-01

The major problem of Digital Holographic Microscopy (DHM) long term live cell imaging is that over time most of the tracked cells move out of the image area and other ones move in. Therefore, most of the cells are lost for the evaluation of individual cellular processes. Here, we present an effective solution for this crucial problem of long-term microscopic live cell analysis. We have generated functionalized slides containing areas of 250 μm per 200 μm. These micropatterned biointerfaces consist of passivating polyaclrylamide brushes (PAAm). Inner areas are backfilled with octadecanthiol (ODT), which allows cell attachment. The fouling properties of these surfaces are highly controllable and therefore the defined areas designed for the size our microscopic image areas were effective in keeping all cells inside the rectangles over the selected imaging period.

Therapeutic modulation of growth factors and cytokines in regenerative medicine.

PubMed

Ioannidou, Effie

2006-01-01

Regeneration that takes place in the human body is limited throughout life. Therefore, when organs are irreparably damaged, they are usually replaced with an artificial device or donor organ. The term "regenerative medicine" covers the restoration or replacement of cells, tissues, and organs. Stem cells play a major role in regenerative medicine by providing the way to repopulate organs damaged by disease. Stem cells have the ability to self renew and to regenerate cells of diverse lineages within the tissue in which they reside. Stem cells could originate from embryos or adult tissues. Growth factors are proteins that may act locally or systemically to affect the growth of cells in several ways. Various cell activities, including division, are influenced by growth factors. Cytokines are a family of low-molecular-weight proteins that are produced by numerous cell types and are responsible for regulating the immune response, inflammation, tissue remodeling and cellular differentiation. Target cells of growth factors and cytokines are mesenchymal, epithelial and endothelial cells. These molecules frequently have overlapping activities and can act in an autocrine or paracrine fashion. A complex network of growth factors and cytokines guides cellular differentiation and regeneration in all organs and tissues. The aim of this paper is to review the role of growth factors and cytokines in different organs or systems and explore their therapeutic application in regenerative medicine. The role of stem cells combined with growth factors and cytokines in the regeneration of vascular and hematopoietic, neural, skeletal, pancreatic, periodontal, and mucosal tissue is reviewed. There is evidence that supports the use of growth factors and cytokines in the treatment of neurological diseases, diabetes, cardiovascular disease, periodontal disease, cancer and its complication, oral mucositis. After solving the ethical issues and establishing clear and reasonable regulations, regenerative medicine through stem cell application combined with specific growth factors and cytokines will have great potential in curing a variety of human diseases.

Cell phones and brain tumors: a review including the long-term epidemiologic data.

PubMed

Khurana, Vini G; Teo, Charles; Kundi, Michael; Hardell, Lennart; Carlberg, Michael

2009-09-01

The debate regarding the health effects of low-intensity electromagnetic radiation from sources such as power lines, base stations, and cell phones has recently been reignited. In the present review, the authors attempt to address the following question: is there epidemiologic evidence for an association between long-term cell phone usage and the risk of developing a brain tumor? Included with this meta-analysis of the long-term epidemiologic data are a brief overview of cell phone technology and discussion of laboratory data, biological mechanisms, and brain tumor incidence. In order to be included in the present meta-analysis, studies were required to have met all of the following criteria: (i) publication in a peer-reviewed journal; (ii) inclusion of participants using cell phones for > or = 10 years (ie, minimum 10-year "latency"); and (iii) incorporation of a "laterality" analysis of long-term users (ie, analysis of the side of the brain tumor relative to the side of the head preferred for cell phone usage). This is a meta-analysis incorporating all 11 long-term epidemiologic studies in this field. The results indicate that using a cell phone for > or = 10 years approximately doubles the risk of being diagnosed with a brain tumor on the same ("ipsilateral") side of the head as that preferred for cell phone use. The data achieve statistical significance for glioma and acoustic neuroma but not for meningioma. The authors conclude that there is adequate epidemiologic evidence to suggest a link between prolonged cell phone usage and the development of an ipsilateral brain tumor.

From Cell to Module: Fabrication and Long-term Stability of Dye-sensitized Solar Cells

NASA Astrophysics Data System (ADS)

Nursam, N. M.; Hidayat, J.; Muliani, L.; Anggraeni, P. N.; Retnaningsih, L.; Idayanti, N.

2017-07-01

Dye-sensitized solar cell (DSSC), which has been firstly developed by Graetzel et al back in 1991, has attracted a considerable interest since its discovery. However, two of the main challenges that the DSSC technology will have to overcome towards commercialization involve device scale-up and long-term stability. In our group, the fabrication technology of DSSC has been developed from laboratory to module scale over the past few years, nevertheless, the long-term stability has still became a major concern. In this contribution, the long-term DSSC performance in relation to their scale-up from cell to module is investigated. The photoelectrode of the DSSCs were fabricated using nanocrystalline titanium dioxide materials that were subsequently sensitized using ruthenium-based dye. Additionally, TiCl4 pre- and post-treatment were carried out to enhance the overall device efficiency. When fabricated as cells, the DSSC prototypes showed relatively stable performance during repeated tests over three months. In order to increase the output power of the solar cells, the DSSCs were then connected in a Z-type series connection to obtain sub-module panels. The DSSC sub-modules exhibit poor stability, particularly as indicated by the significant decrease in the short circuit current (ISC ). Herein, the effect of photoelectrode and sealant materials as well as module design are investigated, highlighting their profound influence upon the DSSC efficiency and long-term stability.

Maintenance of the HIV Reservoir Is Antagonized by Selective BCL2 Inhibition

PubMed Central

Cummins, Nathan W.; Sainski-Nguyen, Amy M.; Natesampillai, Sekar; Aboulnasr, Fatma; Kaufmann, Scott

2017-01-01

ABSTRACT Decay of the HIV reservoir is slowed over time in part by expansion of the pool of HIV-infected cells. This expansion reflects homeostatic proliferation of infected cells by interleukin-7 (IL-7) or antigenic stimulation, as well as new rounds of infection of susceptible target cells. As novel therapies are being developed to accelerate the decay of the latent HIV reservoir, it will be important to identify interventions that prevent expansion and/or repopulation of the latent HIV reservoir. Our previous studies showed that HIV protease cleaves the host protein procaspase 8 to generate Casp8p41, which can bind and activate Bak to induce apoptosis of infected cells. In circumstances where expression of the anti-apoptotic protein BCL2 is high, Casp8p41 instead binds BCL2, and cell death does not occur. This effect can be overcome by treating cells with the clinically approved BCL2 antagonist venetoclax, which prevents Casp8p41 from binding BCL2, thereby allowing Casp8p41 to bind Bak and kill the infected cell. Here we assess whether the events that maintain the HIV reservoir are also antagonized by venetoclax. Using the J-Lat 10.6 model of persistent infection, we demonstrate that proliferation and HIV expression are countered by the use of venetoclax, which causes preferential killing of the HIV-expressing cells. Similarly, during new rounds of infection of primary CD4 T cells, venetoclax causes selective killing of HIV-infected cells, resulting in decreased numbers of HIV DNA-containing cells. IMPORTANCE Cure of HIV infection requires an intervention that reduces the HIV reservoir size. A variety of approaches are being tested for their ability to impact HIV reservoir size. Even if successful, however, these approaches will need to be combined with additional complementary approaches that prevent replenishment or repopulation of the HIV reservoir. Our previous studies have shown that the FDA-approved BCL2 antagonist venetoclax has a beneficial effect on the HIV reservoir size following HIV reactivation. Here we demonstrate that venetoclax also has a beneficial effect on HIV reservoir size in a model of homeostatic proliferation of HIV as well as in acute spreading infection of HIV in primary CD4 T cells. These results suggest that venetoclax, either alone or in combination with other approaches to reducing HIV reservoir size, is a compound worthy of further study for its effects on HIV reservoir size. PMID:28331083

Fast stack activation procedure and effective long-term storage for high-performance polymer electrolyte membrane fuel cell

NASA Astrophysics Data System (ADS)

Yang, Seung Yong; Seo, Dong-Jun; Kim, Myeong-Ri; Seo, Min Ho; Hwang, Sun-Mi; Jung, Yong-Min; Kim, Beom-Jun; Yoon, Young-Gi; Han, Byungchan; Kim, Tae-Young

2016-10-01

Time-saving stack activation and effective long-term storage are one of most important issues that must be resolved for the commercialization of polymer electrolyte membrane fuel cell (PEMFC). Herein, we developed the cost-effective stack activation method to finish the whole activation within 30 min and the long-term storage method by using humidified N2 without any significant decrease in cell's performance for 30 days. Specifically, the pre-activation step with the direct injection of DI water into the stack and storage at 65 or 80 °C for 2 h increases the distinctive phase separation between the hydrophobic and hydrophilic regions in Nafion membrane, which significantly reduces the total activation time within 30 min. Additionally, the long-term storage with humidified N2 has no effect on the Pt oxidation and drying of Nafion membrane for 30 days due to its exergonic reaction in the cell. As a result, the high water content in Nafion membrane and the decrease of Pt oxidation are the critical factors that have a strong influence on the activation and long-term storage for high-performance PEMFC.

PW1 gene/paternally expressed gene 3 (PW1/Peg3) identifies multiple adult stem and progenitor cell populations

PubMed Central

Besson, Vanessa; Smeriglio, Piera; Wegener, Amélie; Relaix, Frédéric; Nait Oumesmar, Brahim; Sassoon, David A.; Marazzi, Giovanna

2011-01-01

A variety of markers are invaluable for identifying and purifying stem/progenitor cells. Here we report the generation of a murine reporter line driven by Pw1 that reveals cycling and quiescent progenitor/stem cells in all adult tissues thus far examined, including the intestine, blood, testis, central nervous system, bone, skeletal muscle, and skin. Neurospheres generated from the adult PW1-reporter mouse show near 100% reporter-gene expression following a single passage. Furthermore, epidermal stem cells can be purified solely on the basis of reporter-gene expression. These cells are clonogenic, repopulate the epidermal stem-cell niches, and give rise to new hair follicles. Finally, we demonstrate that only PW1 reporter-expressing epidermal cells give rise to follicles that are capable of self-renewal following injury. Our data demonstrate that PW1 serves as an invaluable marker for competent self-renewing stem cells in a wide array of adult tissues, and the PW1-reporter mouse serves as a tool for rapid stem cell isolation and characterization. PMID:21709251

Critical issues for engineering cord blood stem cells to produce insulin.

PubMed

Denner, Larry; Urban, Randall J

2008-09-01

The objectives of using cord blood stem cells for treating type 1 diabetes are simple in principle yet complex in biological and molecular mechanisms. These are defined by the complexity of the insulin-producing unit of the pancreas, the islet. Islets are composed of various cell types that arise from diverse lineages and communicate by hormones, growth factors and small-molecule mediators. These processes are regulated by integration of signal transduction pathways. While advances have been made to engineer umbilical cord blood stem cells to produce insulin, these studies only illuminate the potential of such cells to fulfil a necessary, but not sufficient, requirement for transplantation. The challenges ahead demand detailed understanding of molecular mechanisms to move from an opportunistic, phenotypic approach to transplantation and amelioration of blood glucose, to an orderly and logical approach to a biologically and medically meaningful solution. The issues include expansion to generate large numbers of cells, self-renewal to regulate the destiny of cord blood stem cells to repopulate the hematopoietic system, and multipotency of stem cells to generate the distinct cell types of an islet.

High Temperature Steam Electrolysis: Demonstration of Improved Long-Term Performance

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

J. E. O'Brien; X. Zhang; R. C. O'Brien

2011-11-01

Long-term performance is an ongoing issue for hydrogen production based on high-temperature steam electrolysis (HTSE). For commercial deployment, solid-oxide electrolysis stacks must achieve high performance with long-term degradation rates of {approx}0.5%/1000 hours or lower. Significant progress has been achieved toward this goal over the past few years. This paper will provide details of progress achieved under the Idaho National Laboratory high temperature electrolysis research program. Recent long-term stack tests have achieved high initial performance with degradation rates less than 5%/khr. These tests utilize internally manifolded stacks with electrode-supported cells. The cell material sets are optimized for the electrolysis mode ofmore » operation. Details of the cells and stacks will be provided along with details of the test apparatus, procedures, and results.« less

A new method for cryopreserving adipose-derived stem cells: an attractive and suitable large-scale and long-term cell banking technology.

PubMed

De Rosa, Alfredo; De Francesco, Francesco; Tirino, Virginia; Ferraro, Giuseppe A; Desiderio, Vincenzo; Paino, Francesca; Pirozzi, Giuseppe; D'Andrea, Francesco; Papaccio, Gianpaolo

2009-12-01

Recent studies have shown potential ways for improving stem cell cryopreservation. The major need for autologous stem cell use is a long-term storage: this arises from the humans' hope of future use of their own cells. Therefore, it is important to evaluate the cell potential of vitality and differentiation before and after cryopreservation. Although several studies have shown a long-term preservation of adipose tissue, a few of them focused their attention to stem cells. The aim of this study was to evaluate the fate of cryopreserved stem cells collected from adipose tissue and stored at low a temperature in liquid nitrogen through an optimal cryopreservation solution (using slowly cooling in 6% threalose, 4% dimethyl sulfoxide, and 10% fetal bovine serum) and to develop a novel approach to efficiently preserve adipose-derived stem cells (ASCs) for future clinical applications. Results showed that stem cells, after being thawed, are still capable of differentiation and express all surface antigens detected before storage, confirming the integrity of their biology. In particular, ASCs differentiated into adipocytes, showed diffuse positivity for PPARgamma and adiponectin, and were also able to differentiate into endothelial cells without addition of angiogenic factors. Therefore, ASCs can be long-term cryopreserved, and this, due to their great numbers, is an attractive tool for clinical applications as well as of impact for the derived market.

[Development of an incubation system for an inverted microscopy for long-term observation of cell cultures using chamber slides].

PubMed

Feicht, W; Buchner, A; Riesenberg, R

2001-05-01

Trifunctional bispecific antibodies open up new immunological possibilities in tumour treatment. Prior to clinical application, comprehensive investigations using animal models and in vitro examinations need to be done. To investigate long-term interactions between various immunologically active blood cells and individual tumour cells in the presence of antibodies, we developed an incubation system for experimental cell cultures on an inverted microscope. The system consists of a perspex box with a central moisture chamber with integrated water reservoir, external air circulation heating, and a CO2 supply. The sterile cell cultures are located in the wells of a slide positioned within a depression in the water reservoir. The newly developed incubation system enables continuous observation over the long term of experiments under optimal cell cultures conditions in combination with modern video techniques.

Tubular organ epithelialisation

PubMed Central

Saksena, Rhea; Gao, Chuanyu; Wicox, Mathew; de Mel, Achala

2016-01-01

Hollow, tubular organs including oesophagus, trachea, stomach, intestine, bladder and urethra may require repair or replacement due to disease. Current treatment is considered an unmet clinical need, and tissue engineering strategies aim to overcome these by fabricating synthetic constructs as tissue replacements. Smart, functionalised synthetic materials can act as a scaffold base of an organ and multiple cell types, including stem cells can be used to repopulate these scaffolds to replace or repair the damaged or diseased organs. Epithelial cells have not yet completely shown to have efficacious cell–scaffold interactions or good functionality in artificial organs, thus limiting the success of tissue-engineered grafts. Epithelial cells play an essential part of respective organs to maintain their function. Without successful epithelialisation, hollow organs are liable to stenosis, collapse, extensive fibrosis and infection that limit patency. It is clear that the source of cells and physicochemical properties of scaffolds determine the successful epithelialisation. This article presents a review of tissue engineering studies on oesophagus, trachea, stomach, small intestine, bladder and urethral constructs conducted to actualise epithelialised grafts. PMID:28228931

RAD51 Is a Selective DNA Repair Target to Radiosensitize Glioma Stem Cells.

PubMed

King, Harry O; Brend, Tim; Payne, Helen L; Wright, Alexander; Ward, Thomas A; Patel, Karan; Egnuni, Teklu; Stead, Lucy F; Patel, Anjana; Wurdak, Heiko; Short, Susan C

2017-01-10

Patients with glioblastoma die from local relapse despite surgery and high-dose radiotherapy. Resistance to radiotherapy is thought to be due to efficient DNA double-strand break (DSB) repair in stem-like cells able to survive DNA damage and repopulate the tumor. We used clinical samples and patient-derived glioblastoma stem cells (GSCs) to confirm that the DSB repair protein RAD51 is highly expressed in GSCs, which are reliant on RAD51-dependent DSB repair after radiation. RAD51 expression and RAD51 foci numbers fall when these cells move toward astrocytic differentiation. In GSCs, the small-molecule RAD51 inhibitors RI-1 and B02 prevent RAD51 focus formation, reduce DNA DSB repair, and cause significant radiosensitization. We further demonstrate that treatment with these agents combined with radiation promotes loss of stem cells defined by SOX2 expression. This indicates that RAD51-dependent repair represents an effective and specific target in GSCs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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.

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

Bone Marrow Cells Expressing Clara Cell Secretory Protein Increase Epithelial Repair After Ablation of Pulmonary Clara Cells

PubMed Central

Bustos, Martha L; Mura, Marco; Marcus, Paula; Hwang, David; Ludkovski, Olga; Wong, Amy P; Waddell, Thomas K

2013-01-01

We have previously reported a subpopulation of bone marrow cells (BMC) that express Clara cell secretory protein (CCSP), generally felt to be specific to lung Clara cells. Ablation of lung Clara cells has been reported using a transgenic mouse that expresses thymidine kinase under control of the CCSP promoter. Treatment with ganciclovir results in permanent elimination of CCSP+ cells, failure of airway regeneration, and death. To determine if transtracheal delivery of wild-type bone marrow CCSP+ cells is beneficial after ablation of lung CCSP+ cells, transgenic mice were treated with ganciclovir followed by transtracheal administration of CCSP+ or CCSP− BMC. Compared with mice administered CCSP− cells, mice treated with CCSP+ cells had more donor cells lining the airway epithelium, where they expressed epithelial markers including CCSP. Although donor CCSP+ cells did not substantially repopulate the airway, their administration resulted in increased host ciliated cells, better preservation of airway epithelium, reduction of inflammatory cells, and an increase in animal survival time. Administration of CCSP+ BMC is beneficial after permanent ablation of lung Clara cells by increasing bronchial epithelial repair. Therefore, CCSP+ BMC could be important for treatment of lung diseases where airways re-epithelialization is compromised. PMID:23609017

Effect of Lipopolysaccharide on Progesterone Production during Luteinization of Granulosa and Theca cells In Vitro.

PubMed

Shimizu, Takashi; Echizenya, Riku; Miyamoto, Akio

2016-04-01

The aim of this study is to examine the effect of lipopolysaccharide (LPS) on progesterone production during luteinization of granulosa and theca cells isolated from bovine large follicles. Granulosa and theca cells isolated from large follicles of bovine ovaries were exposed to LPS under appropriate hormone conditions in vitro. Progesterone (P4) production in theca cells, but not granulosa cells, was decreased by long-term exposure of LPS. Long-term exposure of LPS suppressed the gene expression of luteinizing hormone receptor in theca cells. Although long-term exposure of LPS did not affect the expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxy-steroid dehydrogenase (3β-HSD) genes, it did inhibit the protein expression of StAR and 3β-HSD in theca cells. These findings suggest that theca cells, rather than granulosa cells, are susceptible to LPS during luteinization and that LPS inhibits P4 production by decreasing protein levels of StAR during luteinization of theca cells. © 2016 Wiley Periodicals, Inc.

Investigation of bacterial repopulation after sinus surgery and perioperative antibiotics.

PubMed

Hauser, Leah J; Ir, Diana; Kingdom, Todd T; Robertson, Charles E; Frank, Daniel N; Ramakrishnan, Vijay R

2016-01-01

Endoscopic sinus surgery (ESS) enjoys high success rates, but repopulation with pathogenic bacteria is 1 of the hallmarks of poorer outcomes. There are many hypothesized sources of repopulating bacteria; however, this process remains largely unexplored. This study examined changes in the sinus microbiome after ESS and medical therapies to identify potential sources for postsurgical microbial repopulation. Samples from the anterior nares, ethmoid sinus, and nasopharynx were taken at the time of surgery from 13 subjects undergoing ESS for chronic rhinosinusitis (CRS). Patients were treated postoperatively with 2 weeks of oral antibiotics and saline rinses. The ethmoid sinus was sampled at 2 and 6 weeks postoperatively; microbiota were characterized using quantitative polymerase chain reaction (qPCR) and 16S ribosomal RNA (rRNA) gene sequencing. The Morisita-Horn beta-diversity index (M-H) was used to compare similarity between samples. The bacterial burden of the ethmoid was higher 2 weeks postoperatively than 6 weeks postoperatively (p = 0.01). The 6-week samples most closely represented the anterior nares and ethmoid at surgery (M-H = 0.58 and 0.59, respectively), and were least similar to the nasopharynx (M-H = 0.28). Principal coordinates analysis (PCoA) plots illustrate that the ethmoid microbiota temporarily shifted after surgery and antibiotics but returned toward baseline in many subjects. Bacterial communities colonizing the ethmoid 6 weeks postoperatively were most similar to anterior nasal cavity and pretreatment sinus microbial profiles, indicating a high degree of resilience in the sinonasal microbiome of most subjects. Interestingly, surgery and postoperative antibiotic therapy does not appear to reduce bacterial burden, but rather, shifts the microbial consortia. © 2015 ARS-AAOA, LLC.

Human Endothelial Cells: Use of Heparin in Cloning and Long-Term Serial Cultivation

NASA Astrophysics Data System (ADS)

Thornton, Susan C.; Mueller, Stephen N.; Levine, Elliot M.

1983-11-01

Endothelial cells from human blood vessels were cultured in vitro, with doubling times of 17 to 21 hours for 42 to 79 population doublings. Cloned human endothelial cell strains were established for the first time and had similar proliferative capacities. This vigorous cell growth was achieved by addition of heparin to culture medium containing reduced concentrations of endothelial cell growth factor. The routine cloning and long-term culture of human endothelial cells will facilitate studying the human endothelium in vitro.

Biochemical Changes in Erythrocytes as a Molecular Marker of Cell Damage during Long-Term Simvastatin Treatment.

PubMed

Mikashinovich, Z I; Belousova, E S

2016-08-01

Long-term administration of simvastatin to rats, irrespective of the baseline cholesterol levels, induced biochemical changes in erythrocytes attesting to hypoxic damage (accumulation of lactate and 2,3-diphosphoglycerate), disturbances in ATP-dependent mechanisms of ion homeostasis regulation (decrease in total ATPase and Ca(2+)-ATPase activities), and antioxidant enzymes system imbalance. These changes can be considered as a sensitive indicator and molecular basis of cell damage during long-term administration of statins.

Density-Dependent Recycling Promotes the Long-Term Survival of Bacterial Populations during Periods of Starvation.

PubMed

Takano, Sotaro; Pawlowska, Bogna J; Gudelj, Ivana; Yomo, Tetsuya; Tsuru, Saburo

2017-02-07

The amount of natural resources in the Earth's environment is in flux, which can trigger catastrophic collapses of ecosystems. How populations survive under nutrient-poor conditions is a central question in ecology. Curiously, some bacteria persist for a long time in nutrient-poor environments. Although this survival may be accomplished through cell death and the recycling of dead cells, the importance of these processes and the mechanisms underlying the survival of the populations have not been quantitated. Here, we use microbial laboratory experiments and mathematical models to demonstrate that death and recycling are essential activities for the maintenance of cell survival. We also show that the behavior of the survivors is governed by population density feedback, wherein growth is limited not only by the available resources but also by the population density. The numerical simulations suggest that population density-dependent recycling could be an advantageous behavior under starvation conditions. How organisms survive after exhaustion of resources is a central question in ecology. Starving Escherichia coli constitute a model system to understand survival mechanisms during long-term starvation. Although death and the recycling of dead cells might play a key role in the maintenance of long-term survival, their mechanisms and importance have not been quantitated. Here, we verified the significance of social recycling of dead cells for long-term survival. We also show that the survivors restrained their recycling and did not use all available nutrients released from dead cells, which may be advantageous under starvation conditions. These results indicate that not only the utilization of dead cells but also restrained recycling coordinate the effective utilization of limited resources for long-term survival under starvation. Copyright © 2017 Takano et al.

Application of micro-porous polycarbonate membranes in dye-sensitized solar cells: Cell performance and long-term stability

NASA Astrophysics Data System (ADS)

Lue, Shingjiang Jessie; Lo, Pei Wen; Hung, Ling-Yung; Tung, Yung Liang

This research investigates the cell performance and long-term stability of dye-sensitized solar cells (DSSCs) containing micro-porous polycarbonate (PC) film as the frame work material to stabilize the electrolyte solution. The track-etched PC film has cylindrical pore geometry, which is beneficial for ion transport in the electrolyte trapped inside the PC film. The photovoltaic efficiency of the DSSC with 0.2-μm PC membrane is 5.75 ± 0.73% under irradiation of 100 mW cm -2, which is slightly lower than that (6.34 ± 0.44%) of cells without PC film. The differences in fill factor and open-circuit voltage between the DSSCs with and without PC film are not statistically significant. The long-term cell performance is carried out at continuous illumination of 100 mW cm -2 (1 sun) and in darkness at 60 °C for up to 1000 h. There is no significant efficiency difference between the cells with and without PC film in light soaking (4.33% vs. 4.52%) for 960 h. In darkness, however, the cells with PC film demonstrate much higher efficiency (at 2.37%) than cells without PC (0.85%) after 1000 h. The improved long-term efficiency data and the higher percentage of working cells confirm the superior lifetime and performance using the micro-porous PC film.

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

Sugano, Yasutaka; Mizuta, Masahiro; Takao, Seishin

Purpose: Radiotherapy of solid tumors has been performed with various fractionation regimens such as multi- and hypofractionations. However, the ability to optimize the fractionation regimen considering the physical dose distribution remains insufficient. This study aims to optimize the fractionation regimen, in which the authors propose a graphical method for selecting the optimal number of fractions (n) and dose per fraction (d) based on dose–volume histograms for tumor and normal tissues of organs around the tumor. Methods: Modified linear-quadratic models were employed to estimate the radiation effects on the tumor and an organ at risk (OAR), where the repopulation of themore » tumor cells and the linearity of the dose-response curve in the high dose range of the surviving fraction were considered. The minimization problem for the damage effect on the OAR was solved under the constraint that the radiation effect on the tumor is fixed by a graphical method. Here, the damage effect on the OAR was estimated based on the dose–volume histogram. Results: It was found that the optimization of fractionation scheme incorporating the dose–volume histogram is possible by employing appropriate cell surviving models. The graphical method considering the repopulation of tumor cells and a rectilinear response in the high dose range enables them to derive the optimal number of fractions and dose per fraction. For example, in the treatment of prostate cancer, the optimal fractionation was suggested to lie in the range of 8–32 fractions with a daily dose of 2.2–6.3 Gy. Conclusions: It is possible to optimize the number of fractions and dose per fraction based on the physical dose distribution (i.e., dose–volume histogram) by the graphical method considering the effects on tumor and OARs around the tumor. This method may stipulate a new guideline to optimize the fractionation regimen for physics-guided fractionation.« less

Decrease in calcitonin and parathyroid hormone mRNA levels and hormone secretion under long-term hypervitaminosis D3 in rats.

PubMed

Fernández-Santos, J M; Utrilla, J C; Conde, E; Hevia, A; Loda, M; Martín-Lacave, I

2001-04-01

In calcium homeostasis, vitamin D3 is a potent serum calcium-raising agent which in vivo regulates both calcitonin (CT) and parathyroid hormone (PTH) gene expression. Serum calcium is the major secretagogue for CT, a hormone product whose biosynthesis is the main biological activity of thyroid C-cells. Taking advantage of this regulatory mechanism, long-term vitamin D3-induced hypercalcemia has been extensively used as a model to produce hyperactivation, hyperplasia and even proliferative lesions of C-cells, supposedly to reduce the sustained high calcium serum concentrations. We have recently demonstrated that CT serum levels did not rise after long-term hypervitaminosis D3. Moreover, C-cells did not have a proliferative response, rather a decrease in CT-producing C-cell number was observed. In order to confirm the inhibitory effect of vitamin D3 on C-cells, Wistar rats were administered vitamin D3 chronically (25,000 IU/d) with or without calcium chloride (CaCl2). Under these long-term vitamin D3-hypercalcemic conditions, calcium, active metabolites of vitamin D3, CT and PTH serum concentrations were determined by RIA; CT and PTH mRNA levels were analysed by Northern blot and in situ hybridization; and, finally, the ultrastructure of calciotrophic hormone-producing cells was analysed by electron microscopy. Our results show, that, in rats, long term administration of vitamin D3 results in a decrease in hormone biosynthetic activities of both PTH and CT-producing cells, albeit at different magnitudes. Based upon these results, we conclude that hypervitaminosis D3-based methods do not stimulate C-cell activity and can not be used to induce proliferative lesions of calcitonin-producing cells.

Hepatic progenitor cells in canine and feline medicine: potential for regenerative strategies

PubMed Central

2014-01-01

New curative therapies for severe liver disease are urgently needed in both the human and veterinary clinic. It is important to find new treatment modalities which aim to compensate for the loss of parenchymal tissue and to repopulate the liver with healthy hepatocytes. A prime focus in regenerative medicine of the liver is the use of adult liver stem cells, or hepatic progenitor cells (HPCs), for functional recovery of liver disease. This review describes recent developments in HPC research in dog and cat and compares these findings to experimental rodent studies and human pathology. Specifically, the role of HPCs in liver regeneration, key components of the HPC niche, and HPC activation in specific types of canine and feline liver disease will be reviewed. Finally, the potential applications of HPCs in regenerative medicine of the liver are discussed and a potential role is suggested for dogs as first target species for HPC-based trials. PMID:24946932

Solid organ fabrication: comparison of decellularization to 3D bioprinting.

PubMed

Jung, Jangwook P; Bhuiyan, Didarul B; Ogle, Brenda M

2016-01-01

Solid organ fabrication is an ultimate goal of Regenerative Medicine. Since the introduction of Tissue Engineering in 1993, functional biomaterials, stem cells, tunable microenvironments, and high-resolution imaging technologies have significantly advanced efforts to regenerate in vitro culture or tissue platforms. Relatively simple flat or tubular organs are already in (pre)clinical trials and a few commercial products are in market. The road to more complex, high demand, solid organs including heart, kidney and lung will require substantive technical advancement. Here, we consider two emerging technologies for solid organ fabrication. One is decellularization of cadaveric organs followed by repopulation with terminally differentiated or progenitor cells. The other is 3D bioprinting to deposit cell-laden bio-inks to attain complex tissue architecture. We reviewed the development and evolution of the two technologies and evaluated relative strengths needed to produce solid organs, with special emphasis on the heart and other tissues of the cardiovascular system.

Decreased heterotopic osteogenesis in vitamin-D-deficient, but normocalcemic guinea pigs

NASA Technical Reports Server (NTRS)

Dziedzic-Goclawska, A.; Toverud, S. U.; Kaminski, A.; Boass, A.; Yamauchi, M.

1992-01-01

The effect of vitamin D deficiency unhampered by hypocalcemia on de novo bone formation was studied in guinea pigs. Heterotopic induction of osteogenesis was evaluated 4 weeks after intramuscular transplantation of allogenic urinary bladder transitional epithelium from vitamin-D-repleted (+D) donors into +D and -D recipients. In -D recipients the frequency of osteogenesis and the amount of induced bone were significantly diminished; induced bone was less mature, scantly cellular woven bone poorly repopulated with bone marrow. No effect of vitamin D deficiency on orthotopic bone growth and on mineralization of orthotopic and heterotopically induced bone was observed. It is proposed that in addition to inducing factors (BMPs, growth factors) which may be responsible for transformation of mesenchymal cells to osteoprogenitor cells, normal concentrations of 1,25-(OH)2D3 may be required for proliferation and further differentiation of these cells into osteoblasts and for expression of genes engaged in extracellular matrix formation and maturation.

Cell-substrate impedance fluctuations of single amoeboid cells encode cell-shape and adhesion dynamics

NASA Astrophysics Data System (ADS)

Leonhardt, Helmar; Gerhardt, Matthias; Höppner, Nadine; Krüger, Kirsten; Tarantola, Marco; Beta, Carsten

2016-01-01

We show systematic electrical impedance measurements of single motile cells on microelectrodes. Wild-type cells and mutant strains were studied that differ in their cell-substrate adhesion strength. We recorded the projected cell area by time-lapse microscopy and observed irregular oscillations of the cell shape. These oscillations were correlated with long-term variations in the impedance signal. Superposed to these long-term trends, we observed fluctuations in the impedance signal. Their magnitude clearly correlated with the adhesion strength, suggesting that strongly adherent cells display more dynamic cell-substrate interactions.

Organ reconstruction: Dream or reality for the future.

PubMed

Stoltz, J-F; Zhang, L; Ye, J S; De Isla, N

2017-01-01

The relevance of research on reconstructed organs is justified by the lack of organs available for transplant and the growing needs for the ageing population. The development of a reconstructed organ involves two parallel complementary steps: de-cellularization of the organ with the need to maintain the structural integrity of the extracellular matrix and vascular network and re-cellularization of the scaffold with stem cells or resident cells.Whole organ engineering for liver, heart, lung or kidneys, is particularly difficult because of the structural complexity of organs and heterogeneity of cells. Rodent, porcine and rhesus monkey organs have been de-cellularized to obtain a scaffold with preserved extracellular matrix and vascular network. As concern the cells for re-cellularization, embryonic, foetal, adult, progenitor stem cells and also iPS have been proposed.Heart construction could be an alternative option for the treatment of cardiac insufficiency. It is based on the use of an extra-cellular matrix coming from an animal's heart and seeded with cells likely to reconstruct a normal cardiac function. Though de-cellularization techniques now seem controlled, the issues posed by the selection of cells capable of generating the various components of cardiac tissue are not settled yet. In addition, the recolonisation of the matrix does not only depend on the phenotype of cells that are used, but it is also impacted by the nature of biochemical signals emitted.Recent researches have shown that it is possible to use decellularized whole liver treated by detergents as scaffold, which keeps the entire network of blood vessels and the integrated extracellular matrix (ECM). Beside of decellularized whole organ scaffold seeding cells selected to repopulate a decellularized liver scaffold are critical for the function of the bioengineered liver. At present, potential cell sources are hepatocyte, and mesenchymal stem cells.Pulmonary regeneration using engineering approaches is complex. In fact, several types of local progenitor cells that contribute to cell repair have been described at different levels of the respiratory tract. Moving towards the alveoles, one finds bronchioalveolar stem cells as well as epithelial cells and pneumocytes. A promising option to increase the donor organ pool is to use allogeneic or xenogeneic decellularized lungs as a scaffold to engineer functional lung tissue ex vivo.The kidney is certainly one of the most difficult organs to reconstruct due to its complex nature and the heterogeneous nature of the cells. There is relatively little research on auto-construction, and experiments have been performed on rats, pigs and monkeys.Nevertheless, before these therapeutic approaches can be applied in clinical practice, many researches are necessary to understand and in particular the behaviour of cells on the decellularized organs as well as the mechanisms of their interaction with the microenvironment. Current knowledges allow optimism for the future but definitive answers can only be given after long term animal studies and controlled clinical studies.

In vitro and in vivo cell invasion and systemic spreading of Mycoplasma agalactiae in the sheep infection model

PubMed Central

Hegde, Shivanand; Hegde, Shrilakshmi; Spergser, Joachim; Brunthaler, René; Rosengarten, Renate; Chopra-Dewasthaly, Rohini

2014-01-01

Generally regarded as extracellular pathogens, molecular mechanisms of mycoplasma persistence, chronicity and disease spread are largely unknown. Mycoplasma agalactiae, an economically important pathogen of small ruminants, causes chronic infections that are difficult to eradicate. Animals continue to shed the agent for several months and even years after the initial infection, in spite of long antibiotic treatment. However, little is known about the strategies that M. agalactiae employs to survive and spread within an immunocompetent host to cause chronic disease. Here, we demonstrate for the first time its ability to invade cultured human (HeLa) and ruminant (BEND and BLF) host cells. Presence of intracellular mycoplasmas is clearly substantiated using differential immunofluorescence technique and quantitative gentamicin invasion assays. Internalized M. agalactiae could survive and exit the cells in a viable state to repopulate the extracellular environment after complete removal of extracellular bacteria with gentamicin. Furthermore, an experimental sheep intramammary infection was carried out to evaluate its systemic spread to organs and host niches distant from the site of initial infection. Positive results obtained via PCR, culture and immunohistochemistry, especially the latter depicting the presence of M. agalactiae in the cytoplasm of mammary duct epithelium and macrophages, clearly provide the first formal proof of M. agalactiae's capability to translocate across the mammary epithelium and systemically disseminate to distant inner organs. Altogether, the findings of these in vitro and in vivo studies indicate that M. agalactiae is capable of entering host cells and this might be the strategy that it employs at a population level to ward off the host immune response and antibiotic action, and to disseminate to new and safer niches to later egress and once again proliferate upon the return of favorable conditions to cause persistent chronic infections. PMID:25129554

Bone Marrow Transplantation Results in Human Donor Blood Cells Acquiring and Displaying Mouse Recipient Class I MHC and CD45 Antigens on Their Surface

PubMed Central

Yamanaka, Nobuko; Wong, Christine J.; Gertsenstein, Marina; Casper, Robert F.; Nagy, Andras; Rogers, Ian M.

2009-01-01

Background Mouse models of human disease are invaluable for determining the differentiation ability and functional capacity of stem cells. The best example is bone marrow transplants for studies of hematopoietic stem cells. For organ studies, the interpretation of the data can be difficult as transdifferentiation, cell fusion or surface antigen transfer (trogocytosis) can be misinterpreted as differentiation. These events have not been investigated in hematopoietic stem cell transplant models. Methodology/Principal Findings In this study we investigated fusion and trogocytosis involving blood cells during bone marrow transplantation using a xenograft model. We report that using a standard SCID repopulating assay almost 100% of the human donor cells appear as hybrid blood cells containing both mouse and human surface antigens. Conclusion/Significance Hybrid cells are not the result of cell-cell fusion events but appear to be due to efficient surface antigen transfer, a process referred to as trogocytosis. Antigen transfer appears to be non-random and includes all donor cells regardless of sub-type. We also demonstrate that irradiation preconditioning enhances the frequency of hybrid cells and that trogocytosis is evident in non-blood cells in chimera mice. PMID:20046883

Nicotine Uses Neuron-Glia Communication to Enhance Hippocampal Synaptic Transmission and Long-term Memory

PubMed Central

López-Hidalgo, Mónica; Salgado-Puga, Karla; Alvarado-Martínez, Reynaldo; Medina, Andrea Cristina; Prado-Alcalá, Roberto A.; García-Colunga, Jesús

2012-01-01

Nicotine enhances synaptic transmission and facilitates long-term memory. Now it is known that bi-directional glia-neuron interactions play important roles in the physiology of the brain. However, the involvement of glial cells in the effects of nicotine has not been considered until now. In particular, the gliotransmitter D-serine, an endogenous co-agonist of NMDA receptors, enables different types of synaptic plasticity and memory in the hippocampus. Here, we report that hippocampal long-term synaptic plasticity induced by nicotine was annulled by an enzyme that degrades endogenous D-serine, or by an NMDA receptor antagonist that acts at the D-serine binding site. Accordingly, both effects of nicotine: the enhancement of synaptic transmission and facilitation of long-term memory were eliminated by impairing glial cells with fluoroacetate, and were restored with exogenous D-serine. Together, these results show that glial D-serine is essential for the long-term effects of nicotine on synaptic plasticity and memory, and they highlight the roles of glial cells as key participants in brain functions. PMID:23185511

Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity.

PubMed

Krajňáková, Jana; Sutela, Suvi; Aronen, Tuija; Gömöry, Dušan; Vianello, Angelo; Häggman, Hely

2011-08-01

In coniferous species, including Greek fir (Abies cephalonica Loud), the involvement of somatic embryo plants in breeding and reforestation programs is dependent on the success of long-term cryostorage of embryogenic cultures during clonal field testing. In the present study on Greek fir, we assayed the recovery, morphological characteristics and genetic fidelity of embryogenic cell lines 6 and 8 during proliferation and maturation after long-term cryostorage. Our results indicate successful recovery of both cell lines after 6 years in cryostorage. In the maturation phase, both cell lines were capable of producing somatic embryos although some differences were detected among experiments. However, these changes were more dependent on the differences in the components of the maturation media or in the experimental set-up than on the long-term cryostorage. During both proliferation and maturation phases, the morphological fidelity of the embryogenic cultures as well as of the somatic embryos were alike before and after cryopreservation. The genetic fidelity of the cryopreserved cell line 6 that was assayed by random amplified polymorphic DNA (i.e. RAPD) markers demonstrated some changes in the RAPD profiles. The results indicate possible genetic aberrations caused by long-term cryopreservation or somaclonal variation during the proliferation stage. However, in spite of these changes the embryogenic cultures did not lose their proliferation or maturation abilities. Copyright © 2011 Elsevier Inc. All rights reserved.

Mechanisms of High Temperature Resistance of Synechocystis sp. PCC 6803: An Impact of Histidine Kinase 34.

PubMed

Červený, Jan; Sinetova, Maria A; Zavřel, Tomáš; Los, Dmitry A

2015-03-02

Synechocystis sp. PCC 6803 is a widely used model cyanobacterium for studying responses and acclimation to different abiotic stresses. Changes in transcriptome, proteome, lipidome, and photosynthesis in response to short term heat stress are well studied in this organism, and histidine kinase 34 (Hik34) is shown to play an important role in mediating such response. Corresponding data on long term responses, however, are fragmentary and vary depending on parameters of experiments and methods of data collection, and thus are hard to compare. In order to elucidate how the early stress responses help cells to sustain long-term heat stress, as well as the role of Hik34 in prolonged acclimation, we examined the resistance to long-term heat stress of wild-type and ΔHik34 mutant of Synechocystis. In this work, we were able to precisely control the long term experimental conditions by cultivating Synechocystis in automated photobioreactors, measuring selected physiological parameters within a time range of minutes. In addition, morphological and ultrastructural changes in cells were analyzed and western blotting of individual proteins was used to study the heat stress-affected protein expression. We have shown that the majority of wild type cell population was able to recover after 24 h of cultivation at 44 °C. In contrast, while ΔHik34 mutant cells were resistant to heat stress within its first hours, they could not recover after 24 h long high temperature treatment. We demonstrated that the early induction of HspA expression and maintenance of high amount of other HSPs throughout the heat incubation is critical for successful adaptation to long-term stress. In addition, it appears that histidine kinase Hik34 is an essential component for the long term high temperature resistance.

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

Long-term tracking of budding yeast cells in brightfield microscopy: CellStar and the Evaluation Platform.

PubMed

Versari, Cristian; Stoma, Szymon; Batmanov, Kirill; Llamosi, Artémis; Mroz, Filip; Kaczmarek, Adam; Deyell, Matt; Lhoussaine, Cédric; Hersen, Pascal; Batt, Gregory

2017-02-01

With the continuous expansion of single cell biology, the observation of the behaviour of individual cells over extended durations and with high accuracy has become a problem of central importance. Surprisingly, even for yeast cells that have relatively regular shapes, no solution has been proposed that reaches the high quality required for long-term experiments for segmentation and tracking (S&T) based on brightfield images. Here, we present CellStar , a tool chain designed to achieve good performance in long-term experiments. The key features are the use of a new variant of parametrized active rays for segmentation, a neighbourhood-preserving criterion for tracking, and the use of an iterative approach that incrementally improves S&T quality. A graphical user interface enables manual corrections of S&T errors and their use for the automated correction of other, related errors and for parameter learning. We created a benchmark dataset with manually analysed images and compared CellStar with six other tools, showing its high performance, notably in long-term tracking. As a community effort, we set up a website, the Yeast Image Toolkit, with the benchmark and the Evaluation Platform to gather this and additional information provided by others. © 2017 The Authors.

Long-term tracking of budding yeast cells in brightfield microscopy: CellStar and the Evaluation Platform

PubMed Central

Versari, Cristian; Stoma, Szymon; Batmanov, Kirill; Llamosi, Artémis; Mroz, Filip; Kaczmarek, Adam; Deyell, Matt

2017-01-01

With the continuous expansion of single cell biology, the observation of the behaviour of individual cells over extended durations and with high accuracy has become a problem of central importance. Surprisingly, even for yeast cells that have relatively regular shapes, no solution has been proposed that reaches the high quality required for long-term experiments for segmentation and tracking (S&T) based on brightfield images. Here, we present CellStar, a tool chain designed to achieve good performance in long-term experiments. The key features are the use of a new variant of parametrized active rays for segmentation, a neighbourhood-preserving criterion for tracking, and the use of an iterative approach that incrementally improves S&T quality. A graphical user interface enables manual corrections of S&T errors and their use for the automated correction of other, related errors and for parameter learning. We created a benchmark dataset with manually analysed images and compared CellStar with six other tools, showing its high performance, notably in long-term tracking. As a community effort, we set up a website, the Yeast Image Toolkit, with the benchmark and the Evaluation Platform to gather this and additional information provided by others. PMID:28179544

GH Mediates Exercise-Dependent Activation of SVZ Neural Precursor Cells in Aged Mice

PubMed Central

Blackmore, Daniel G.; Vukovic, Jana; Waters, Michael J.; Bartlett, Perry F.

2012-01-01

Here we demonstrate, both in vivo and in vitro, that growth hormone (GH) mediates precursor cell activation in the subventricular zone (SVZ) of the aged (12-month-old) brain following exercise, and that GH signaling stimulates precursor activation to a similar extent to exercise. Our results reveal that both addition of GH in culture and direct intracerebroventricular infusion of GH stimulate neural precursor cells in the aged brain. In contrast, no increase in neurosphere numbers was observed in GH receptor null animals following exercise. Continuous infusion of a GH antagonist into the lateral ventricle of wild-type animals completely abolished the exercise-induced increase in neural precursor cell number. Given that the aged brain does not recover well after injury, we investigated the direct effect of exercise and GH on neural precursor cell activation following irradiation. This revealed that physical exercise as well as infusion of GH promoted repopulation of neural precursor cells in irradiated aged animals. Conversely, infusion of a GH antagonist during exercise prevented recovery of precursor cells in the SVZ following irradiation. PMID:23209615

Long-term plasticity in identified hippocampal GABAergic interneurons in the CA1 area in vivo.

PubMed

Lau, Petrina Yau-Pok; Katona, Linda; Saghy, Peter; Newton, Kathryn; Somogyi, Peter; Lamsa, Karri P

2017-05-01

Long-term plasticity is well documented in synapses between glutamatergic principal cells in the cortex both in vitro and in vivo. Long-term potentiation (LTP) and -depression (LTD) have also been reported in glutamatergic connections to hippocampal GABAergic interneurons expressing parvalbumin (PV+) or nitric oxide synthase (NOS+) in brain slices, but plasticity in these cells has not been tested in vivo. We investigated synaptically-evoked suprathreshold excitation of identified hippocampal neurons in the CA1 area of urethane-anaesthetized rats. Neurons were recorded extracellularly with glass microelectrodes, and labelled with neurobiotin for anatomical analyses. Single-shock electrical stimulation of afferents from the contralateral CA1 elicited postsynaptic action potentials with monosynaptic features showing short delay (9.95 ± 0.41 ms) and small jitter in 13 neurons through the commissural pathway. Theta-burst stimulation (TBS) generated LTP of the synaptically-evoked spike probability in pyramidal cells, and in a bistratified cell and two unidentified fast-spiking interneurons. On the contrary, PV+ basket cells and NOS+ ivy cells exhibited either LTD or LTP. An identified axo-axonic cell failed to show long-term change in its response to stimulation. Discharge of the cells did not explain whether LTP or LTD was generated. For the fast-spiking interneurons, as a group, no correlation was found between plasticity and local field potential oscillations (1-3 or 3-6 Hz components) recorded immediately prior to TBS. The results demonstrate activity-induced long-term plasticity in synaptic excitation of hippocampal PV+ and NOS+ interneurons in vivo. Physiological and pathological activity patterns in vivo may generate similar plasticity in these interneurons.

Sepsis induces long-term metabolic and mitochondrial muscle stem cell dysfunction amenable by mesenchymal stem cell therapy

PubMed Central

Rocheteau, P.; Chatre, L.; Briand, D.; Mebarki, M.; Jouvion, G.; Bardon, J.; Crochemore, C.; Serrani, P.; Lecci, P. P.; Latil, M.; Matot, B.; Carlier, P. G.; Latronico, N.; Huchet, C.; Lafoux, A.; Sharshar, T.; Ricchetti, M.; Chrétien, F.

2015-01-01

Sepsis, or systemic inflammatory response syndrome, is the major cause of critical illness resulting in admission to intensive care units. Sepsis is caused by severe infection and is associated with mortality in 60% of cases. Morbidity due to sepsis is complicated by neuromyopathy, and patients face long-term disability due to muscle weakness, energetic dysfunction, proteolysis and muscle wasting. These processes are triggered by pro-inflammatory cytokines and metabolic imbalances and are aggravated by malnutrition and drugs. Skeletal muscle regeneration depends on stem (satellite) cells. Herein we show that mitochondrial and metabolic alterations underlie the sepsis-induced long-term impairment of satellite cells and lead to inefficient muscle regeneration. Engrafting mesenchymal stem cells improves the septic status by decreasing cytokine levels, restoring mitochondrial and metabolic function in satellite cells, and improving muscle strength. These findings indicate that sepsis affects quiescent muscle stem cells and that mesenchymal stem cells might act as a preventive therapeutic approach for sepsis-related morbidity. PMID:26666572

Sepsis induces long-term metabolic and mitochondrial muscle stem cell dysfunction amenable by mesenchymal stem cell therapy.

PubMed

Rocheteau, P; Chatre, L; Briand, D; Mebarki, M; Jouvion, G; Bardon, J; Crochemore, C; Serrani, P; Lecci, P P; Latil, M; Matot, B; Carlier, P G; Latronico, N; Huchet, C; Lafoux, A; Sharshar, T; Ricchetti, M; Chrétien, F

2015-12-15

Sepsis, or systemic inflammatory response syndrome, is the major cause of critical illness resulting in admission to intensive care units. Sepsis is caused by severe infection and is associated with mortality in 60% of cases. Morbidity due to sepsis is complicated by neuromyopathy, and patients face long-term disability due to muscle weakness, energetic dysfunction, proteolysis and muscle wasting. These processes are triggered by pro-inflammatory cytokines and metabolic imbalances and are aggravated by malnutrition and drugs. Skeletal muscle regeneration depends on stem (satellite) cells. Herein we show that mitochondrial and metabolic alterations underlie the sepsis-induced long-term impairment of satellite cells and lead to inefficient muscle regeneration. Engrafting mesenchymal stem cells improves the septic status by decreasing cytokine levels, restoring mitochondrial and metabolic function in satellite cells, and improving muscle strength. These findings indicate that sepsis affects quiescent muscle stem cells and that mesenchymal stem cells might act as a preventive therapeutic approach for sepsis-related morbidity.

Degradation modeling of high temperature proton exchange membrane fuel cells using dual time scale simulation

NASA Astrophysics Data System (ADS)

Pohl, E.; Maximini, M.; Bauschulte, A.; vom Schloß, J.; Hermanns, R. T. E.

2015-02-01

HT-PEM fuel cells suffer from performance losses due to degradation effects. Therefore, the durability of HT-PEM is currently an important factor of research and development. In this paper a novel approach is presented for an integrated short term and long term simulation of HT-PEM accelerated lifetime testing. The physical phenomena of short term and long term effects are commonly modeled separately due to the different time scales. However, in accelerated lifetime testing, long term degradation effects have a crucial impact on the short term dynamics. Our approach addresses this problem by applying a novel method for dual time scale simulation. A transient system simulation is performed for an open voltage cycle test on a HT-PEM fuel cell for a physical time of 35 days. The analysis describes the system dynamics by numerical electrochemical impedance spectroscopy. Furthermore, a performance assessment is performed in order to demonstrate the efficiency of the approach. The presented approach reduces the simulation time by approximately 73% compared to conventional simulation approach without losing too much accuracy. The approach promises a comprehensive perspective considering short term dynamic behavior and long term degradation effects.

The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture

NASA Technical Reports Server (NTRS)

Schatten, H.; Hedrick, J.; Chakrabarti, A.

1998-01-01

Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

Sulfur poisoning of Ni/Gadolinium-doped ceria anodes: A long-term study outlining stable solid oxide fuel cell operation

NASA Astrophysics Data System (ADS)

Riegraf, Matthias; Zekri, Atef; Knipper, Martin; Costa, Rémi; Schiller, Günter; Friedrich, K. Andreas

2018-03-01

This work presents an analysis of the long-term behavior of nickel/gadolinium-doped ceria (CGO) anode-based solid oxide fuel cells (SOFC) under sulfur poisoning conditions. A parameter study of sulfur-induced irreversible long-term degradation of commercial, high-performance single cells was carried out at 900 °C for different H2/N2/H2S fuel gas atmospheres, current densities and Ni/CGO anodes. The poisoning periods of the cells varied from 200 to 1500 h. The possibility of stable long-term Ni/CGO anode operation under sulfur exposure is established and the critical operating regime is outlined. Depending on the operating conditions, two degradation phenomena can be observed. Small degradation of the ohmic resistance was witnessed for sulfur exposure times of approximately 1000 h. Moreover, degradation of the anode charge transfer resistance was observed to be triggered by the combination of a small anodic potential step and high sulfur coverage on Ni. The microstructural evolution of altered Ni/CGO anodes was examined post-mortem by means of SEM and FIB/SEM, and is correlated to the anode performance degradation under critical operating conditions, establishing Ni depletion, porosity increase and a tripe phase boundary density decrease in the anode functional layer. It is shown that short-term sulfur poisoning behavior can be used to assess long-term stability.

Immunomodulatory effect of ganoderma lucidum polysaccharides (GLP) on long-term heavy-load exercising mice.

PubMed

Shi, Yali; Cai, Dehua; Wang, Xiaojie; Liu, Xinshen

2012-12-01

Long-term heavy-load exercise can lead to a decrease in the organism's immune response. In this study, we used 100 Kunming (KM) mice to investigate the immune-regulatory effects of Ganoderma lucidum polysaccharides (GLP) on long-term heavy-load exercising mice. Peripheral white blood cells (WBC), the absolute value of neutrophils (NEUT), the phagocytic function of macrophages, serum agglutination valence, and the number of plaque-forming cells (PFC) were evaluated 4 weeks after gavaging long-term heavy-load exercising mice with GLP. After exercise, the WBC count in peripheral blood, absolute neutrophil count, macrophage phagocytic index, serum agglutination valence, and the number of plaque-forming cells were significantly reduced in the mice not fed GLP. Both medium and high doses of GLP drastically increased peripheral WBC, absolute neutrophil count, macrophage phagocytic index, serum agglutination valence, and the number of plaque-forming cells in long-term heavy-load exercising mice. High doses of GLP increased peritoneal macrophage phagocytic rate considerably. With this study, we demonstrate that 4 weeks of heavy-load exercise can lead to exercise-induced immunosuppression in mice. A supplement of GLP fed to these mice improves both non-specific and specific immune responses among these mice. The effect for the high-dose GLP treatment is especially significant.

Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche

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

Katano, Takahito; Ootani, Akifumi; Department of Internal Medicine, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501

2013-03-22

Highlights: ► We established a 3D culture system to allow long-term culture of stomach cells. ► In this culture system, gastric epithelial cells grew for about 3 months. ► The cultured cells differentiated into multi-units of the stomach. ► This culture method should be useful for elucidating the cause of gastric diseases. -- Abstract: Compared to the small intestine and colon, little is known about stem cells in the stomach because of a lack of specific stem cell markers and an in vitro system that allows long-term culture. Here we describe a long-term three-dimensional (3D) primary gastric culture system withinmore » the stem cell niche. Glandular stomach cells from neonatal mice cultured in collagen gel yielded expanding sphere-like structures for 3 months. The wall of the gastrospheres consisted of a highly polarized epithelial monolayer with an outer lining of myofibroblasts. The epithelial cells showed a tall columnar cell shape, basal round nuclei, and mucus-filled cytoplasm as well as expression of MUC5AC, indicating differentiation into gastric surface mucous cells. These cells demonstrated the features of fully differentiated gastric surface mucous cells such as microvilli, junctional complexes, and glycogen and secretory granules. Fewer than 1% of cultured epithelial cells differentiated into enteroendocrine cells. Active proliferation of the epithelial cells and many apoptotic cells in the inner lumen revealed the rapid cell turnover in gastrospheres in vitro. This method enables us to investigate the role of signaling between cell–cell and epithelial–mesenchymal interactions in an environment that is extremely similar to the in vivo environment.« less

Modelling Potential Consequences of Different Geo-Engineering Treatments for the Baltic Sea Ecosystem

NASA Astrophysics Data System (ADS)

Schrum, C.; Daewel, U.

2017-12-01

From 1950 onwards, the Baltic Sea ecosystem suffered increasingly from eutrophication. The most obvious reason for the eutrophication is the huge amount of nutrients (nitrogen and phosphorus) reaching the Baltic Sea from human activities. However, although nutrient loads have been decreasing since 1980, the hypoxic areas have not decreased accordingly. Thus, geo-engineering projects were discussed and evaluated to artificially ventilate the Baltic Sea deep water and suppress nutrient release from the sediments. Here, we aim at understanding the consequences of proposed geo-engineering projects in the Baltic Sea using long-term scenario modelling. For that purpose, we utilize a 3d coupled ecosystem model ECOSMO E2E, a novel NPZD-Fish model approach that resolves hydrodynamics, biogeochemical cycling and lower and higher trophic level dynamics. We performed scenario modelling that consider proposed geo-engineering projects such as artificial ventilation of Baltic Sea deep waters and phosphorus binding in sediments with polyaluminium chlorides. The model indicates that deep-water ventilation indeed suppresses phosphorus release in the first 1-4 years of treatment. Thereafter macrobenthos repopulates the formerly anoxic bottom regions and nutrients are increasingly recycled in the food web. Consequently, overall system productivity and fish biomass increases and toxic algae blooms decrease. However, deep-water ventilation has no long-lasting effect on the ecosystem: soon after completion of the ventilation process, the system turns back into its original state. Artificial phosphorus binding in sediments in contrast decreases overall ecosystem productivity through permanent removal of phosphorus. As expected it decreases bacterial production and toxic algae blooms, but it also decreases fish production substantially. Contrastingly to deep water ventilation, artificial phosphorus binding show a long-lasting effect over decades after termination of the treatment.

T-Cell-Mediated Immune Responses in Patients with Cutaneous or Mucosal Leishmaniasis: Long-Term Evaluation after Therapy

PubMed Central

Da-Cruz, Alda Maria; Bittar, Rita; Mattos, Marise; Oliveira-Neto, Manuel P.; Nogueira, Ricardo; Pinho-Ribeiro, Vanessa; Azeredo-Coutinho, Rilza Beatriz; Coutinho, Sergio G.

2002-01-01

T-cell immune responses in patients with cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) were studied during the active disease, at the end of therapy, and 1 to 17 years posttherapy (long-term follow-up). Lymphocyte proliferative responses, phenotypic characterization of CD4+ and CD8+ Leishmania-reactive T cells, and cytokine production were assayed. Patients with active ML and CL showed higher proportions of CD4+ than CD8+ T cells. In CL, the healing process was associated with a decrease of CD4+ and an increase of CD8+, leading to similar CD4+ and CD8+ proportions. This pattern was only seen in ML after long-term therapy. Long-term follow-up of patients with CL showed a positive CD4+/CD8+ ratio as observed during the active disease, although the percentages of these T cell subsets were significantly lower. Patients with CL did not show significant differences between gamma interferon (IFN-γ) and interleukin-5 (IL-5) production during the period of study. Patients with active ML presented higher IFN-γ and IL-5 levels compared to patients with active CL. IL-4 was only detected during active disease. Patients long term after cure from ML showed increasing production of IFN-γ, significant decrease of IL-5, and no IL-4 production. Two apparently beneficial immunological parameters were detected in tegumentary leishmaniasis: (i) decreasing proportions of CD4+ Leishmania-reactive T cells in the absence of IL-4 production associated with cure of CL and ML and (ii) decreasing levels of IL-5 long after cure, better detected in patients with ML. The observed T-cell responses maintained for a long period in healed patients could be relevant for immunoprotection against reinfection and used as a parameter for determining the prognosis of patients and selecting future vaccine preparations. PMID:11874860

Genetic modification of stem cells for improved therapy of the infarcted myocardium.

PubMed

Haider, Husnain Kh; Mustafa, Anique; Feng, Yuliang; Ashraf, Muhammad

2011-10-03

The conventional treatment modalities for ischemic heart disease only provide symptomatic relief to the patient without repairing and regenerating the damaged myocardium. Stem cell transplantation has emerged as a promising alternative therapeutic approach for cardiovascular diseases. Stem cells possess the potential of differentiation to adopt morphofunctional cardiac and vasculogenic phenotypes to repopulate the scar tissue and restore regional blood flow in the ischemic myocardium. These beneficial therapeutic effects make stem cell transplantation the method of choice for the treatment of ischemic heart disease. The efficacy of stem cell transplantation may be augmented by genetic manipulation of the cells prior to transplantation. Not only will insertion of therapeutic transgene(s) into the stem cells support the survival and differentiation of cells in the unfavorable microenvironment of the ischemic myocardium, but also the genetically manipulated stem cells will serve as a source of the transgene expression product in the heart for therapeutic benefits. We provide an overview of the extensively studied stem cell types for cardiac regeneration, the various methods in which these cells have been genetically manipulated and rationale of genetic modification of stem cells for use in regenerative cardiovascular therapeutics.

Long-term surveillance plan for the Green River, Utah, disposal site

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

NONE

1997-06-01

The long-term surveillance plan (LTSP) for the Green River, Utah, Uranium Mill Tailings Remedial Action (UMTRA) Project disposal site describes the surveillance activities for the Green River disposal cell. The U.S. Department of Energy (DOE) will carry out these activities to ensure that the disposal cell continues to function as designed. This final LTSP was prepared as a requirement for acceptance under the U.S. Nuclear Regulatory Commission (NRC) general license for custody and long-term care of residual radioactive materials (RRM). This LTSP documents whether the land and interests are owned by the United States or an Indian tribe and detailsmore » how the long-term care of the disposal site will be carried out. The Green River, Utah, LTSP is based on the DOE`s Guidance for Implementing the UMTRA Project Long-term Surveillance Program (DOE, 1992a).« less

[Establishment of immortal lymphoblastoid cell bank of keloids pedigree].

PubMed

Song, Mei; Gao, Jian-hua; Yan, Xin; Liu, Xiao-jun; Chen, Yang

2006-11-01

To provide perpetual research materials for long term studies by establishing immortal lymphoblastoid cell bank of keloids pedigree. The immortal lymphoblastoid cell lines of keloids pedigree were established by Epstein-Barr virus transformation of peripheral blood B lymphocytes. 27 immortal lymphoblastoid cell lines of keloids pedigree were obtained successfully, all of the immortal lymphoblastoid cell lines were successfully revivificated after been frozen in liquid nitrogen. It is important to establish immortal lymphoblastoid cell bank of keloids pedigree and provide long-term DNA materials for deep study of keloids in the future.

Hyaline cartilage regeneration by combined therapy of microfracture and long-term bone morphogenetic protein-2 delivery.

PubMed

Yang, Hee Seok; La, Wan-Geun; Bhang, Suk Ho; Kim, Hak-Jun; Im, Gun-Il; Lee, Haeshin; Park, Jung-Ho; Kim, Byung-Soo

2011-07-01

Microfracture of cartilage induces migration of bone-marrow-derived mesenchymal stem cells. However, this treatment often results in fibrocartilage regeneration. Growth factors such as bone morphogenetic protein (BMP)-2 induce the differentiation of bone-marrow-derived mesenchymal stem cells into chondrocytes, which can be used for hyaline cartilage regeneration. Here, we tested the hypothesis that long-term delivery of BMP-2 to cartilage defects subjected to microfracture results in regeneration of high-quality hyaline-like cartilage, as opposed to short-term delivery of BMP-2 or no BMP-2 delivery. Heparin-conjugated fibrin (HCF) and normal fibrin were used as carriers for the long- and short-term delivery of BMP-2, respectively. Rabbit articular cartilage defects were treated with microfracture combined with one of the following: no treatment, fibrin, short-term delivery of BMP-2, HCF, or long-term delivery of BMP-2. Eight weeks after treatment, histological analysis revealed that the long-term delivery of BMP-2 group (microfracture + HCF + BMP-2) showed the most staining with alcian blue. A biochemical assay, real-time polymerase chain reaction assay and Western blot analysis all revealed that the long-term delivery of BMP-2 group had the highest glucosaminoglycan content as well as the highest expression level of collagen type II. Taken together, the long-term delivery of BMP-2 to cartilage defects subjected to microfracture resulted in regeneration of hyaline-like cartilage, as opposed to short-term delivery or no BMP-2 delivery. Therefore, this method could be more convenient for hyaline cartilage regeneration than autologous chondrocyte implantation due to its less invasive nature and lack of cell implantation.

Use of autologous human mesenchymal stromal cell/fibrin clot constructs in upper limb non-unions: long-term assessment.

PubMed

Giannotti, Stefano; Trombi, Luisa; Bottai, Vanna; Ghilardi, Marco; D'Alessandro, Delfo; Danti, Serena; Dell'Osso, Giacomo; Guido, Giulio; Petrini, Mario

2013-01-01

Tissue engineering appears to be an attractive alternative to the traditional approach in the treatment of fracture non-unions. Mesenchymal stromal cells (MSCs) are considered an appealing cell source for clinical intervention. However, ex vivo cell expansion and differentiation towards the osteogenic lineage, together with the design of a suitable scaffold have yet to be optimized. Major concerns exist about the safety of MSC-based therapies, including possible abnormal overgrowth and potential cancer evolution. We examined the long-term efficacy and safety of ex vivo expanded bone marrow MSCs, embedded in autologous fibrin clots, for the healing of atrophic pseudarthrosis of the upper limb. Our research work relied on three main issues: use of an entirely autologous context (cells, serum for ex vivo cell culture, scaffold components), reduced ex vivo cell expansion, and short-term MSC osteoinduction before implantation. Bone marrow MSCs isolated from 8 patients were expanded ex vivo until passage 1 and short-term osteo-differentiated in autologous-based culture conditions. Tissue-engineered constructs designed to embed MSCs in autologous fibrin clots were locally implanted with bone grafts, calibrating their number on the extension of bone damage. Radiographic healing was evaluated with short- and long-term follow-ups (range averages: 6.7 and 76.0 months, respectively). All patients recovered limb function, with no evidence of tissue overgrowth or tumor formation. Our study indicates that highly autologous treatment can be effective and safe in the long-term healing of bone non-unions. This tissue engineering approach resulted in successful clinical and functional outcomes for all patients.

Loss of memory B cells impairs maintenance of long-term serologic memory during HIV-1 infection.

PubMed

Titanji, Kehmia; De Milito, Angelo; Cagigi, Alberto; Thorstensson, Rigmor; Grützmeier, Sven; Atlas, Ann; Hejdeman, Bo; Kroon, Frank P; Lopalco, Lucia; Nilsson, Anna; Chiodi, Francesca

2006-09-01

Circulating memory B cells are severely reduced in the peripheral blood of HIV-1-infected patients. We investigated whether dysfunctional serologic memory to non-HIV antigens is related to disease progression by evaluating the frequency of memory B cells, plasma IgG, plasma levels of antibodies to measles, and Streptococcus pneumoniae, and enumerating measles-specific antibody-secreting cells in patients with primary, chronic, and long-term nonprogressive HIV-1 infection. We also evaluated the in vitro production of IgM and IgG antibodies against measles and S pneumoniae antigens following polyclonal activation of peripheral blood mononuclear cells (PBMCs) from patients. The percentage of memory B cells correlated with CD4+ T-cell counts in patients, thus representing a marker of disease progression. While patients with primary and chronic infection had severe defects in serologic memory, long-term nonprogressors had memory B-cell frequency and levels of antigen-specific antibodies comparable with controls. We also evaluated the effect of antiretroviral therapy on these serologic memory defects and found that antiretroviral therapy did not restore serologic memory in primary or in chronic infection. We suggest that HIV infection impairs maintenance of long-term serologic immunity to HIV-1-unrelated antigens and this defect is initiated early in infection. This may have important consequences for the response of HIV-infected patients to immunizations.

Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia.

PubMed

Shlush, Liran I; Zandi, Sasan; Mitchell, Amanda; Chen, Weihsu Claire; Brandwein, Joseph M; Gupta, Vikas; Kennedy, James A; Schimmer, Aaron D; Schuh, Andre C; Yee, Karen W; McLeod, Jessica L; Doedens, Monica; Medeiros, Jessie J F; Marke, Rene; Kim, Hyeoung Joon; Lee, Kwon; McPherson, John D; Hudson, Thomas J; Brown, Andrew M K; Yousif, Fouad; Trinh, Quang M; Stein, Lincoln D; Minden, Mark D; Wang, Jean C Y; Dick, John E

2014-02-20

In acute myeloid leukaemia (AML), the cell of origin, nature and biological consequences of initiating lesions, and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukaemic phase. Here, highly purified haematopoietic stem cells (HSCs), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3A mutations (DNMT3A(mut)) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3A(mut)-bearing HSCs showed a multilineage repopulation advantage over non-mutated HSCs in xenografts, establishing their identity as pre-leukaemic HSCs. Pre-leukaemic HSCs were found in remission samples, indicating that they survive chemotherapy. Therefore DNMT3A(mut) arises early in AML evolution, probably in HSCs, leading to a clonally expanded pool of pre-leukaemic HSCs from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukaemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance.

Role of casein kinase 1A1 in the biology and targeted therapy of del(5q) MDS

PubMed Central

Schneider, Rebekka K.; Ademà, Vera; Heckl, Dirk; Järås, Marcus; Mallo, Mar; Lord, Allegra M.; Chu, Lisa P.; McConkey, Marie E.; Kramann, Rafael; Mullally, Ann; Bejar, Rafael; Solé, Francesc; Ebert, Benjamin L.

2014-01-01

Summary The Casein kinase 1A1 gene (CSNK1A1) is a putative tumor suppressor gene located in the common deleted region for del(5q) myelodysplastic syndrome (MDS). We generated a murine model with conditional inactivation of Csnk1a1 and found that Csnk1a1 haploinsufficiency induces hematopoietic stem cell expansion and a competitive repopulation advantage whereas homozygous deletion induces hematopoietic stem cell failure. Based on this finding, we found that heterozygous inactivation of Csnk1a1 sensitizes cells to a CSNK1 inhibitor relative to cells with two intact alleles. In addition, we identified recurrent somatic mutations in CSNK1A1 on the non-deleted allele of patients with del(5q) MDS. These studies demonstrate that CSNK1A1 plays a central role in the biology of del(5q) MDS and is a promising therapeutic target. PMID:25242043

Exploiting the unique regenerative capacity of the liver to underpin cell and gene therapy strategies for genetic and acquired liver disease.

PubMed

Logan, Grant J; de Alencastro, Gustavo; Alexander, Ian E; Yeoh, George C

2014-11-01

The number of genetic or acquired diseases of the liver treatable by organ transplantation is ever-increasing as transplantation techniques improve placing additional demands on an already limited organ supply. While cell and gene therapies are distinctly different modalities, they offer a synergistic alternative to organ transplant due to distinct architectural and physiological properties of the liver. The hepatic blood supply and fenestrated endothelial system affords relatively facile accessibility for cell and/or gene delivery. More importantly, however, the remarkable capacity of hepatocytes to proliferate and repopulate the liver creates opportunities for new treatments based on emerging technologies. This review will summarise current understanding of liver regeneration, describe clinical and experimental cell and gene therapeutic modalities and discuss critical challenges to translate these new technologies to wider clinical utility. This article is part of a Directed Issue entitled: "Regenerative Medicine: the challenge of translation". Copyright © 2014 Elsevier Ltd. All rights reserved.

Long-term load duration induces N-cadherin down-regulation and loss of cell phenotype of nucleus pulposus cells in a disc bioreactor culture.

PubMed

Li, Pei; Zhang, Ruijie; Wang, Liyuan; Gan, Yibo; Xu, Yuan; Song, Lei; Luo, Lei; Zhao, Chen; Zhang, Chengmin; Ouyang, Bin; Tu, Bing; Zhou, Qiang

2017-04-30

Long-term exposure to a mechanical load causes degenerative changes in the disc nucleus pulposus (NP) tissue. A previous study demonstrated that N-cadherin (N-CDH)-mediated signalling can preserve the NP cell phenotype. However, N-CDH expression and the resulting phenotype alteration in NP cells under mechanical compression remain unclear. The present study investigated the effects of the compressive duration on N-CDH expression and on the phenotype of NP cells in an ex vivo disc organ culture. Porcine discs were organ cultured in a self-developed mechanically active bioreactor for 7 days. The discs were subjected to different dynamic compression durations (1 and 8 h at a magnitude of 0.4 MPa and frequency of 1.0 Hz) once per day. Discs that were not compressed were used as controls. The results showed that long-term compression duration (8 h) significantly down-regulated the expression of N-CDH and NP-specific molecule markers (Brachyury, Laminin, Glypican-3 and Keratin 19), attenuated Alcian Blue staining intensity, decreased glycosaminoglycan (GAG) and hydroxyproline (HYP) contents and decreased matrix macromolecule (aggrecan and collagen II) expression compared with the short-term compression duration (1 h). Taken together, these findings demonstrate that long-term load duration can induce N-CDH down-regulation, loss of normal cell phenotype and result in attenuation of NP-related matrix synthesis in NP cells. © 2017 The Author(s).

STAT3 Controls the Long-Term Survival and Phenotype of Repair Schwann Cells during Nerve Regeneration.

PubMed

Benito, Cristina; Davis, Catherine M; Gomez-Sanchez, Jose A; Turmaine, Mark; Meijer, Dies; Poli, Valeria; Mirsky, Rhona; Jessen, Kristjan R

2017-04-19

After nerve injury, Schwann cells convert to a phenotype specialized to promote repair. But during the slow process of axonal regrowth, these repair Schwann cells gradually lose their regeneration-supportive features and eventually die. Although this is a key reason for the frequent regeneration failures in humans, the transcriptional mechanisms that control long-term survival and phenotype of repair cells have not been studied, and the molecular signaling underlying their decline is obscure. We show, in mice, that Schwann cell STAT3 has a dual role. It supports the long-term survival of repair Schwann cells and is required for the maintenance of repair Schwann cell properties. In contrast, STAT3 is less important for the initial generation of repair Schwann cells after injury. In repair Schwann cells, we find that Schwann cell STAT3 activation by Tyr705 phosphorylation is sustained during long-term denervation. STAT3 is required for maintaining autocrine Schwann cell survival signaling, and inactivation of Schwann cell STAT3 results in a striking loss of repair cells from chronically denervated distal stumps. STAT3 inactivation also results in abnormal morphology of repair cells and regeneration tracks, and failure to sustain expression of repair cell markers, including Shh, GDNF, and BDNF. Because Schwann cell development proceeds normally without STAT3, the function of this factor appears restricted to Schwann cells after injury. This identification of transcriptional mechanisms that support long-term survival and differentiation of repair cells will help identify, and eventually correct, the failures that lead to the deterioration of this important cell population. SIGNIFICANCE STATEMENT Although injured peripheral nerves contain repair Schwann cells that provide signals and spatial clues for promoting regeneration, the clinical outcome after nerve damage is frequently poor. A key reason for this is that, during the slow growth of axons through the proximal parts of injured nerves repair, Schwann cells gradually lose regeneration-supporting features and eventually die. Identification of signals that sustain repair cells is therefore an important goal. We have found that in mice the transcription factor STAT3 protects these cells from death and contributes to maintaining the molecular and morphological repair phenotype that promotes axonal regeneration. Defining the molecular mechanisms that maintain repair Schwann cells is an essential step toward developing therapeutic strategies that improve nerve regeneration and functional recovery. Copyright © 2017 Benito, Davis et al.

Bovine central memory T cells are highly proliferative in response to bovine tuberculosis infection

USDA-ARS?s Scientific Manuscript database

Long-term (i.e., 14 days) cultured IFN-gamma ELISPOT assays measure central memory T cell (Tcm) responses in both humans and cattle. With bovine tuberculosis, vaccine-elicited long-term IFN-gamma ELISPOT responses correlate with protection. In other species, Tcm’s pose low activation threshold and a...

Acute and long-term in vitro effects of zinc oxide nanoparticles.

PubMed

Annangi, Balasubramanyam; Rubio, Laura; Alaraby, Mohamed; Bach, Jordi; Marcos, Ricard; Hernández, Alba

2016-09-01

Since most of the toxic studies of zinc oxide nanoparticles (ZnO NPs) focused on acute and high-dose exposure conditions, the aim of the present study was to fill the existing knowledge gap of long-term effects of ZnO NPs at sub-toxic doses. To overcome this point, we have evaluated the toxic, genotoxic, and carcinogenic effects of ZnO NPs under long-term treatments (12 weeks), using a sub-toxic dose (1 µg/mL) according to acute 48-h exposure. Preliminarily, oxidative stress and genotoxic/oxidative DNA damage were determined under acute exposure and high-dose conditions. To determine the role of oxidative DNA damage, a wild-type mouse embryonic fibroblast (MEF Ogg1 (+/+)) and its isogenic 8-oxo-guanine DNA glycosylase 1 (Ogg1) knockout partner (MEF Ogg1 (-/-)) cell lines were used. Although short-term exposure (24-h) experiments demonstrated that ZnO NPs were able to induce ROS, genotoxicity, and oxidative DNA damage in both cell lines, no effects were obtained under long-term exposure scenario. Thus, 1 µg/mL exposure over 12 weeks was unable to induce genotoxicity as well as cellular transformation in both cell types, as indicated by the lack of observed morphological cell changes, variations in the secretion of matrix metalloproteinases, and anchorage-independent cell growth ability, regarded as cancer-like phenotypic hallmarks. Our results indicate that short-term effects of ZnO NP exposure are not replicated under long-term and sub-toxic dose conditions. All together, the lack of genotoxic/carcinogenic effects after chronic treatments seem to indicate a reduced risk associated with ZnO NP exposure.

Re-epithelialization of cutaneous wounds in adult zebrafish combines mechanisms of wound closure in embryonic and adult mammals.

PubMed

Richardson, Rebecca; Metzger, Manuel; Knyphausen, Philipp; Ramezani, Thomas; Slanchev, Krasimir; Kraus, Christopher; Schmelzer, Elmon; Hammerschmidt, Matthias

2016-06-15

Re-epithelialization of cutaneous wounds in adult mammals takes days to complete and relies on numerous signalling cues and multiple overlapping cellular processes that take place both within the epidermis and in other participating tissues. Re-epithelialization of partial- or full-thickness skin wounds of adult zebrafish, however, is extremely rapid and largely independent of the other processes of wound healing. Live imaging after treatment with transgene-encoded or chemical inhibitors reveals that re-epithelializing keratinocytes repopulate wounds by TGF-β- and integrin-dependent lamellipodial crawling at the leading edges of the epidermal tongue. In addition, re-epithelialization requires long-range epithelial rearrangements, involving radial intercalations, flattening and directed elongation of cells - processes that are dependent on Rho kinase, JNK and, to some extent, planar cell polarity within the epidermis. These rearrangements lead to a massive recruitment of keratinocytes from the adjacent epidermis and make re-epithelialization independent of keratinocyte proliferation and the mitogenic effect of FGF signalling, which are only required after wound closure, allowing the epidermis outside the wound to re-establish its normal thickness. Together, these results demonstrate that the adult zebrafish is a valuable in vivo model for studying and visualizing the processes involved in cutaneous wound closure, facilitating the dissection of direct from indirect and motogenic from mitogenic effects of genes and molecules affecting wound re-epithelialization. © 2016. Published by The Company of Biologists Ltd.

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.

Rejuvenation of the aged muscle stem cell population restores strength to injured aged muscles

PubMed Central

Cosgrove, Benjamin D.; Gilbert, Penney M.; Porpiglia, Ermelinda; Mourkioti, Foteini; Lee, Steven P.; Corbel, Stephane Y.; Llewellyn, Michael E.; Delp, Scott L.; Blau, Helen M.

2014-01-01

The aged suffer from progressive muscle weakness and regenerative failure. We demonstrate that muscle regeneration is impaired with aging due in part to a cell-autonomous functional decline in skeletal muscle stem cells (MuSCs). Two-thirds of aged MuSCs are intrinsically defective relative to young MuSCs, with reduced capacity to repair myofibers and repopulate the stem cell reservoir in vivo following transplantation due to a higher incidence of cells that express senescence markers and that have elevated p38α/β MAPK activity. We show that these limitations cannot be overcome by transplantation into the microenvironment of young recipient muscles. In contrast, subjecting the aged MuSC population to transient inhibition of p38α/β in conjunction with culture on soft hydrogel substrates rapidly expands the residual functional aged MuSC population, rejuvenating its potential for regeneration, serial transplantation, and strengthening damaged muscles of aged mice. These findings reveal a synergy between biophysical and biochemical cues that provides a paradigm for a localized autologous muscle stem cell therapy in aged individuals. PMID:24531378

Cryopreservation of testicular tissue before long-term testicular cell culture does not alter in vitro cell dynamics.

PubMed

Baert, Yoni; Braye, Aude; Struijk, Robin B; van Pelt, Ans M M; Goossens, Ellen

2015-11-01

To assess whether testicular cell dynamics are altered during long-term culture after testicular tissue cryopreservation. Experimental basic science study. Reproductive biology laboratory. Testicular tissue with normal spermatogenesis was obtained from six donors. None. Detection and comparison of testicular cells from fresh and frozen tissues during long-term culture. Human testicular cells derived from fresh (n = 3) and cryopreserved (n = 3) tissues were cultured for 2 months and analyzed with quantitative reverse-transcription polymerase chain reaction and immunofluorescence. Spermatogonia including spermatogonial stem cells (SSCs) were reliably detected by combining VASA, a germ cell marker, with UCHL1, a marker expressed by spermatogonia. The established markers STAR, ACTA2, and SOX9 were used to analyze the presence of Leydig cells, peritubular myoid cells, and Sertoli cells, respectively. No obvious differences were found between the cultures initiated from fresh or cryopreserved tissues. Single or small groups of SSCs (VASA(+)/UCHL1(+)) were detected in considerable amounts up to 1 month of culture, but infrequently after 2 months. SSCs were found attached to the feeder monolayer, which expressed markers for Sertoli cells, Leydig cells, and peritubular myoid cells. In addition, VASA(-)/UCHL1(+) cells, most likely originating from the interstitium, also contributed to this monolayer. Apart from Sertoli cells, all somatic cell types could be detected throughout the culture period. Testicular tissue can be cryopreserved before long-term culture without modifying its outcome, which encourages implementation of testicular tissue banking for fertility preservation. However, because of the limited numbers of SSCs available after 2 months, further exploration and optimization of the culture system is needed. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Growth factors and medium hyperglycemia induce Sox9+ ductal cell differentiation into β cells in mice with reversal of diabetes

PubMed Central

Zhang, Mingfeng; Lin, Qing; Qi, Tong; Wang, Tiankun; Chen, Ching-Cheng; Riggs, Arthur D.; Zeng, Defu

2016-01-01

We previously reported that long-term administration of a low dose of gastrin and epidermal growth factor (GE) augments β-cell neogenesis in late-stage diabetic autoimmune mice after eliminating insulitis by induction of mixed chimerism. However, the source of β-cell neogenesis is still unknown. SRY (sex-determining region Y)-box 9+ (Sox9+) ductal cells in the adult pancreas are clonogenic and can give rise to insulin-producing β cells in an in vitro culture. Whether Sox9+ ductal cells in the adult pancreas can give rise to β cells in vivo remains controversial. Here, using lineage-tracing with genetic labeling of Insulin- or Sox9-expressing cells, we show that hyperglycemia (>300 mg/dL) is required for inducing Sox9+ ductal cell differentiation into insulin-producing β cells, and medium hyperglycemia (300–450 mg/dL) in combination with long-term administration of low-dose GE synergistically augments differentiation and is associated with normalization of blood glucose in nonautoimmune diabetic C57BL/6 mice. Short-term administration of high-dose GE cannot augment differentiation, although it can augment preexisting β-cell replication. These results indicate that medium hyperglycemia combined with long-term administration of low-dose GE represents one way to induce Sox9+ ductal cell differentiation into β cells in adult mice. PMID:26733677

Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo - Implications for Tissue Engineering and Clinical Applications.

PubMed

Szabó, Dóra Júlia; Noer, Agate; Nagymihály, Richárd; Josifovska, Natasha; Andjelic, Sofija; Veréb, Zoltán; Facskó, Andrea; Moe, Morten C; Petrovski, Goran

2015-01-01

Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.

Splenectomy Fails to Provide Long-Term Protection Against Ischemic Stroke.

PubMed

Ran, Yuanyuan; Liu, Zongjian; Huang, Shuo; Shen, Jiamei; Li, Fengwu; Zhang, Wenxiu; Chen, Chen; Geng, Xiaokun; Ji, Zhili; Du, Huishan; Hu, Xiaoming

2018-06-01

Splenectomy before or immediately after stroke provides early brain protection. This study aims to explore the effect of splenectomy on long-term neurological recovery after stroke, which is currently lacking in the field. Adult male rats were randomized into splenectomy or sham groups and then subjected to 90 min of middle cerebral artery occlusion (MCAO). Spleen was removed right upon reperfusion or 3d after MCAO. Infarct volume, neurological functions, and peripheral immune cell populations were assessed up to 28d after stroke. The results show that delayed removal of spleen did not reduce brain tissue loss and showed no effect on sensorimotor function (Rotarod, beam balance, forelimb placing, grid walk, and adhesive removal tests) or cognitive function (Morris water maze). Spleen removal immediately upon reperfusion, although significantly reduced the infarct size and immune cell infiltration 3d after MCAO, also failed to promote long-term recovery. Flow cytometry analysis demonstrated that immediate splenectomy after MCAO resulted in a prolonged decrease in the percentage of CD3 + CD4 + and CD3 + CD8 + T cells in total lymphocytes as compared to non-splenectomy MCAO rats. In contrast, the percentage of CD3 - CD45RA + B cells was significantly elevated after splenectomy. As a result, the ratio of T/B cells was significantly reduced in stroke rats with splenectomy. In conclusion, delayed splenectomy failed to provide long-term protection to the ischemic brain or improve functional recovery. The acute neuroprotective effect achieved by early splenectomy after stroke cannot last for long term. This loss of neuroprotection might be related to the prolonged disturbance in the T cell to B cell ratio.

Harnessing the potential of lung stem cells for regenerative medicine.

PubMed

McQualter, Jonathan L; Anthony, Desiree; Bozinovski, Steven; Prêle, Cecilia M; Laurent, Geoffrey J

2014-11-01

In response to recurrent exposure to environmental insults such as allergens, pollution, irritants, smoke and viral/bacterial infection, the epithelium of the lung is continually damaged. Homeostasis of the lung requires a balance between immune regulation and promotion of tissue regeneration, which requires the co-ordinated proliferation and differentiation of stem and progenitor cells. In this review we reflect on the current understanding of lung epithelial stem and progenitor cells and advocate a model hierarchy in which self-renewing multipotent lung epithelial stem cells give rise to lineage restricted progenitor cells that repopulate airway and alveolar epithelial cell lineages during homeostasis and repair. We also discuss the role of mesenchymal progenitor cells in maintaining the structural integrity of the lung and propose a model in which mesenchymal cells act as the quintessential architects of lung regeneration by providing molecular signals, such as FGF-10, to regulate the fate and specificity of epithelial stem and progenitor cells. Moreover, we discuss the current status and future prospects for translating lung stem cell therapies to the clinic to replace, repair, or regenerate diseased lung tissue. This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Alkaline fuel cells for the regenerative fuel cell energy storage system

NASA Technical Reports Server (NTRS)

Martin, R. E.

1983-01-01

The development of the alkaline Regenerative Fuel Cell System, whose fuel cell module would be a derivative of the 12-kW fuel cell power plant currently being produced for the Space Shuttle Orbiter, is reviewed. Long-term endurance testing of full-size fuel cell modules has demonstrated: (1) the extended endurance capability of potassium titanate matrix cells, (2) the long-term performance stability of the anode catalyst, and (3) the suitability of a lightweight graphite structure for use at the anode. These approaches, developed in the NASA-sponsored fuel cell technology advancement program, would also reduce cell weight by nearly one half.

Impact of dose escalation and adaptive radiotherapy for cervical cancers on tumour shrinkage—a modelling study

NASA Astrophysics Data System (ADS)

Røthe Arnesen, Marius; Paulsen Hellebust, Taran; Malinen, Eirik

2017-03-01

Tumour shrinkage occurs during fractionated radiotherapy and is regulated by radiation induced cellular damage, repopulation of viable cells and clearance of dead cells. In some cases additional tumour shrinkage during external beam therapy may be beneficial, particularly for locally advanced cervical cancer where a small tumour volume may simplify and improve brachytherapy. In the current work, a mathematical tumour model is utilized to investigate how local dose escalation affects tumour shrinkage, focusing on implications for brachytherapy. The iterative two-compartment model is based upon linear-quadratic radiation response, a doubling time for viable cells and a half-time for clearance of dead cells. The model was individually fitted to clinical tumour volume data from fractionated radiotherapy of 25 cervical cancer patients. Three different fractionation patterns for dose escalation, all with an additional dose of 12.2 Gy, were simulated and compared to standard fractionation in terms of tumour shrinkage. An adaptive strategy where dose escalation was initiated after one week of treatment was also considered. For 22 out of 25 patients, a good model fit was achieved to the observed tumour shrinkage. A large degree of inter-patient variation was seen in predicted volume reduction following dose escalation. For the 10 best responding patients, a mean tumour volume reduction of 34  ±  3% (relative to standard treatment) was estimated at the time of brachytherapy. Timing of initiating dose escalation had a larger impact than the number of fractions applied. In conclusion, the model was found useful in evaluating the impact from dose escalation on tumour shrinkage. The results indicate that dose escalation could be conducted from the start of external beam radiotherapy in order to obtain additional tumour shrinkage before brachytherapy.

Microgravity

NASA Image and Video Library

1998-10-10

Isolation of human mammary epithelial cells (HMEC) from breast cancer susceptible tissue; A: Duct element recovered from breast tissue digest. B: Outgrowth of cells from duct element in upper right corner cultured in a standard dish; most cells spontaneousely die during early cell divisions, but a few will establish long-term growth. C: Isolate of long-term frowth HMEC from outgrowth of duct element; cells shown soon after isolation and in early full-cell contact growth in culture in a dish. D: same long-term growth HMEC, but after 3 weeks in late full-cell contact growth in a continuous culture in a dish. Note attempts to reform duct elements but this in two demensions in a dish rather than in three dimensions in tissue. NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Robert Richmond, NASA/Marshall Space Flight Center (MSFC).

[Isolation,culture and identification of adipose-derived stem cells from SD rat adipose tissues subjected to long-term cryopreservation].

PubMed

Liu, Qin; Wang, Liping; Chen, Fang; Zhang, Yi

2017-02-01

To study the feasibility of isolation and culture of adipose-derived stem cells( ADSCs) from SD rat adipose tissues subjected to long-term cryopreservation. We took inguinal fat pads from healthy SD rats. Adipose tissues were stored with 100 m L / L dimethyl sulfoxide( DMSO) combined with 900 m L / L fetal bovine serum( FBS) in liquid nitrogen. Three months later,the adipose tissues were resuscitated for the isolation and culture of ADSCs. The growth status and morphology were observed. The growth curve and cell surface markers CD29,CD45,CD90 of the 3rd passage cells were analyzed respectively by CCK-8 assay and immunocytochemistry. The 3rd passage cells were induced towards adipogenic lineages and osteogenic lineages by different inducers,and the resulting cells were examined separately by oil red O staining and alizarin red staining. The ADSCs obtained from SD rat adipose tissues subjected to long-term cryopreservation showed a spindle-shape appearance and had a good proliferation ability. The cell growth curve was typical "S " curve.Immunocytochemistry showed that the 3rd passage cells were positive for CD29 and CD90,while negative for CD45. The cells were positive for oil red O staining after adipogenic induction,and also positive for alizarin red staining after osteogenic induction. The ADSCs can be isolated from SD rat adipose tissues subjected to long-term cryopreservation.

Cell wars: regulation of cell survival and proliferation by cell competition

PubMed Central

Vivarelli, Silvia; Wagstaff, Laura; Piddini, Eugenia

2012-01-01

During cell competition fitter cells take over the tissue at the expense of viable, but less fit, cells, which are eliminated by induction of apoptosis or senescence. This probably acts as a quality-control mechanism to eliminate suboptimal cells and safeguard organ function. Several experimental conditions have been shown to trigger cell competition, including differential levels in ribosomal activity or in signalling pathway activation between cells, although it is unclear how those differences are sensed and translated into fitness levels. Many of the pathways implicated in cell competition have been previously linked with cancer, and this has led to the hypothesis that cell competition could play a role in tumour formation. Cell competition could be co-opted by cancer cells to kill surrounding normal cells and boost their own tissue colonization. However, in some cases, cell competition could have a tumour suppressor role, as cells harbouring mutations in a subset of tumour suppressor genes are killed by wild-type cells. Originally described in developing epithelia, competitive interactions have also been observed in some stem cell niches, where they play a role in regulating stem cell selection, maintenance and tissue repopulation. Thus competitive interactions could be relevant to the maintenance of tissue fitness and have a protective role against aging. PMID:22928509

Effect of long-term culture of mouse embryonic stem cells under low oxygen concentration as well as on glycosaminoglycan hyaluronan on cell proliferation and differentiation.

PubMed

Ramírez, M Á; Pericuesta, E; Yáñez-Mó, M; Palasz, A; Gutiérrez-Adán, A

2011-02-01

Maintaining undifferentiated stem cells in defined conditions is of critical importance to improve their in vitro culture. We have evaluated the effects of culturing mouse stem (mES) cells under physiological oxygen concentration as well as by replacing fibroblast feeder layer (mEF) with gelatin or glycosaminoglycan hyaluronan (HA), on cell proliferation and differentiation. After 3 days culture or after long-term cell culture under different conditions, levels of apoptotic cell death were determined by cell cycle and TUNEL (TdT-mediated dUTP nick end labelling) assays and levels of cell proliferation by CFSE (5-(and-6)-carboxyfluorescein diacetate succinimidyl ester) labelling. We assessed spontaneous differentiation into cardiomyocytes and mRNA expression of pluripotency and differentiation biomarkers. After 3 days culture under hypoxic conditions, levels of proliferation and apoptosis of mES cells were higher, in correlation with increase in intracellular reactive oxygen species. However, when cells were continuously grown for 1 month under those conditions, the level of apoptosis was, in all cases, under 4%. Hypoxia reduced spontaneous differentiation of mES into cardiomyocytes. Long-term culture on HA was more effective in maintaining the pluripotent state of the mES cells when compared to that on gelatin. Level of terminal differentiation was highest on mEF, intermediate on HA and lowest on gelatin. Our data suggest that hypoxia is not necessary for maintaining pluripotency of mES cells and appeared to be detrimental during ES differentiation. Moreover, HA may offer a valuable alternative for long-term culture of mES cells in vitro. © 2010 Blackwell Publishing Ltd.

EdU induces DNA damage response and cell death in mESC in culture.

PubMed

Kohlmeier, Fanni; Maya-Mendoza, Apolinar; Jackson, Dean A

2013-03-01

Recently, a novel DNA replication precursor analogue called 5-ethynyl-2'-deoxyuridine (EdU) has been widely used to monitor DNA synthesis as an alternative to bromodeoxyuridine. Use of EdU benefits from simplicity and reproducibility and the simple chemical detection systems allows excellent preservation of nuclear structure. However, the alkyne moiety is highly reactive, raising the possibility that incorporation might compromise genome stability. To assess the extent of possible DNA damage, we have analysed the effect of EdU incorporation into DNA during short- and long-term cell culture using a variety of cell lines. We show that EdU incorporation has no measurable impact on the rate of elongation of replication forks during synthesis. However, using different cell lines we find that during long-term cell culture variable responses to EdU incorporation are seen, which range from delayed cell cycle progression to complete cell cycle arrest. The most profound phenotypes were seen in mouse embryonic stem cells, which following incorporation of EdU accumulated in the G2/M-phase of the cell cycle before undergoing apoptosis. In long-term cell culture, EdU incorporation also triggered a DNA damage response in all cell types analysed. Our study shows that while EdU is extremely useful to tag sites of on-going replication, for long-term studies (i.e. beyond the cell cycle in which labelling is performed), a careful analysis of cell cycle perturbations must be performed in order to ensure that any conclusions made after EdU treatment are not a direct consequence of EdU-dependent activation of cell stress responses.

Fanconi Anemia Mesenchymal Stromal Cells-Derived Glycerophospholipids Skew Hematopoietic Stem Cell Differentiation Through Toll-Like Receptor Signaling.

PubMed

Amarachintha, Surya; Sertorio, Mathieu; Wilson, Andrew; Li, Xiaoli; Pang, Qishen

2015-11-01

Fanconi anemia (FA) patients develop bone marrow (BM) failure or leukemia. One standard care for these devastating complications is hematopoietic stem cell transplantation. We identified a group of mesenchymal stromal cells (MSCs)-derived metabolites, glycerophospholipids, and their endogenous inhibitor, 5-(tetradecyloxy)-2-furoic acid (TOFA), as regulators of donor hematopoietic stem and progenitor cells. We provided two pieces of evidence that TOFA could improve hematopoiesis-supporting function of FA MSCs: (a) limiting-dilution cobblestone area-forming cell assay revealed that TOFA significantly increased cobblestone colonies in Fanca-/- or Fancd2-/- cocultures compared to untreated cocultures. (b) Competitive repopulating assay using output cells collected from cocultures showed that TOFA greatly alleviated the abnormal expansion of the donor myeloid (CD45.2+Gr1+Mac1+) compartment in both peripheral blood and BM of recipient mice transplanted with cells from Fanca-/- or Fancd2-/- cocultures. Furthermore, mechanistic studies identified Tlr4 signaling as the responsible pathway mediating the effect of glycerophospholipids. Thus, targeting glycerophospholipid biosynthesis in FA MSCs could be a therapeutic strategy to improve hematopoiesis and stem cell transplantation. © 2015 AlphaMed Press.

Tumor Heterogeneity, Single-Cell Sequencing, and Drug Resistance.

PubMed

Schmidt, Felix; Efferth, Thomas

2016-06-16

Tumor heterogeneity has been compared with Darwinian evolution and survival of the fittest. The evolutionary ecosystem of tumors consisting of heterogeneous tumor cell populations represents a considerable challenge to tumor therapy, since all genetically and phenotypically different subpopulations have to be efficiently killed by therapy. Otherwise, even small surviving subpopulations may cause repopulation and refractory tumors. Single-cell sequencing allows for a better understanding of the genomic principles of tumor heterogeneity and represents the basis for more successful tumor treatments. The isolation and sequencing of single tumor cells still represents a considerable technical challenge and consists of three major steps: (1) single cell isolation (e.g., by laser-capture microdissection), fluorescence-activated cell sorting, micromanipulation, whole genome amplification (e.g., with the help of Phi29 DNA polymerase), and transcriptome-wide next generation sequencing technologies (e.g., 454 pyrosequencing, Illumina sequencing, and other systems). Data demonstrating the feasibility of single-cell sequencing for monitoring the emergence of drug-resistant cell clones in patient samples are discussed herein. It is envisioned that single-cell sequencing will be a valuable asset to assist the design of regimens for personalized tumor therapies based on tumor subpopulation-specific genetic alterations in individual patients.

Phenotype and specificity of T cells in primary human cytomegalovirus infection during pregnancy: IL-7Rpos long-term memory phenotype is associated with protection from vertical transmission.

PubMed

Mele, Federico; Fornara, Chiara; Jarrossay, David; Furione, Milena; Arossa, Alessia; Spinillo, Arsenio; Lanzavecchia, Antonio; Gerna, Giuseppe; Sallusto, Federica; Lilleri, Daniele

2017-01-01

Congenital human cytomegalovirus (HCMV) infection is the major cause of birth defects and a precise definition of the HCMV-specific T-cell response in primary infection may help define reliable correlates of immune protection during pregnancy. In this study, a high throughput method was used to define the frequency of CD4+ and CD8+ T cells specific for four HCMV proteins in the naïve compartment of seronegative subjects and the effector/memory compartments of subjects with primary/remote HCMV infection. The naïve repertoire displayed comparable frequencies of T cells that were reactive with HCMV structural (pp65, gB and the pentamer gHgLpUL128L) and non-structural (IE-1) proteins. Whereas, following natural infection, the majority of effector/memory CD4+ and CD8+ T cells recognized either gB or IE-1, respectively, and pp65. The pattern of T cell reactivity was comparable at early and late stages of infection and in pregnant women with primary HCMV infection transmitting or not transmitting the virus to the fetus. At an early stage of primary infection, about 50% of HCMV-reactive CD4+ T cells were long-term IL-7Rpos memory cells, while 6-12 months later, the frequency of these cells increased to 70%, approaching 100% in remote infections. In contrast, only 10-20% of HCMV-specific CD8+ T cells were long-term memory cells up to 12 months after infection onset, thereafter increasing to 70% in remote infections. Interestingly, a significantly higher frequency of HCMV-specific CD4+ T cells with a long-term IL-7Rpos memory phenotype was observed in non-transmitting compared to transmitting women. These findings indicate that immunodominance in HCMV infection is not predetermined in the naïve compartment, but is the result of virus-host interactions and suggest that prompt control of HCMV infection in pregnancy is associated with the rapid development of long-term IL-7Rpos memory HCMV-specific CD4+ T cells and a low risk of virus transmission to the fetus.

Long term imaging of living brain cancer cells

NASA Astrophysics Data System (ADS)

Farias, Patricia M. A.; Galembeck, André; Milani, Raquel; Andrade, Arnaldo C. D. S.; Stingl, Andreas

2018-02-01

QDs synthesized in aqueous medium and functionalized with polyethylene glycol were used as fluorescent probes. They label and monitor living healthy and cancer brain glial cells in culture. Physical-chemical characterization was performed. Toxicological studies were performed by in vivo short and long-term inhalation in animal models. Healthy and cancer glial living cells were incubated in culture media with highly controlled QDs. Specific features of glial cancer cells were enhanced by QD labelling. Cytoplasmic labelling pattern was clearly distinct for healthy and cancer cells. Labelled cells kept their normal activity for same period as non-labelled control samples.

Continuous activation of Nrf2 and its target antioxidant enzymes leads to arsenite-induced malignant transformation of human bronchial epithelial cells

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

Yang, Xu; Wang, Dapeng; Department of Toxicology, School of Public Health, Guizhou Medical University, Guiyang, Guizhou

Long-term exposure to arsenite leads to human lung cancer, but the underlying mechanisms of carcinogenesis remain obscure. The transcription factor of nuclear factor-erythroid-2 p45-related factor (Nrf2)-mediated antioxidant response represents a critical cellular defense mechanism and protection against various diseases. Paradoxically, emerging data suggest that the constitutive activation of Nrf2 is associated with cancer development, progression and chemotherapy resistance. However, the role of Nrf2 in the occurrence of cancer induced by long-term arsenite exposure remains to be fully understood. By establishing transformed human bronchial epithelial (HBE) cells via chronic low-dose arsenite treatment, we showed that, in acquiring this malignant phenotype, continuousmore » low level of ROS and sustained enhancement of Nrf2 and its target antioxidant enzyme levels were observed in the later-stage of arsenite-induced cell transformation. The downregulation of Keap1 level may be responsible for the over-activation of Nrf2 and its target enzymes. To validate these observations, Nrf2 was knocked down in arsenite-transformed HBE cells by SiRNA transfection, and the levels of Nrf2 and its target antioxidant enzymes, ROS, cell proliferation, migration, and colony formation were determined following these treatments. Results showed that blocked Nrf2 expression significantly reduced Nrf2 and its target antioxidant enzyme levels, restored ROS levels, and eventually suppressed cell proliferation, migration, and colony formation of the transformed cells. In summary, the results of the study strongly suggested that the continuous activation of Nrf2 and its target antioxidant enzymes led to the over-depletion of intracellular ROS levels, which contributed to arsenite-induced HBE cell transformation. - Highlights: • Low level, long term arsenite exposure induces malignant transformation in vitro. • Long term arsenite exposure reduces ROS and MDA levels. • Long term arsenite exposure enhances Nrf2-mediated antioxidant levels. • Knockdown of Nrf2 reduces malignant degree of arsenite-transformed cells.« less

Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver

PubMed Central

Huch, Meritxell; Gehart, Helmuth; van Boxtel, Ruben; Hamer, Karien; Blokzijl, Francis; Verstegen, Monique M.A.; Ellis, Ewa; van Wenum, Martien; Fuchs, Sabine A.; de Ligt, Joep; van de Wetering, Marc; Sasaki, Nobuo; Boers, Susanne J.; Kemperman, Hans; de Jonge, Jeroen; Ijzermans, Jan N.M.; Nieuwenhuis, Edward E.S.; Hoekstra, Ruurdtje; Strom, Stephen; Vries, Robert R.G.; van der Laan, Luc J.W.; Cuppen, Edwin; Clevers, Hans

2015-01-01

Summary Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy. PMID:25533785

Focal cutaneous squamous cell carcinoma following radium-223 extravasation.

PubMed

Benjegerdes, Katie E; Brown, Shannon C; Housewright, Chad D

2017-01-01

Long-term sequelae due to extravasation of intravenous radioisotopes resulting in radiation injuries are rarely reported. As the use of radioactive isotopes for the treatment of osteoblastic metastases increases, information regarding the prevention, treatment, and long-term monitoring of suspected extravasation injury will become increasingly important. We present a patient with no previous history of skin cancer who developed an aggressive cutaneous squamous cell carcinoma at the site of prior radium-223 extravasation. We recommend that patients who experience extravasation of therapeutic radioisotopes be monitored by dermatologists for long-term sequelae. Cutaneous squamous cell carcinoma should be recognized as a rare but potential adverse event following cutaneous extravasation of radium-223 and is likely a side effect that is severely underreported.

IL-7 promotes long-term in vitro survival of unique long-lived memory subset generated from mucosal effector memory CD4+ T cells in chronic colitis mice.

PubMed

Takahara, Masahiro; Nemoto, Yasuhiro; Oshima, Shigeru; Matsuzawa, Yu; Kanai, Takanori; Okamoto, Ryuichi; Tsuchiya, Kiichiro; Nakamura, Tetsuya; Yamamoto, Kazuhide; Watanabe, Mamoru

2013-01-01

Colitogenic memory CD4(+) T cells are important in the pathogenesis of inflammatory bowel disease (IBD). Although memory stem cells with high survival and self-renewal capacity were recently identified in both mice and humans, it is unclear whether a similar subset is present in chronic colitis mice. We sought to identify and purify a long-lived subset of colitogenic memory CD4(+) T cells, which may be targets for treatment of IBD. A long-lived subset of colitogenic memory CD4(+) T cells was purified using a long-term culture system. The characteristics of these cells were assessed. Interleukin (IL)-7 promoted the in vitro survival for >8 weeks of lamina propria (LP) CD4(+) T cells from colitic SCID mice previously injected with CD4(+)CD45RB(high) T cells. These cells were in a quiescent state and divided a maximum of 5 times in 4 weeks. LP CD4(+) T cells expressed higher levels of Bcl-2, integrin-α4β7, CXCR3 and CD25 after than before culture, as well as secreting high concentrations of IL-2 and low concentrations of IFN-γ and IL-17 in response to intestinal bacterial antigens. LP CD4(+) T cells from colitic mice cultured with IL-7 for 8 weeks induced more severe colitis than LP CD4(+) T cells cultured for 4 weeks. We developed a novel culture system to purify a long-lived, highly pathogenic memory subset from activated LP CD4(+) T cells. IL-7 promoted long-term in vitro survival of this subset in a quiescent state. This subset will be a novel, effective target for the treatment of IBD. Copyright © 2013 Elsevier B.V. All rights reserved.

Induction of three-dimensional assembly of human liver cells by simulated microgravity

NASA Technical Reports Server (NTRS)

Khaoustov, V. I.; Darlington, G. J.; Soriano, H. E.; Krishnan, B.; Risin, D.; Pellis, N. R.; Yoffe, B.

1999-01-01

The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.

Microbial Repopulation Following In Situ STAR Remediation

NASA Astrophysics Data System (ADS)

Gerhard, J.; Overbeeke, G.; Edwards, E.; Lomheim, L.; Grant, G.

2016-12-01

STAR (Self-sustaining Treatment for Active Remediation) is an emerging remediation technology that employs a self-sustaining smouldering reaction to destroy nonaqueous phase liquids (NAPLs) in the subsurface. The reaction front travels outwards from an ignition well at approximately 0.5 per day and subjects the soil to temperatures of 400°C-1000°C. The objectives of this work were to monitor re-saturation of the soil over time and quantify the microbial repopulation of the treated zone. STAR is currently being applied as a full scale, in situ remedy for coal tar beneath a former creosol manufacturing facility in New Jersey, USA. This study analyzed soil cores taken at regular intervals following STAR treatment, allowing time for groundwater to re-infiltrate and for microbial populations to potentially reestablish. Soil and groundwater were analyzed for total number of microorganisms via quantitative Polymerase Chain Reaction (qPCR), as well as microbial diversity via amplicon sequencing. Results demonstrate that microbes rapidly repopulated over a 2 month period to 106 gene copies/g of soil. However, concentrations in the treated zone did not rise above this concentration over 6 months post-STAR, indicating a low carrying capacity of the treated soil. To examine the system in more detail and consider the effects of bio-stimulation, a bench top column study using site soil and artificial groundwater explored the rate at which STAR-treated soil is repopulated with naturally occurring microorganisms in the presence and absence of lactate and a terminal electron acceptor. Results demonstrated that biostimulation did not increase the carrying capacity of the STAR treated sol, but rather shifted the microbial community to reflect the TEA provided, in this case, promoting sulfate reducers. Overall, the work illustrates that microbial populations in STAR treated soil do recover via groundwater infiltration but robust communities will take time to naturally establish.

A unique role of the cholera toxin A1-DD adjuvant for long-term plasma and memory B cell development.

PubMed

Bemark, Mats; Bergqvist, Peter; Stensson, Anneli; Holmberg, Anna; Mattsson, Johan; Lycke, Nils Y

2011-02-01

Adjuvants have traditionally been appreciated for their immunoenhancing effects, whereas their impact on immunological memory has largely been neglected. In this paper, we have compared three mechanistically distinct adjuvants: aluminum salts (Alum), Ribi (monophosphoryl lipid A), and the cholera toxin A1 fusion protein CTA1-DD. Their influence on long-term memory development was dramatically different. Whereas a single immunization i.p. with 4-hydroxy-3-nitrophenyl acetyl (NP)-chicken γ-globulin and adjuvant stimulated serum anti-NP IgG titers that were comparable at 5 wk, CTA1-DD-adjuvanted responses were maintained for >16 mo with a half-life of anti-NP IgG ∼36 wk, but <15 wk after Ribi or Alum. A CTA1-DD dose-dependent increase in germinal center (GC) size and numbers was found, with >60% of splenic B cell follicles hosting GC at an optimal CTA1-DD dose. Roughly 7% of these GC were NP specific. This GC-promoting effect correlated well with the persistence of long-term plasma cells in the bone marrow and memory B cells in the spleen. CTA1-DD also facilitated increased somatic hypermutation and affinity maturation of NP-specific IgG Abs in a dose-dependent fashion, hence arguing that large GC not only promotes higher Ab titers but also high-quality Ab production. Adoptive transfer of splenic CD80(+), but not CD80(-), B cells, at 1 y after immunization demonstrated functional long-term anti-NP IgG and IgM memory cells. To our knowledge, this is the first report to specifically compare and document that adjuvants can differ considerably in their support of long-term immune responses. Differential effects on the GC reaction appear to be the basis for these differences.

Fanconi anemia mesenchymal stromal cells-derived glycerophospholipids skew hematopoietic stem cell differentiation through Toll-like receptor signaling

PubMed Central

Amarachintha, Surya; Sertorio, Mathieu; Wilson, Andrew; Li, Xiaoli; Pang, Qishen

2015-01-01

Fanconi anemia (FA) patients develop bone marrow (BM) failure or leukemia. One standard care for these devastating complications is hematopoietic stem cell transplantation. We identified a group of mesenchymal stromal cells (MSCs)-derived metabolites, glycerophospholipids and their endogenous inhibitor, 5-(Tetradecyloxy)-2-furoic acid (TOFA), as regulators of donor hematopoietic stem and progenitor cells (HSPCs). We provided two pieces of evidence that TOFA could improve hematopoiesis-supporting function of FA MSCs: (1) limiting-dilution CAFC assay revealed that TOFA significantly increased cobblestone colonies in Fanca−/− or Fancd2−/− co-cultures compared to untreated co-cultures. (2) Competitive repopulating assay using output cells collected from co-cultures showed that TOFA greatly alleviated the abnormal expansion of the donor myeloid (CD45.2+Gr1+Mac1+) compartment in both peripheral blood and BM of recipient mice transplanted with cells from Fanca−/− or Fancd2−/− co-cultures. Further, mechanistic studies identified Tlr4 signaling as the responsible pathway mediating the effect of glycerophospholipids. Thus, targeting Glycerophospholipid biosynthesis in FA MSCs could be a therapeutic strategy to improve hematopoiesis and stem cell transplantation. PMID:26212365

Egf Signaling Directs Neoblast Repopulation by Regulating Asymmetric Cell Division in Planarians.

PubMed

Lei, Kai; Thi-Kim Vu, Hanh; Mohan, Ryan D; McKinney, Sean A; Seidel, Chris W; Alexander, Richard; Gotting, Kirsten; Workman, Jerry L; Sánchez Alvarado, Alejandro

2016-08-22

A large population of proliferative stem cells (neoblasts) is required for physiological tissue homeostasis and post-injury regeneration in planarians. Recent studies indicate that survival of a few neoblasts after sublethal irradiation results in the clonal expansion of the surviving stem cells and the eventual restoration of tissue homeostasis and regenerative capacity. However, the precise mechanisms regulating the population dynamics of neoblasts remain largely unknown. Here, we uncovered a central role for epidermal growth factor (EGF) signaling during in vivo neoblast expansion mediated by Smed-egfr-3 (egfr-3) and its putative ligand Smed-neuregulin-7 (nrg-7). Furthermore, the EGF receptor-3 protein localizes asymmetrically on the cytoplasmic membrane of neoblasts, and the ratio of asymmetric to symmetric cell divisions decreases significantly in egfr-3(RNAi) worms. Our results not only provide the first molecular evidence of asymmetric stem cell divisions in planarians, but also demonstrate that EGF signaling likely functions as an essential regulator of neoblast clonal expansion. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Long-term drug modification to the surface of mesenchymal stem cells by the avidin-biotin complex method.

PubMed

Takayama, Yukiya; Kusamori, Kosuke; Hayashi, Mika; Tanabe, Noriko; Matsuura, Satoru; Tsujimura, Mari; Katsumi, Hidemasa; Sakane, Toshiyasu; Nishikawa, Makiya; Yamamoto, Akira

2017-12-05

Mesenchymal stem cells (MSCs) have various functions, making a significant contribution to tissue repair. On the other hand, the viability and function of MSCs are not lasting after an in vivo transplant, and the therapeutic effects of MSCs are limited. Although various chemical modification methods have been applied to MSCs to improve their viability and function, most of conventional drug modification methods are short-term and unstable and cause cytotoxicity. In this study, we developed a method for long-term drug modification to C3H10T1/2 cells, murine mesenchymal stem cells, without any damage, using the avidin-biotin complex method (ABC method). The modification of NanoLuc luciferase (Nluc), a reporter protein, to C3H10T1/2 cells by the ABC method lasted for at least 14 days in vitro without major effects on the cellular characteristics (cell viability, cell proliferation, migration ability, and differentiation ability). Moreover, in vivo, the surface Nluc modification to C3H10T1/2 cells by the ABC method lasted for at least 7 days. Therefore, these results indicate that the ABC method may be useful for long-term surface modification of drugs and for effective MSC-based therapy.

Formation of resting cells by non-spore-forming microorganisms as a strategy of long-term survival in the environment

NASA Astrophysics Data System (ADS)

Mulyukin, Andrei L.; Soina, Vera S.; Demkina, Elena V.; Kozlova, Alla N.; Suzina, Natalia E.; Dmitriev, Vladimir V.; Duda, Vitalii I.; El'-Registan, Galina I.

2003-01-01

Non-spore-forming bacteria of the genera Micrococcus and Arthrobacter, including the isolates from permafrost sediments, were found to be able to form cystlike cells under special conditions. Cystlike cells maintained the viability during long-term storage (for up to several years), had undetectable respiratory activity and the elevated resistance to heating and other unfavorable conditions, possessed the specific fine structure and morphology, and were formed in the life cycles of the microorganism. These properties allow cystlike cells to be attributed to a new type of resting microbial forms. Furthermore, the distinctive feature of resting cystlike cells was their low P/S ratios and high Ca/K ratios in comparison to vegetative cells as shown by X-ray microanalysis. The experimentally obtained bacterial cystlike cells with thickened and laminated cell walls and altered texture of the cytoplasm were similar to the cells abundant in native microbial populations isolated from permafrost sediments and ancient soils of the Kolyma lowland (Siberia, Russia). Due to the inherent elevated resistance to adverse conditions and maintenance of viability for prolonged periods, resting cystlike cells are likely to ensure long-term survival of non-spore-forming bacteria in cold environments.

CD8+ T Cells Primed in the Periphery Provide Time-Bound Immune-Surveillance to the Central Nervous System

PubMed Central

Young, Kevin G.; MacLean, Susanne; Dudani, Renu; Krishnan, Lakshmi; Sad, Subash

2016-01-01

After vaccination, memory CD8+ T cells migrate to different organs to mediate immune surveillance. In most nonlymphoid organs, following an infection, CD8+ T cells differentiate to become long-lived effector-memory cells, thereby providing long-term protection against a secondary infection. In this study, we demonstrated that Ag-specific CD8+ T cells that migrate to the mouse brain following a systemic Listeria infection do not display markers reminiscent of long-term memory cells. In contrast to spleen and other nonlymphoid organs, none of the CD8+ T cells in the brain reverted to a memory phenotype, and all of the cells were gradually eliminated. These nonmemory phenotype CD8+ T cells were found primarily within the choroid plexus, as well as in the cerebrospinal fluid-filled spaces. Entry of these CD8+ T cells into the brain was governed primarily by CD49d/VCAM-1, with the majority of entry occurring in the first week postinfection. When CD8+ T cells were injected directly into the brain parenchyma, cells that remained in the brain retained a highly activated (CD69hi) phenotype and were gradually lost, whereas those that migrated out to the spleen were CD69low and persisted long-term. These results revealed a mechanism of time-bound immune surveillance to the brain by CD8+ T cells that do not reside in the parenchyma. PMID:21715683

Long-term stabilization of place cell remapping produced by a fearful experience

PubMed Central

Wang, Melissa E.; Wann, Ellen G.; Yuan, Robin K.; Ramos Álvarez, Manuel M.; Stead, Squire M.; Muzzio, Isabel A.

2012-01-01

Fear is an emotional response to danger that is highly conserved throughout evolution because it is critical for survival. Accordingly, episodic memory for fearful locations is widely studied using contextual fear conditioning, a hippocampus-dependent task (Kim and Fanselow, 1992; Phillips and LeDoux, 1992). The hippocampus has been implicated in episodic emotional memory and is thought to integrate emotional stimuli within a spatial framework. Physiological evidence supporting the role of the hippocampus in contextual fear indicates that pyramidal cells in this region, which fire in specific locations as an animal moves through an environment, shift their preferred firing locations shortly after the presentation of an aversive stimulus (Moita et al., 2004). However, the long-term physiological mechanisms through which emotional memories are encoded by the hippocampus are unknown. Here we show that during and directly after a fearful experience, new hippocampal representations are established and persist in the long term. We recorded from the same place cells in mouse hippocampal area CA1 over several days during predator odor contextual fear conditioning and found that a subset of cells changed their preferred firing locations in response to the fearful stimulus. Furthermore, the newly formed representations of the fearful context stabilized in the long term. Our results demonstrate that place cells respond to the presence of an aversive stimulus, modify their firing patterns during emotional learning, and stabilize a long-term spatial representation in response to a fearful encounter. The persistent nature of these representations may contribute to the enduring quality of emotional memories. PMID:23136419

Mechanical characterization and numerical simulation of a subcutaneous implantable 3D printed cell encapsulation system.

PubMed

Adamo, Federica; Farina, Marco; Thekkedath, Usha R; Grattoni, Alessandro; Sesana, Raffaella

2018-06-01

Cell transplantation in bioengineered scaffolds and encapsulation systems has shown great promise in regenerative medicine. Depending on the site of implantation, type of cells and their expected function, these systems are designed to provide cells with a physiological-like environment while providing mechanical support and promoting long-term viability and function of the graft. A minimally invasive 3D printed system termed neovascularized implantable cell homing and encapsulation (NICHE) was developed in polylactic acid for subcutaneous transplantation of endocrine cells, including pancreatic islets. The suitability of the NICHE for long term in vivo deployment is investigated by assessing mechanical behavior of both fresh devices under simulated subcutaneous conditions and NICHE retrieved from subcutaneous implantation in pigs. Both experimental and numerical studies were performed with a focus on validating the constitutive material model used in the numerical analysis for accuracy and reliability. Notably, homogeneous isotropic constitutive material model calibrated by means of uniaxial testing well suited experimental results. The results highlight the long term durability for in vivo applications and the potential applicability of the model to predict the mechanical behavior of similar devices in various physiological settings. Copyright © 2018 Elsevier Ltd. All rights reserved.

Memory retrieval by activating engram cells in mouse models of early Alzheimer's disease.

PubMed

Roy, Dheeraj S; Arons, Autumn; Mitchell, Teryn I; Pignatelli, Michele; Ryan, Tomás J; Tonegawa, Susumu

2016-03-24

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive memory decline and subsequent loss of broader cognitive functions. Memory decline in the early stages of AD is mostly limited to episodic memory, for which the hippocampus has a crucial role. However, it has been uncertain whether the observed amnesia in the early stages of AD is due to disrupted encoding and consolidation of episodic information, or an impairment in the retrieval of stored memory information. Here we show that in transgenic mouse models of early AD, direct optogenetic activation of hippocampal memory engram cells results in memory retrieval despite the fact that these mice are amnesic in long-term memory tests when natural recall cues are used, revealing a retrieval, rather than a storage impairment. Before amyloid plaque deposition, the amnesia in these mice is age-dependent, which correlates with a progressive reduction in spine density of hippocampal dentate gyrus engram cells. We show that optogenetic induction of long-term potentiation at perforant path synapses of dentate gyrus engram cells restores both spine density and long-term memory. We also demonstrate that an ablation of dentate gyrus engram cells containing restored spine density prevents the rescue of long-term memory. Thus, selective rescue of spine density in engram cells may lead to an effective strategy for treating memory loss in the early stages of AD.

Long-term (6-wk) hindlimb suspension inhibits spermatogenesis in adult male rats

NASA Technical Reports Server (NTRS)

Tash, Joseph S.; Johnson, Donald C.; Enders, George C.

2002-01-01

The International Space Station will allow extended habitation in space and long-term exposure to microgravity (microG). A concern is the impact of long-term microG exposure on the ability of species to reproduce. The model often used to simulate microG is rat hindlimb suspension (HLS), where the hindlimbs are elevated above the cage floor with a tail harness. Experiments described here are the first to examine the effect of long-term HLS on testicular function in adult male rats. Free-roaming (controls), animals with only the tail harnessed but hindlimbs in contact with the cage floor (TO), and HLS animals were tested for 6 wk. Cryptorchidism was prevented in TO and HLS animals by partial constriction of the inguinal canal with sutures. All parameters were compared at the end of the 6-wk experiment. Testicular weights and spermatogenesis were significantly reduced by HLS, such that no spermatogenic cells beyond round spermatids were present and epididymides were devoid of mature sperm. In many tubules, loss of all germ cells, except a few spermatogonia, resulting in histopathology similar to the Sertoli cell, was observed. Spermatogenesis appeared unaffected in control and TO animals. Sertoli and Leydig cell appearance, testosterone, luteinizing hormone, and follicle-stimulating hormone levels, and epididymal and seminal vesicle weight were unchanged by HLS. Cortisone was not elevated by HLS; thus stress may not be a factor. These results demonstrate that spermatogenesis is severely inhibited by long-term HLS, whereas testicular androgen production is not. These results have significant implications regarding serious effects of long-term exposure to microG on the reproductive capability of scrotal mammals, including humans.

Competitive fitness during feast and famine: how SOS DNA polymerases influence physiology and evolution in Escherichia coli.

PubMed

Corzett, Christopher H; Goodman, Myron F; Finkel, Steven E

2013-06-01

Escherichia coli DNA polymerases (Pol) II, IV, and V serve dual roles by facilitating efficient translesion DNA synthesis while simultaneously introducing genetic variation that can promote adaptive evolution. Here we show that these alternative polymerases are induced as cells transition from exponential to long-term stationary-phase growth in the absence of induction of the SOS regulon by external agents that damage DNA. By monitoring the relative fitness of isogenic mutant strains expressing only one alternative polymerase over time, spanning hours to weeks, we establish distinct growth phase-dependent hierarchies of polymerase mutant strain competitiveness. Pol II confers a significant physiological advantage by facilitating efficient replication and creating genetic diversity during periods of rapid growth. Pol IV and Pol V make the largest contributions to evolutionary fitness during long-term stationary phase. Consistent with their roles providing both a physiological and an adaptive advantage during stationary phase, the expression patterns of all three SOS polymerases change during the transition from log phase to long-term stationary phase. Compared to the alternative polymerases, Pol III transcription dominates during mid-exponential phase; however, its abundance decreases to <20% during long-term stationary phase. Pol IV transcription dominates as cells transition out of exponential phase into stationary phase and a burst of Pol V transcription is observed as cells transition from death phase to long-term stationary phase. These changes in alternative DNA polymerase transcription occur in the absence of SOS induction by exogenous agents and indicate that cell populations require appropriate expression of all three alternative DNA polymerases during exponential, stationary, and long-term stationary phases to attain optimal fitness and undergo adaptive evolution.

An Alternative Method for Long-Term Culture of Chicken Embryonic Stem Cell In Vitro.

PubMed

Zhang, Li; Wu, Yenan; Li, Xiang; Wei, Shao; Xing, Yiming; Lian, Zhengxing; Han, Hongbing

2018-01-01

Chicken embryonic stem cells (cESCs) obtained from stage X embryos provide a novel model for the study of avian embryonic development. A new way to maintain cESCs for a long period in vitro still remains unexplored. We found that the cESCs showed stem cell-like properties in vitro for a long term with the support of DF-1 feeder and basic culture medium supplemented with human basic fibroblast growth factor (hbFGF), mouse stem cell factor (mSCF), and human leukemia inhibitory factor (hLIF). During the long culture period, the cESCs showed typical ES cell morphology and expressed primitive stem cell markers with a relatively stable proliferation rate and high telomerase activity. These cells also exhibited the capability to differentiate into cardiac myocytes, smooth muscle cells, neural cells, osteoblast, and adipocyte in vitro . Chimera chickens were produced by cESCs cultured for 25 passages with this new culture system. The experiments showed that DF-1 was the optimal feeder and hbFGF was an important factor for maintaining the pluripotency of cESCs in vitro .

Simulated predator stimuli reduce brain cell proliferation in two electric fish species, Brachyhypopomus gauderio and Apteronotus leptorhynchus.

PubMed

Dunlap, Kent D; Keane, Geoffrey; Ragazzi, Michael; Lasky, Elise; Salazar, Vielka L

2017-07-01

The brain structure of many animals is influenced by their predators, but the cellular processes underlying this brain plasticity are not well understood. Previous studies showed that electric fish ( Brachyhypopomus occidentalis ) naturally exposed to high predator ( Rhamdia quelen ) density and tail injury had reduced brain cell proliferation compared with individuals facing few predators and those with intact tails. However, these field studies described only correlations between predator exposure and cell proliferation. Here, we used a congener Brachyhypopomus gauderio and another electric fish Apteronotus leptorhynchus to experimentally test the hypothesis that exposure to a predator stimulus and tail injury causes alterations in brain cell proliferation. To simulate predator exposure, we either amputated the tail followed by short-term (1 day) or long-term (17-18 days) recovery or repeatedly chased intact fish with a plastic rod over a 7 day period. We measured cell proliferation (PCNA+ cell density) in the telencephalon and diencephalon, and plasma cortisol, which commonly mediates stress-induced changes in brain cell proliferation. In both species, either tail amputation or simulated predator chase decreased cell proliferation in the telencephalon in a manner resembling the effect of predators in the field. In A. leptorhynchus , cell proliferation decreased drastically in the short term after tail amputation and partially rebounded after long-term recovery. In B. gauderio , tail amputation elevated cortisol levels, but repeated chasing had no effect. In A. leptorhynchus , tail amputation elevated cortisol levels in the short term but not in the long term. Thus, predator stimuli can cause reductions in brain cell proliferation, but the role of cortisol is not clear. © 2017. Published by The Company of Biologists Ltd.

Breast Cancer Research at NASA

NASA Technical Reports Server (NTRS)

1998-01-01

Isolation of human mammary epithelial cells (HMEC) from breast cancer susceptible tissue; A: Duct element recovered from breast tissue digest. B: Outgrowth of cells from duct element in upper right corner cultured in a standard dish; most cells spontaneousely die during early cell divisions, but a few will establish long-term growth. C: Isolate of long-term frowth HMEC from outgrowth of duct element; cells shown soon after isolation and in early full-cell contact growth in culture in a dish. D: same long-term growth HMEC, but after 3 weeks in late full-cell contact growth in a continuous culture in a dish. Note attempts to reform duct elements but this in two demensions in a dish rather than in three dimensions in tissue. NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Robert Richmond, NASA/Marshall Space Flight Center (MSFC).

Postmitotic human dermal fibroblasts preserve intact feeder properties for epithelial cell growth after long-term cryopreservation.

PubMed

Limat, A; Hunziker, T; Boillat, C; Noser, F; Wiesmann, U

1990-07-01

In vitro, human dermal fibroblasts (HDF) differentiate through morphologically and biochemically identified compartments. In the course of this spontaneous differentiation through mitotic and postmitotic states, a tremendous increase in cellular and nuclear size occurs. Induction of postmitotic states can be accelerated by chemical (e.g., mitomycin C) or physical (e.g., x-ray) treatments. Such experimentally induced postmitotic HDF cells support very efficiently the growth of cutaneous epithelial cells, i.e. interfollicular keratinocytes and follicular outer root sheath cells, especially in primary cultures starting from very low cell seeding densities. The HDF feeder system provides more fundamental and also practical advantages, i.e. use of initially diploid human fibroblasts from known anatomic locations, easy handling and excellent reproducibility, and the possibility of long-term storage by incubation at 37 degrees C. Conditions for the cryogenic storage of postmitotic HDF cells in liquid nitrogen are presented and related to the feeder capacity for epithelial cell growth. Because postmitotic HDF cells preserve intact feeder properties after long-term storage, the immediate availability of feeder cells and the possibility to repeat experiments with identical materials further substantiate the usefulness of this feeder system.

Age and CD4 count at initiation of antiretroviral therapy in HIV-infected children: effects on long-term T-cell reconstitution.

PubMed

Lewis, Joanna; Walker, A Sarah; Castro, Hannah; De Rossi, Anita; Gibb, Diana M; Giaquinto, Carlo; Klein, Nigel; Callard, Robin

2012-02-15

Effective therapies and reduced AIDS-related morbidity and mortality have shifted the focus in pediatric human immunodeficiency virus (HIV) from minimizing short-term disease progression to maintaining optimal long-term health. We describe the effects of children's age and pre-antiretroviral therapy (ART) CD4 count on long-term CD4 T-cell reconstitution. CD4 counts in perinatally HIV-infected, therapy-naive children in the Paediatric European Network for the Treatment of AIDS 5 trial were monitored following initiation of ART for a median 5.7 years. In a substudy, naive and memory CD4 counts were recorded. Age-standardized measurements were analyzed using monophasic, asymptotic nonlinear mixed-effects models. One hundred twenty-seven children were studied. Older children had lower age-adjusted CD4 counts in the long term and at treatment initiation (P < .001). At all ages, lower counts before treatment were associated with impaired recovery (P < .001). Age-adjusted naive CD4 counts increased on a timescale comparable to overall CD4 T-cell reconstitution, whereas age-adjusted memory CD4 counts increased less, albeit on a faster timescale. It appears the immature immune system can recover well from HIV infection via the naive pool. However, this potential is progressively damaged with age and/or duration of infection. Current guidelines may therefore not optimize long-term immunological health.

Sealed-cell nickel-cadmium battery applications manual

NASA Technical Reports Server (NTRS)

Scott, W. R.; Rusta, D. W.

1979-01-01

The design, procurement, testing, and application of aerospace quality, hermetically sealed nickel-cadmium cells and batteries are presented. Cell technology, cell and battery development, and spacecraft applications are emphasized. Long term performance is discussed in terms of the effect of initial design, process, and application variables. Design guidelines and practices are given.

Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors.

PubMed

Kochenderfer, James N; Rosenberg, Steven A

2013-05-01

Most B-cell malignancies express CD19, and a majority of patients with B-cell malignancies are not cured by current standard therapies. Chimeric antigen receptors (CARs) are fusion proteins consisting of antigen recognition moieties and T-cell activation domains. T cells can be genetically modified to express CARs, and adoptive transfer of anti-CD19 CAR T cells is now being tested in clinical trials. Effective clinical treatment with anti-CD19 CAR T cells was first reported in 2010 after a patient with advanced-stage lymphoma treated at the NCI experienced a partial remission of lymphoma and long-term eradication of normal B cells. Additional patients have subsequently obtained long-term remissions of advanced-stage B-cell malignancies after infusions of anti-CD19 CAR T cells. Long-term eradication of normal CD19(+) B cells from patients receiving infusions of anti-CD19 CAR T cells demonstrates the potent antigen-specific activity of these T cells. Some patients treated with anti-CD19 CAR T cells have experienced acute adverse effects, which were associated with increased levels of serum inflammatory cytokines. Although anti-CD19 CAR T cells are at an early stage of development, the potent antigen-specific activity observed in patients suggests that infusions of anti-CD19 CAR T cells might become a standard therapy for some B-cell malignancies.

Dynamic equilibrium of reconstituting hematopoietic stem cell populations.

PubMed

O'Quigley, John

2010-12-01

Clonal dominance in hematopoietic stem cell populations is an important question of interest but not one we can directly answer. Any estimates are based on indirect measurement. For marked populations, we can equate empirical and theoretical moments for binomial sampling, in particular we can use the well-known formula for the sampling variation of a binomial proportion. The empirical variance itself cannot always be reliably estimated and some caution is needed. We describe the difficulties here and identify ready solutions which only require appropriate use of variance-stabilizing transformations. From these we obtain estimators for the steady state, or dynamic equilibrium, of the number of hematopoietic stem cells involved in repopulating the marrow. The calculations themselves are not too involved. We give the distribution theory for the estimator as well as simple approximations for practical application. As an illustration, we rework on data recently gathered to address the question as to whether or not reconstitution of marrow grafts in the clinical setting might be considered to be oligoclonal.

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 stem cells. Environ Health Perspect 124:957-965; http://dx.doi.org/10.1289/ehp.1509820.

Neuronal differentiation and long-term culture of the human neuroblastoma line SH-SY5Y.

PubMed

Constantinescu, R; Constantinescu, A T; Reichmann, H; Janetzky, B

2007-01-01

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder in industrialized countries. Present cell culture models for PD rely on either primary cells or immortal cell lines, neither of which allow for long-term experiments on a constant population, a crucial requisite for a realistic model of slowly progressing neurodegenerative diseases. We differentiated SH-SY5Y human dopaminergic neuroblastoma cells to a neuronal-like state in a perfusion culture system using a combination of retinoic acid and mitotic inhibitors. The cells could be cultivated for two months without the need for passage. We show, by various means, that the differentiated cells exhibit, at the molecular level, many neuronal properties not characteristic to the starting line. This approach opens the possibility to develop chronic models, in which the effect of perturbations and putative counteracting strategies can be monitored over long periods of time in a quasi-stable cell population.

Centering Single Cells in Microgels via Delayed Crosslinking Supports Long-Term 3D Culture by Preventing Cell Escape.

PubMed

Kamperman, Tom; Henke, Sieger; Visser, Claas Willem; Karperien, Marcel; Leijten, Jeroen

2017-06-01

Single-cell-laden microgels support physiological 3D culture conditions while enabling straightforward handling and high-resolution readouts of individual cells. However, their widespread adoption for long-term cultures is limited by cell escape. In this work, it is demonstrated that cell escape is predisposed to off-center encapsulated cells. High-speed microscopy reveals that cells are positioned at the microgel precursor droplets' oil/water interface within milliseconds after droplet formation. In conventional microencapsulation strategies, the droplets are typically gelled immediately after emulsification, which traps cells in this off-center position. By delaying crosslinking, driving cells toward the centers of microgels is succeeded. The centering of cells in enzymatically crosslinked microgels prevents their escape during at least 28 d. It thereby uniquely enables the long-term culture of individual cells within <5-µm-thick 3D uniform hydrogel coatings. Single cell analysis of mesenchymal stem cells in enzymatically crosslinked microgels reveals unprecedented high cell viability (>90%), maintained metabolic activity (>70%), and multilineage differentiation capacity (>60%) over a period of 28 d. The facile nature of this microfluidic cell-centering method enables its straightforward integration into many microencapsulation strategies and significantly enhances control, reproducibility, and reliability of 3D single cell cultures. © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Effect of the Purinergic Inhibitor Oxidized ATP in a Model of Islet Allograft Rejection

PubMed Central

Vergani, Andrea; Fotino, Carmen; D’Addio, Francesca; Tezza, Sara; Podetta, Michele; Gatti, Francesca; Chin, Melissa; Bassi, Roberto; Molano, Ruth D.; Corradi, Domenico; Gatti, Rita; Ferrero, Maria E.; Secchi, Antonio; Grassi, Fabio; Ricordi, Camillo; Sayegh, Mohamed H.; Maffi, Paola; Pileggi, Antonello; Fiorina, Paolo

2013-01-01

The lymphocytic ionotropic purinergic P2X receptors (P2X1R-P2X7R, or P2XRs) sense ATP released during cell damage-activation, thus regulating T-cell activation. We aim to define the role of P2XRs during islet allograft rejection and to establish a novel anti-P2XRs strategy to achieve long-term islet allograft function. Our data demonstrate that P2X1R and P2X7R are induced in islet allograft-infiltrating cells, that only P2X7R is increasingly expressed during alloimmune response, and that P2X1R is augmented in both allogeneic and syngeneic transplantation. In vivo short-term P2X7R targeting (using periodate-oxidized ATP [oATP]) delays islet allograft rejection, reduces the frequency of Th1/Th17 cells, and induces hyporesponsiveness toward donor antigens. oATP-treated mice displayed preserved islet grafts with reduced Th1 transcripts. P2X7R targeting and rapamycin synergized in inducing long-term islet function in 80% of transplanted mice and resulted in reshaping of the recipient immune system. In vitro P2X7R targeting using oATP reduced T-cell activation and diminished Th1/Th17 cytokine production. Peripheral blood mononuclear cells obtained from long-term islet-transplanted patients showed an increased percentage of P2X7R+CD4+ T cells compared with controls. The beneficial effects of oATP treatment revealed a role for the purinergic system in islet allograft rejection, and the targeting of P2X7R is a novel strategy to induce long-term islet allograft function. PMID:23315496

KHYG-1 and NK-92 represent different subtypes of LFA-1-mediated NK cell adhesiveness.

PubMed

Suck, Garnet; Tan, Suet-Mien; Chu, Sixian; Niam, Madelaine; Vararattanavech, Ardcharaporn; Lim, Tsyr Jong; Koh, Mickey B C

2011-01-01

Novel cancer cellular therapy approaches involving long-term ex vivo IL-2 stimulated highly cytotoxic natural killer (NK) cells are emerging. However, adhesion properties of such NK cells are not very well understood. Herein, we describe the novel observation of permanently activated alphaLbeta2 integrin leukocyte function-associated antigen (LFA)-1 adhesion receptor in long-term IL-2 activated NK cells and the permanent NK cell lines KHYG-1 and NK-92. We show that such cytokine activated NK effectors constitutively adhered to the LFA-1-ligand ICAM-1, whereas binding to the lower affinity ligand ICAM-3 required additional exogenous activating conditions. The results demonstrate an extended conformation and an intermediate affinity state for the LFA-1 population expressed by the NK cells. Interestingly, adhesion to ICAM-1 or K562 induced pronounced cell spreading in KHYG-1, but not in NK-92, and partially in long-term IL-2 stimulated primary NK cells. It is conceivable that such differential adhesion characteristics may impact motility potential of such NK effectors with relevance to clinical tumor targeting. KHYG-1 could be a useful model in planning future targeted therapeutic approaches involving NK effectors with augmented functions.

Rebuilding the Injured Lung

PubMed Central

2015-01-01

The 57th annual Thomas L. Petty Aspen Lung Conference, entitled “Rebuilding the Injured Lung,” was held from June 4 to 7, 2014 at the Gant Conference Center in Aspen, Colorado. Investigators from a wide range of disciplines and perspectives convened to discuss the biology of lung injury, how the lung repairs itself, how and why repair fails, and how the repair process can be enhanced. Among the challenges identified in the course of the conference was how to develop more predictive experimental models that capture the multidimensional complexity of lung injury and repair in a tractable manner. From such approaches that successfully fuse the biological and physical sciences, the group envisioned that new therapies for acute and chronic lung injury would emerge. The discussion of experimental therapeutics ranged from pharmaceuticals and cells that interdict fibrosis and enhance repair to a de novo lung derived from stem cells repopulating a decellularized matrix. PMID:25830839

Can cell kinetic parameters predict the response of tumours to radiotherapy?

PubMed

McNally, N J

1989-11-01

Three potential predictive assays of the repopulation component in tumour response to therapy are considered. (1) The DNA index can easily be measured. It is of prognostic value for cancers of certain sites, aneuploidy being a bad prognostic indicator. It is not strictly an indicator of cell proliferation. (2) The in vitro labelling index is of predictive value in early stage operable breast cancer and in head and neck cancer. In the former a high pretreatment labelling index can identify patients who could benefit from adjuvant chemotherapy. (3) The tumour potential doubling time (Tpot) can be measured rapidly following in vivo labelling with bromodeoxyuridine or iododeoxyuridine. We have measured Tpot in over 100 solid tumours with a success rate of about 75 per cent. Nearly 50 per cent of the tumours have a pre-treatment potential doubling time of 5 days or less. These would be suitable candidates for accelerated fractionation.

Development and manufacture of an investigational human living dermal equivalent (ICX-SKN).

PubMed

Flasza, Marzena; Kemp, Paul; Shering, David; Qiao, Jizeng; Marshall, Damian; Bokta, Ausha; Johnson, Penny A

2007-11-01

To design and manufacture an investigational living skin graft replacement (ICX-SKN) that is able to incorporate into the host, providing healing by primary intent without the need for a second intervention. The ICX-SKN skin graft replacement has been designed as an allogeneic dermal substitute comprising an extracellular matrix composed largely of human collagen and human dermal fibroblast cells (HDFs). ICX-SKN is first formed by casting a provisional matrix of fibrin, into which HDFs are seeded. Through a process of maturation, HDFs are induced to lay down collagen and other extracellular matrix materials and, as the construct matures, the original fibrin is largely replaced by collagen, which provides tensile strength and flexibility to the construct. In order to design a product and manufacturing system that lends itself to large-scale production the process was developed as a discontinuous process consisting of four stages: 1. batch casting and maturation of the initial construct (pSKN), 2. freeze-drying of pSKN to produce a second intermediate (dSKN), 3. sterilization by gamma-irradiation of dSKN to produce a third intermediate (sSKN), and finally, 4. repopulation of sSKN by fresh HDFs to produce the final product, ICX-SKN skin graft replacement. Preliminary characterization of ICX-SKN and its application in a preclinical model are described. The 7-week maturation period resulted in a construct (pSKN) with robust handling properties, which was composed mainly of human collagen I. Following development of a process for freeze-drying and subsequent sterilization, the matrix was successfully repopulated with fresh HDFs. In addition, it was demonstrated that human keratinocytes attached and differentiated on the matrix. Application of human keratinocytes to the repopulated constructs (ICX-SKN) resulted in expression of markers of basement membranes that was largely dependent on the presence of living HDFs on the constructs. ICX-SKN graft replacements applied to excision wounds in mice healed and were rapidly re-epithelialized. ICX-SKN has been developed as a platform product that can be used as a skin graft replacement and the process by which it is manufactured has been designed for the product to be available to the end-user off-the-shelf and for ease-of-use in practice.

Genetically Encoded Calcium Indicators For Studying Long-Term Calcium Dynamics During Apoptosis

PubMed Central

Garcia, M. Iveth; Chen, Jessica J.; Boehning, Darren

2017-01-01

Intracellular calcium release is essential for regulating almost all cellular functions. Specific spatio-temporal patterns of cytosolic calcium elevations are critical determinants of cell fate in response to pro-apoptotic cellular stressors. As the apoptotic program can take hours or days, measurement of long-term calcium dynamics are essential for understanding the mechanistic role of calcium in apoptotic cell death. Due to the technical limitations of using calcium-sensitive dyes to measure cytosolic calcium little is known about long-term calcium dynamics in living cells after treatment with apoptosis-inducing drugs. Genetically encoded calcium indicators could potentially overcome some of the limitations of calcium-sensitive dyes. Here, we compared the performance of the genetically encoded calcium indicators GCaMP6s and GCaMP6f with the ratiometric dye Fura-2. GCaMP6s performed as well or better than Fura-2 in detecting agonist-induced calcium transients. We then examined the utility of GCaMP6s for continuously measuring apoptotic calcium release over the course of ten hours after treatment with staurosporine. We found that GCaMP6s was suitable for measuring apoptotic calcium release over long time courses and revealed significant heterogeneity in calcium release dynamics in individual cells challenged with staurosporine. Our results suggest GCaMP6s is an excellent indicator for monitoring long-term changes cytosolic calcium during apoptosis. PMID:28073595

Revised Modelling of the Addition of Synchronous Chemotherapy to Radiotherapy in Squamous Cell Carcinoma of the Head and Neck-A Low α/β?

PubMed

Best, James; Fong, Charles; Benghiat, Helen; Mehanna, Hisham; Glaholm, John; Hartley, Andrew

2018-06-13

Background: The effect of synchronous chemotherapy in squamous cell carcinoma of the head and neck (SCCHN) has been modelled as additional Biologically Effective Dose (BED) or as a prolonged tumour cell turnover time during accelerated repopulation. Such models may not accurately predict the local control seen when hypofractionated accelerated radiotherapy is used with synchronous chemotherapy. Methods: For the purposes of this study three isoeffect relationships were assumed: Firstly, from the RTOG 0129 trial, synchronous cisplatin chemotherapy with 70 Gy in 35 fractions over 46 days results in equivalent local control to synchronous cisplatin chemotherapy with 36 Gy in 18# followed by 36 Gy in 24# (2# per day) over a total of 39 days. Secondly, in line with primary local control outcomes from the PET-Neck study, synchronous cisplatin chemotherapy with 70 Gy in 35# over 46 days results in equivalent local control to synchronous cisplatin chemotherapy delivered with 65 Gy in 30# over 39 days. Thirdly, from meta-analysis data, 70 Gy in 35# over 46 days with synchronous cisplatin results in equivalent local control to 84 Gy in 70# over 46 days delivered without synchronous chemotherapy. Using the linear quadratic equation the above isoeffect relationships were expressed algebraically to determine values of α , α/β , and k for SCCHN when treated with synchronous cisplatin using standard parameters for the radiotherapy alone schedule ( α = 0.3 Gy −1 , α/β = 10 Gy, and k = 0.42 Gy 10 day −1 ). Results: The values derived for α/β , α and k were 2 Gy, 0.20 and 0.21 Gy −1 , and 0.65 and 0.71 Gy₂day −1 . Conclusions: Within the limitations of the assumptions made, this model suggests that accelerated repopulation may remain a significant factor when synchronous chemotherapy is delivered with radiotherapy in SCCHN. The finding of a low α/β for SCCHN treated with cisplatin suggests a greater tumour susceptibility to increasing dose per fraction and underlines the importance of the completion of randomized trials examining the role of hypofractionated acceleration in SCCHN.

Nerve growth factor (NGF) immunoreactive neurons in the juvenile rat hippocampus: response to acute and long-term high-light open-field (HL-OF) or forced swim (FS) stress stimulation.

PubMed

Badowska-Szalewska, E; Spodnik, E; Ludkiewicz, B; Klejbor, I; Moryś, J

2011-12-29

This study aimed at examining and comparing the influence of two different stress stimuli on the density (number of cells/mm²) of nerve growth factor (NGF) containing neurons in the hippocampal CA1 and CA3 pyramidal cell layers and the dentate gyrus (DG) granule cell layer in juvenile rats (P28; P-postnatal day). The high-light open-field (HL-OF) test and forced swim (FS) test were employed to investigate the effects of a single, 15-min acute exposure and repeated (15 min daily for 21 days) long-term exposure to stress. In order to detect NGF-ir neurons, immunohistochemical (-ir) techniques were used. In comparison with nonstressed animals, acute and long-term HL-OF or FS stimulation resulted in a marked increase (P<0.001) in the density of NGF-ir containing cells in all the hippocampal structures. The frequency of stress application (acute vs. long-term), however, did not have a substantial impact on the studied parameter, with the exception of the CA3 sector, where a decreased density (P<0.001) of NGF-ir neurons was observed after long-term exposure to FS. It may be concluded that a rise in the density of NGF-ir neurons in the juvenile rat hippocampus after exposure to HL-OF or FS stressors could have affected the activity of the hypothalamic-pituitary-adrenocortical (HPA) stress axis. Prolonged HL-OF or FS stress was probably aggravating enough not to trigger the habituation process. The type of stressor applied (HL-OF vs. FS) was not essentially a factor determining the density of NGF-ir cells in the hippocampus. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Viability of long-term gene therapy in the cochlea.

PubMed

Atkinson, Patrick J; Wise, Andrew K; Flynn, Brianna O; Nayagam, Bryony A; Richardson, Rachael T

2014-04-22

Gene therapy has been investigated as a way to introduce a variety of genes to treat neurological disorders. An important clinical consideration is its long-term effectiveness. This research aims to study the long-term expression and effectiveness of gene therapy in promoting spiral ganglion neuron survival after deafness. Adenoviral vectors modified to express brain derived neurotrophic factor or neurotrophin-3 were unilaterally injected into the guinea pig cochlea one week post ototoxic deafening. After six months, persistence of gene expression and significantly greater neuronal survival in neurotrophin-treated cochleae compared to the contralateral cochleae were observed. The long-term gene expression observed indicates that gene therapy is potentially viable; however the degeneration of the transduced cells as a result of the original ototoxic insult may limit clinical effectiveness. With further research aimed at transducing stable cochlear cells, gene therapy may be an efficacious way to introduce neurotrophins to promote neuronal survival after hearing loss.

Reciprocal Interactions between Multiple Myeloma Cells and Osteoprogenitor Cells Affect Bone Formation and Tumor Growth

DTIC Science & Technology

2015-12-01

cells (HSCs) are multipotent cells that differentiate into myeloid, lymphoid and erythroid lineages, and have short-term or long-term regenerative...All rights reserved Nature Reviews | Rheumatology a b MPP CMP CLP Lymphoid cells NK cellB cell T cell Megakaryocyte and erythrocytes Macrophage and...into other cell types. CLP, common lymphoid progenitor; CMP, common myeloid progenitor; MPP, multipotent progenitor; NK cell , natural killer cell . R E

Investigation of long term storage effects on aerospace nickel-cadmium cell performance

NASA Technical Reports Server (NTRS)

Yi, T. Y.

1986-01-01

A study on evaluation of the long term storage effects on aerospace nickel-cadmium cells currently being performed at NASA/Goddard Space Flight Center (GSFC) is described. A number of cells of 6 Ah and 12 Ah capacities which were stored in shorted condition for 8 to 9 years at the GSFC were selected for this study. These cells will undergo electrical acceptance testing the the GSFC, and life cycling at the NASA Battery Test Facility at the Naval Weapons Facility at the Naval Weapons Support Center (NWSC) in Crane, Indiana; in addition, some cells from the study will undergo destructive analyses.

Investigation of long term storage effects on aerospace nickel-cadmium cell performance

NASA Astrophysics Data System (ADS)

Yi, T. Y.

1986-09-01

A study on evaluation of the long term storage effects on aerospace nickel-cadmium cells currently being performed at NASA/Goddard Space Flight Center (GSFC) is described. A number of cells of 6 Ah and 12 Ah capacities which were stored in shorted condition for 8 to 9 years at the GSFC were selected for this study. These cells will undergo electrical acceptance testing the the GSFC, and life cycling at the NASA Battery Test Facility at the Naval Weapons Facility at the Naval Weapons Support Center (NWSC) in Crane, Indiana; in addition, some cells from the study will undergo destructive analyses.

Long-term survival of donor bone marrow multipotent mesenchymal stromal cells implanted into the periosteum of patients with allogeneic graft failure.

PubMed

Kuzmina, L A; Petinati, N A; Sats, N V; Drize, N J; Risinskaya, N V; Sudarikov, A B; Vasilieva, V A; Drokov, M Y; Michalzova, E D; Parovichnikova, E N; Savchenko, V G

2016-09-01

The present study involved three patients with graft failure following allogeneic hematopoietic stem cell transplantation (allo-HSCT). We obtained multipotent mesenchymal stromal cells (MSCs) from the original hematopoietic cell donors and implanted these cells in the periosteum to treat long-term bone marrow aplasia. The results showed that in all patients endogenous blood formation was recovered 2 weeks after MSC administration. Donor MSCs were found in recipient bone marrow three and 5 months following MSC implantation. Thus, our findings indicate that functional donor MSCs can persist in patient bone marrow.

Long-term temperature effects on GaAs solar cells

NASA Technical Reports Server (NTRS)

Heinbockel, J. H.; Hong, K. H.

1979-01-01

The thermal degradation of AlGaAs solar cells resulting from a long-term operation in a space environment is investigated. The solar cell degradation effects caused by zinc and aluminum diffusion as well as deterioration by arsenic evaporation are presented. Also, the results are presented of experimental testing and measurements of various GaAs solar cell properties while the solar cell was operating in the temperature range of 27 C to 350 C. In particular, the properties of light current voltage curves, dark current voltage curves, and spectral response characteristics are given. Finally, some theoretical models for the annealing of radiation damage over various times and temperatures are included.

Atmospheric Oxygen Inhibits Growth and Differentiation of Marrow-Derived Mouse Mesenchymal Stem Cells via a p53 Dependent Mechanism: Implications for Long-Term Culture Expansion

PubMed Central

Boregowda, Siddaraju; Krishnappa, Veena; Chambers, Jeremy; LoGrasso, Phillip V.; Lai, Wen-Tzu; Ortiz, Luis A.; Phinney, Donald G.

2013-01-01

Large scale expansion of human mesenchymal stem cells (MSCs) is routinely performed for clinical therapy. In contrast, developing protocols for large scale expansion of primary mouse MSCs has been more difficult due to unique aspects of rodent biology. Currently, established methods to isolate mouse MSCs select for rapidly dividing subpopulations that emerge from bone marrow cultures following long-term (months) expansion in atmospheric oxygen. Herein, we demonstrate that exposure to atmospheric oxygen rapidly induced p53, TOP2A and BAX expression and mitochondrial ROS generation in primary mouse MSCs resulting in oxidative stress, reduced cell viability and inhibition of cell proliferation. Alternatively, procurement and culture in 5% oxygen supported more prolific expansion of the CD45−ve/CD44+ve cell fraction in marrow, produced increased MSC yields following immuno-depletion, and supported sustained MSC growth resulting in a 2300-fold increase in cumulative cell yield by 4th passage. MSCs cultured in 5% oxygen also exhibited enhanced tri-lineage differentiation. The oxygen-induced stress response was dependent upon p53 since siRNA mediated knockdown of p53 in wild type cells or exposure of p53−/− MSCs to atmospheric oxygen failed to induce ROS generation, reduce viability, or arrest cell growth. These data indicate that long-term culture expansion of mouse MSCs in atmospheric oxygen selects for clones with absent or impaired p53 function, which allows cells to escape oxygen-induced growth inhibition. In contrast, expansion in 5% oxygen generates large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen, and therefore a functional p53 protein, even after long-term expansion in vitro. PMID:22367737

Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

PubMed

Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

2013-01-01

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology in situ

PubMed Central

Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

2013-01-01

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ. PMID:24223842

Long Term Culture of the A549 Cancer Cell Line Promotes Multilamellar Body Formation and Differentiation towards an Alveolar Type II Pneumocyte Phenotype

PubMed Central

Cooper, James Ross; Abdullatif, Muhammad Bilal; Burnett, Edward C.; Kempsell, Karen E.; Conforti, Franco; Tolley, Howard; Collins, Jane E.; Davies, Donna E.

2016-01-01

Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 ‘alveolar’ cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham’s F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line. PMID:27792742

Identification of Nascent Memory CD8 T Cells and Modeling of Their Ontogeny.

PubMed

Crauste, Fabien; Mafille, Julien; Boucinha, Lilia; Djebali, Sophia; Gandrillon, Olivier; Marvel, Jacqueline; Arpin, Christophe

2017-03-22

Primary immune responses generate short-term effectors and long-term protective memory cells. The delineation of the genealogy linking naive, effector, and memory cells has been complicated by the lack of phenotypes discriminating effector from memory differentiation stages. Using transcriptomics and phenotypic analyses, we identify Bcl2 and Mki67 as a marker combination that enables the tracking of nascent memory cells within the effector phase. We then use a formal approach based on mathematical models describing the dynamics of population size evolution to test potential progeny links and demonstrate that most cells follow a linear naive→early effector→late effector→memory pathway. Moreover, our mathematical model allows long-term prediction of memory cell numbers from a few early experimental measurements. Our work thus provides a phenotypic means to identify effector and memory cells, as well as a mathematical framework to investigate their genealogy and to predict the outcome of immunization regimens in terms of memory cell numbers generated. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

A Kinetic Model for Calcium Dynamics in RAW 264.7 Cells: 2. Knockdown Response and Long-Term Response

PubMed Central

Maurya, Mano Ram; Subramaniam, Shankar

2007-01-01

This article addresses how quantitative models such as the one proposed in the companion article can be used to study cellular network perturbations such as knockdowns and pharmacological perturbations in a predictive manner. Using the kinetic model for cytosolic calcium dynamics in RAW 264.7 cells developed in the companion article, the calcium response to complement 5a (C5a) for the knockdown of seven proteins (C5a receptor; G-β-2; G-α,i-2,3; regulator of G-protein signaling-10; G-protein coupled receptor kinase-2; phospholipase C β-3; arrestin) is predicted and validated against the data from the Alliance for Cellular Signaling. The knockdown responses provide insights into how altered expressions of important proteins in disease states result in intermediate measurable phenotypes. Long-term response and long-term dose response have also been predicted, providing insights into how the receptor desensitization, internalization, and recycle result in tolerance. Sensitivity analysis of long-term response shows that the mechanisms and parameters in the receptor recycle path are important for long-term calcium dynamics. PMID:17483189

Potential of coconut water and soy milk for use as storage media to preserve the viability of periodontal ligament cells: an in vitro study.

PubMed

Moura, Camilla Cristhian Gomes; Soares, Priscilla Barbosa Ferreira; de Paula Reis, Manuella Verdinelli; Fernandes Neto, Alfredo Júlio; Zanetta Barbosa, Darceny; Soares, Carlos José

2014-02-01

There is no consensus regarding the ability of coconut water and soy milk to maintain long-term cell viability. This study investigated the ability of pH-adjusted coconut water and soy milk to maintain the viability of periodontal ligament cells over a short and a longer period and compared these abilities with those of other solutions. Dog premolar teeth were extracted, dried for 30 min, and stored in the following media for 50 min or 24 h: long shelf-life whole milk (SWM), long shelf-life skim milk (SSM), Hank's Balanced Salt Solution (HBSS), soy milk (SM), and pH-adjusted coconut water (CW). The positive and two negative control groups corresponded to 0-min, 30-min (short-term), and 24-h (long-term) dry times, respectively. Cell viability was analyzed by trypan blue exclusion. Data were statistically analyzed using the Kruskal-Wallis test with post-analysis using the Dunn method. In the short-term experiment, the SSM resulted in significantly lower cell viability than SM and CW. At 24 h, SM and CW resulted in higher viability than HBSS and SSM and in comparable performance with the positive control group. Cell viability decreased over time, except in SM and CW. Soy milk and pH-adjusted coconut water showed promising results as storage solutions for avulsed teeth, preserving the viability for up to 24 h. © 2013 John Wiley & Sons A/S.

In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

PubMed

Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

2017-08-01

Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

Memory retrieval by activating engram cells in mouse models of early Alzheimer’s disease

PubMed Central

Roy, Dheeraj S.; Arons, Autumn; Mitchell, Teryn I.; Pignatelli, Michele; Ryan, Tomás J.; Tonegawa, Susumu

2016-01-01

Summary Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive memory decline and subsequent loss of broader cognitive functions1. Memory decline in early stages of Alzheimer’s is mostly limited to episodic memory, for which the hippocampus (HPC) plays a crucial role2. However, it has been uncertain whether the observed amnesia in early stages of Alzheimer’s is due to disrupted encoding and consolidation of episodic information, or an impairment in the retrieval of stored memory information. Here we show that in transgenic mouse models of early Alzheimer’s, direct optogenetic activation of hippocampal memory engram cells results in memory retrieval despite the fact that these mice are amnesic in long-term memory tests when natural recall cues are utilized, revealing a retrieval, rather than a storage impairment. Prior to amyloid plaque deposition, the amnesia in these mice is age-dependent3–5, which correlates with a progressive reduction of spine density of hippocampal dentate gyrus (DG) engram cells. We show that optogenetic induction of long-term potentiation (LTP) at perforant path (PP) synapses of DG engram cells restores both spine density and long-term memory. We also demonstrate that an ablation of DG engram cells containing restored spine density prevents the rescue of long-term memory. Thus, selective rescue of spine density in engram cells may lead to an effective strategy for treating memory loss in early stages of Alzheimer’s disease. PMID:26982728

Dual effects of fluoxetine on mouse early embryonic development

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

Kim, Chang-Woon; Department of Obstetrics and Gynecology, Samsung Changwon Hospital, Sungkyunkwan University, Changwon 630-723; Choe, Changyong

2012-11-15

Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50 μM) for different durations. When late 2-cells were incubated with 5 μM fluoxetine for 6 h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetinemore » (5 μM) over 24 h showed a reduction in blastocyst formation. The addition of fluoxetine (5 μM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K{sup +} channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ∼ 30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating. Highlights: ► Short-term exposure of 2-cells to fluoxetine enhances mouse blastocyst formation. ► The enhancive effect of fluoxetine is resulted from CaMKII activation. ► Long-term exposure of 2-cells to fluoxetine decreases mouse blastocyst formation. ► The inhibitory effect of fluoxetine is mediated through TREK channel gating.« less

Effect of the cellular structure on thermal conductivity of rigid closed-cell foam polymers during long-term aging

NASA Astrophysics Data System (ADS)

Dementyev, A. G.; Dementyev, M. A.; Zinger, P. A.; Metlyakova, I. R.

1999-03-01

The thermal conductivity of rigid closed-cell polyurethane foams during long-term aging has been studied. The similarity between the kinetics of changes in the physical and mechanical characteristics of PU foams on progressive aging is established, which is attributed to the effect of matrix destruction. It is found that rigid foams have cell walls of various strength, whose impact on the kinetics of changes in the physical characteristics of the foams during long-term aging is ascertained. The results of predicting the thermal conductivity of PU foams by the method of temperature-time analogy and establishing the limits of its application are discussed. The research presented is of interest both in determining the foam durability and in replacing freons by alternative, ecologically less harmful blowing agents.

Systematic Prevention of Bubble Formation and Accumulation for Long-Term Culture of Pancreatic Islet Cells in Microfluidic Device

PubMed Central

Wang, Yong; Lee, Dongyoung; Zhang, Lisa; Jeon, Hyojin; Mendoza-Elias, Joshua E.; Harvat, Tricia A.; Hassan, Sarah Z.; Zhou, Amanda; Eddington, David T.; Oberholzer, José

2012-01-01

Reliable long-term cell culture in microfluidic system is limited by air bubble formation and accumulation. In this study, we developed a bubble removal system capable of both trapping and discharging air bubbles in a consistent and reliable manner. Combined with PDMS (Polydimethylsiloxane) hydrophilic surface treatment and vacuum filling, a microfluidic perifusion system equipped with the bubble trap was successfully applied for long-term culture of mouse pancreatic islets with no bubble formation and no flow interruption. In addition to demonstrating normal cell viability and islet morphology, post-cultured islets exhibited normal insulin secretion kinetics, intracellular calcium signaling, and changes in mitochondrial potentials in response to glucose challenge. This design could be easily adapted by other microfluidic systems due to its simple design, ease of fabrication, and portability. PMID:22252566

Wilms Tumor NCAM-Expressing Cancer Stem Cells as Potential Therapeutic Target for Polymeric Nanomedicine.

PubMed

Markovsky, Ela; Vax, Einav; Ben-Shushan, Dikla; Eldar-Boock, Anat; Shukrun, Rachel; Yeini, Eilam; Barshack, Iris; Caspi, Revital; Harari-Steinberg, Orit; Pode-Shakked, Naomi; Dekel, Benjamin; Satchi-Fainaro, Ronit

2017-11-01

Cancer stem cells (CSC) form a specific population within the tumor that has been shown to have self-renewal and differentiation properties, increased ability to migrate and form metastases, and increased resistance to chemotherapy. Consequently, even a small number of cells remaining after therapy can repopulate the tumor and cause recurrence of the disease. CSCs in Wilms tumor, a pediatric renal cancer, were previously shown to be characterized by neural cell adhesion molecule (NCAM) expression. Therefore, NCAM provides a specific biomarker through which the CSC population in this tumor can be targeted. We have recently developed an NCAM-targeted nanosized conjugate of paclitaxel bound to a biodegradable polyglutamic acid polymer. In this work, we examined the ability of the conjugate to inhibit Wilms tumor by targeting the NCAM-expressing CSCs. Results show that the conjugate selectively depleted the CSC population of the tumors and effectively inhibited tumor growth without causing toxicity. We propose that the NCAM-targeted conjugate could be an effective therapeutic for Wilms tumor. Mol Cancer Ther; 16(11); 2462-72. ©2017 AACR . ©2017 American Association for Cancer Research.

Adjudin, a potential male contraceptive, exerts its effects locally in the seminiferous epithelium of mammalian testes.

PubMed

Mok, Ka-Wai; Mruk, Dolores D; Lie, Pearl P Y; Lui, Wing-Yee; Cheng, C Yan

2011-05-01

Adjudin is a derivative of 1H-indazole-3-carboxylic acid that was shown to have potent anti-spermatogenic activity in rats, rabbits, and dogs. It exerts its effects most notably locally in the apical compartment of the seminiferous epithelium, behind the blood-testis barrier, by disrupting adhesion of germ cells, most notably spermatids to the Sertoli cells, thereby inducing release of immature spermatids from the epithelium that leads to infertility. After adjudin is metabolized, the remaining spermatogonial stem cells and spermatogonia repopulate the seminiferous epithelium gradually via spermatogonial self-renewal and differentiation, to be followed by meiosis and spermiogenesis, and thus fertility rebounds. Recent studies in rats have demonstrated unequivocally that the primary and initial cellular target of adjudin in the testis is the apical ectoplasmic specialization, a testis-specific anchoring junction type restricted to the interface between Sertoli cells and elongating spermatids (from step 8 to 19 spermatids). In this review, we highlight some of the recent advances and obstacles regarding the possible use of adjudin as a male contraceptive.

Adjudin, a potential male contraceptive, exerts its effects locally in the seminiferous epithelium of mammalian testes

PubMed Central

Mok, Ka-Wai; Mruk, Dolores D; Lie, Pearl P Y; Lui, Wing-Yee; Cheng, C Yan

2015-01-01

Adjudin is a derivative of 1H-indazole-3-carboxylic acid that was shown to have potent anti-spermatogenic activity in rats, rabbits, and dogs. It exerts its effects most notably locally in the apical compartment of the seminiferous epithelium, behind the blood–testis barrier, by disrupting adhesion of germ cells, most notably spermatids to the Sertoli cells, thereby inducing release of immature spermatids from the epithelium that leads to infertility. After adjudin is metabolized, the remaining spermatogonial stem cells and spermatogonia repopulate the seminiferous epithelium gradually via spermatogonial self-renewal and differentiation, to be followed by meiosis and spermiogenesis, and thus fertility rebounds. Recent studies in rats have demonstrated unequivocally that the primary and initial cellular target of adjudin in the testis is the apical ectoplasmic specialization, a testis-specific anchoring junction type restricted to the interface between Sertoli cells and elongating spermatids (from step 8 to 19 spermatids). In this review, we highlight some of the recent advances and obstacles regarding the possible use of adjudin as a male contraceptive. PMID:21307270

A Milieu Molecule for TGF-β Required for Microglia Function in the Nervous System.

PubMed

Qin, Yan; Garrison, Brian S; Ma, Wenjiang; Wang, Rui; Jiang, Aiping; Li, Jing; Mistry, Meeta; Bronson, Roderick T; Santoro, Daria; Franco, Charlotte; Robinton, Daisy A; Stevens, Beth; Rossi, Derrick J; Lu, Chafen; Springer, Timothy A

2018-06-12

Extracellular proTGF-β is covalently linked to "milieu" molecules in the matrix or on cell surfaces and is latent until TGF-β is released by integrins. Here, we show that LRRC33 on the surface of microglia functions as a milieu molecule and enables highly localized, integrin-αVβ8-dependent TGF-β activation. Lrrc33 -/- mice lack CNS vascular abnormalities associated with deficiency in TGF-β-activating integrins but have microglia with a reactive phenotype and after 2 months develop ascending paraparesis with loss of myelinated axons and death by 5 months. Whole bone marrow transplantation results in selective repopulation of Lrrc33 -/- brains with WT microglia and halts disease progression. The phenotypes of WT and Lrrc33 -/- microglia in the same brain suggest that there is little spreading of TGF-β activated from one microglial cell to neighboring microglia. Our results suggest that interactions between integrin-bearing cells and cells bearing milieu molecule-associated TGF-β provide localized and selective activation of TGF-β. Copyright © 2018 Elsevier Inc. All rights reserved.

Persistent effects of chlorine inhalation on respiratory health

PubMed Central

Hoyle, Gary W.; Svendsen, Erik R.

2016-01-01

Chlorine gas is a toxic respiratory irritant that is considered a chemical threat agent because of the potential for release in industrial accidents or terrorist attacks. Chlorine inhalation damages the respiratory tract, including the airways and distal lung, and can result in acute lung injury. Some individuals exposed to chlorine experience a full recovery from acute injury, whereas others develop persistent adverse effects, such as respiratory symptoms, inflammation, and lung-function decrements. In animal models, chlorine can produce persistent inflammation, remodeling, and obstruction in large or small airways, depending on species. Airways with pseudostratified epithelium are repaired efficiently, with surviving basal epithelial cells serving as progenitor cells that repopulate the complement of differentiated cell types. Distal airways lacking basal cells are repaired less efficiently, leading to chronic inflammation and fibrosis at these sites. Persistent chlorine-induced airway disease in humans is treated with asthma medication to relieve symptoms. However, such treatment does not ameliorate the underlying disease pathogenesis, so treatments that are more effective at preventing initial development of airway disease after irritant gas exposure and at reversing established disease are needed. PMID:27385061

Autologous hematopoietic stem cells for refractory Crohn's disease.

PubMed

DiNicola, C A; Zand, A; Hommes, D W

2017-05-01

Autologous hematopoietic stem cells are gaining ground as an effective and safe treatment for treating severe refractory Crohn's disease (CD). Autologous hematopoietic stem cell therapy (AHSCT) induces resetting of the immune system by de novo regeneration of T-cell repertoire and repopulation of epithelial cells by bone-marrow derived cells to help patients achieve clinical and endoscopic remission. Areas covered: Herein, the authors discuss the use of AHSCT in treating patients with CD. Improvements in disease activity have been seen in patients with severe autoimmune disease and patients with severe CD who underwent AHSCT for a concomitant malignant hematological disease. Clinical and endoscopic remission has been achieved in patients treated with AHSCT for CD. The only randomized trial published to date, the ASTIC Trial, did not support further use of AHSCT to treat CD. Yet, critics of this trial have deemed AHSCT as a promising treatment for severe refractory CD. Expert opinion: Even with the promising evidence presented for HSCT for refractory CD, protocols need to be refined through the collaboration of GI and hemato-oncology professionals. The goal is to incorporate safe AHSCT and restore tolerance by delivering an effective immune 'cease fire' as a treatment option for severe refractory CD.

Enhanced Expansion and Sustained Inductive Function of Skin‐Derived Precursor Cells in Computer‐Controlled Stirred Suspension Bioreactors

PubMed Central

Agabalyan, Natacha A.; Borys, Breanna S.; Sparks, Holly D.; Boon, Kathryn; Raharjo, Eko W.; Abbasi, Sepideh; Kallos, Michael S.

2016-01-01

Abstract Endogenous dermal stem cells (DSCs) reside in the adult hair follicle mesenchyme and can be isolated and grown in vitro as self‐renewing colonies called skin‐derived precursors (SKPs). Following transplantation into skin, SKPs can generate new dermis and reconstitute the dermal papilla and connective tissue sheath, suggesting they could have important therapeutic value for the treatment of skin disease (alopecia) or injury. Controlled cell culture processes must be developed to efficiently and safely generate sufficient stem cell numbers for clinical use. Compared with static culture, stirred‐suspension bioreactors generated fivefold greater expansion of viable SKPs. SKPs from each condition were able to repopulate the dermal stem cell niche within established hair follicles. Both conditions were also capable of inducing de novo hair follicle formation and exhibited bipotency, reconstituting the dermal papilla and connective tissue sheath, although the efficiency was significantly reduced in bioreactor‐expanded SKPs compared with static conditions. We conclude that automated bioreactor processing could be used to efficiently generate large numbers of autologous DSCs while maintaining their inherent regenerative function. Stem Cells Translational Medicine 2017;6:434–443 PMID:28191777

Genomic and Phenotypic Characterization of Yeast Biosensor for Deep-space Radiation

NASA Technical Reports Server (NTRS)

Marina, Diana B.; Santa Maria, Sergio; Bhattacharya, Sharmila

2016-01-01

The BioSentinel mission was selected to launch as a secondary payload onboard NASA Exploration Mission 1 (EM-1) in 2018. In BioSentinel, the budding yeast Saccharomyces cerevisiae will be used as a biosensor to measure the long-term impact of deep-space radiation to living organisms. In the 4U-payload, desiccated yeast cells from different strains will be stored inside microfluidic cards equipped with 3-color LED optical detection system to monitor cell growth and metabolic activity. At different times throughout the 12-month mission, these cards will be filled with liquid yeast growth media to rehydrate and grow the desiccated cells. The growth and metabolic rates of wild-type and radiation-sensitive strains in deep-space radiation environment will be compared to the rates measured in the ground- and microgravity-control units. These rates will also be correlated with measurements obtained from onboard physical dosimeters. In our preliminary long-term desiccation study, we found that air-drying yeast cells in 10% trehalose is the best method of cell preservation in order to survive the entire 18-month mission duration (6-month pre-launch plus 12-month full-mission periods). However, our study also revealed that desiccated yeast cells have decreasing viability over time when stored in payload-like environment. This suggests that the yeast biosensor will have different population of cells at different time points during the long-term mission. In this study, we are characterizing genomic and phenotypic changes in our yeast biosensor due to long-term storage and desiccation. For each yeast strain that will be part of the biosensor, several clones were reisolated after long-term storage by desiccation. These clones were compared to their respective original isolate in terms of genomic composition, desiccation tolerance and radiation sensitivity. Interestingly, clones from a radiation-sensitive mutant have better desiccation tolerance compared to their original isolate without losing radiation sensitivity. We employed Next-Generation Sequencing technology to better understand this phenotypic variation. Current effort is focusing on the analysis of high-throughput sequencing data to look for genomic changes in these reisolated clones compared to their original isolate.

Short- and long-term immunogenicity and protection induced by non-replicating smallpox vaccine candidates in mice and comparison with the traditional 1st generation vaccine.

PubMed

Ferrier-Rembert, Audrey; Drillien, Robert; Tournier, Jean-Nicolas; Garin, Daniel; Crance, Jean-Marc

2008-03-25

This study assessed three non-replicating smallpox vaccine candidates (modified vaccinia Ankara (MVA), NYVAC and HR) for their immunogenicity and ability to protect mice against an intranasal cowpox virus challenge and compared them with the traditional replicating vaccine. A single immunisation with the non-replicating vaccines induced a complete protection from death at short-term, but was not fully protective when mice were challenged 150 days post-vaccination with protection correlated with the specific neutralizing antibodies and CD4(+) T-cells responses. Prime-boost vaccination enabled effective long-term protection from death for mice vaccinated with MVA, but protection from disease and CD4(+) T-cell level were lower than the ones induced by the traditional vaccine over the long-term period. Further investigations are necessary with MVA to determine the optimal conditions of immunisation to induce at long-term immunogenicity and protection observed with the 1st generation smallpox vaccine.

Radiation-Induced Hemopoietic and Immune Dysfunction

DTIC Science & Technology

1991-06-01

the dog. Culture conditions were studied and optimized, and marrow cells were transplanted into otherwise lethally irradiated dogs to investigate stem ... cell survival in long- term cultures. Engraftment was observed only with short-term marrow cultures.

Rituximab does not reset defective early B cell tolerance checkpoints

PubMed Central

Chamberlain, Nicolas; Massad, Christopher; Oe, Tyler; Cantaert, Tineke; Herold, Kevan C.; Meffre, Eric

2015-01-01

Type 1 diabetes (T1D) patients show abnormalities in early B cell tolerance checkpoints, resulting in the accumulation of large numbers of autoreactive B cells in their blood. Treatment with rituximab, an anti-CD20 mAb that depletes B cells, has been shown to preserve β cell function in T1D patients and improve other autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. However, it remains largely unknown how anti–B cell therapy thwarts autoimmunity in these pathologies. Here, we analyzed the reactivity of Abs expressed by single, mature naive B cells from 4 patients with T1D before and 52 weeks after treatment to determine whether rituximab resets early B cell tolerance checkpoints. We found that anti–B cell therapy did not alter the frequencies of autoreactive and polyreactive B cells, which remained elevated in the blood of all patients after rituximab treatment. Moreover, the limited proliferative history of autoreactive B cells after treatment revealed that these clones were newly generated B cells and not self-reactive B cells that had escaped depletion and repopulated the periphery through homeostatic expansion. We conclude that anti–B cell therapy may provide a temporary dampening of autoimmune processes through B cell depletion. However, repletion with autoreactive B cells may explain the relapse that occurs in many autoimmune patients after anti–B cell therapy. PMID:26642366

Does stress remove the HDAC brakes for the formation and persistence of long-term memory?

PubMed

White, André O; Wood, Marcelo A

2014-07-01

It has been known for numerous decades that gene expression is required for long-lasting forms of memory. In the past decade, the study of epigenetic mechanisms in memory processes has revealed yet another layer of complexity in the regulation of gene expression. Epigenetic mechanisms do not only provide complexity in the protein regulatory complexes that control coordinate transcription for specific cell function, but the epigenome encodes critical information that integrates experience and cellular history for specific cell functions as well. Thus, epigenetic mechanisms provide a unique mechanism of gene expression regulation for memory processes. This may be why critical negative regulators of gene expression, such as histone deacetylases (HDACs), have powerful effects on the formation and persistence of memory. For example, HDAC inhibition has been shown to transform a subthreshold learning event into robust long-term memory and also generate a form of long-term memory that persists beyond the point at which normal long-term memory fails. A key question that is explored in this review, from a learning and memory perspective, is whether stress-dependent signaling drives the formation and persistence of long-term memory via HDAC-dependent mechanisms. Copyright © 2013 Elsevier Inc. All rights reserved.

Does stress remove the HDAC brakes for the formation and persistence of long-term memory?

PubMed Central

White, André O.; Wood, Marcelo A.

2013-01-01

It has been known for numerous decades that gene expression is required for long-lasting forms of memory. In the past decade, the study of epigenetic mechanisms in memory processes has revealed yet another layer of complexity in the regulation of gene expression. Epigenetic mechanisms do not only provide complexity in the protein regulatory complexes that control coordinate transcription for specific cell function, but the epigenome encodes critical information that integrates experience and cellular history for specific cell functions as well. Thus, epigenetic mechanisms provide a unique mechanism of gene expression regulation for memory processes. This may be why critical negative regulators of gene expression, such as histone deacetylases (HDACs), have powerful effects on the formation and persistence of memory. For example, HDAC inhibition has been shown to transform a subthreshold learning event into robust long-term memory and also generate a form of long-term memory that persists beyond the point at which normal long-term memory fails. A key question that is explored in this review, from a learning and memory perspective, is whether stress-dependent signaling drives the formation and persistence of long-term memory via HDAC-dependent mechanisms. PMID:24149059

Dual-Function Au@Y2O3:Eu3+ Smart Film for Enhanced Power Conversion Efficiency and Long-Term Stability of Perovskite Solar Cells.

PubMed

Kim, Chang Woo; Eom, Tae Young; Yang, In Seok; Kim, Byung Su; Lee, Wan In; Kang, Yong Soo; Kang, Young Soo

2017-07-28

In the present study, a dual-functional smart film combining the effects of wavelength conversion and amplification of the converted wave by the localized surface plasmon resonance has been investigated for a perovskite solar cell. This dual-functional film, composed of Au nanoparticles coated on the surface of Y 2 O 3 :Eu 3+ phosphor (Au@Y 2 O 3 :Eu 3+ ) nanoparticle monolayer, enhances the solar energy conversion efficiency to electrical energy and long-term stability of photovoltaic cells. Coupling between the Y 2 O 3 :Eu 3+ phosphor monolayer and ultraviolet solar light induces the latter to be converted into visible light with a quantum yield above 80%. Concurrently, the Au nanoparticle monolayer on the phosphor nanoparticle monolayer amplifies the converted visible light by up to 170%. This synergy leads to an increased solar light energy conversion efficiency of perovskite solar cells. Simultaneously, the dual-function film suppresses the photodegradation of perovskite by UV light, resulting in long-term stability. Introducing the hybrid smart Au@Y 2 O 3 :Eu 3+ film in perovskite solar cells increases their overall solar-to-electrical energy conversion efficiency to 16.1% and enhances long-term stability, as compared to the value of 15.2% for standard perovskite solar cells. The synergism between the wavelength conversion effect of the phosphor nanoparticle monolayer and the wave amplification by the localized surface plasmon resonance of the Au nanoparticle monolayer in a perovskite solar cell is comparatively investigated, providing a viable strategy of broadening the solar spectrum utilization.

Enhancement of Immune Memory Responses to Respiratory Infection

DTIC Science & Technology

2017-08-01

Unlimited Distribution 13. SUPPLEMENTARY NOTES 14. ABSTRACT Maintenance of long - term immunological memory against pathogens is crucial for the rapid...highly expressed in memory B cells in mice, and Atg7 is required for maintenance of long - term memory B cells needed to protect against influenza...AWARD NUMBER: W81XWH-16-1-0360 TITLE: Enhancement of Immune Memory Responses to Respiratory Infection PRINCIPAL INVESTIGATORs: Dr Min Chen PhD

Admission white blood cell count predicts short-term clinical outcomes in patients with uncomplicated Stanford type B acute aortic dissection.

PubMed

Chen, Zhao-Ran; Huang, Bi; Lu, Hai-Song; Zhao, Zhen-Hua; Hui, Ru-Tai; Yang, Yan-Min; Fan, Xiao-Han

2017-01-01

Inflammation has been shown to be related with acute aortic dissection (AAD). The present study aimed to evaluate the association of white blood cell counts (WBCc) on admission with both in-hospital and long-term all-cause mortality in patients with uncomplicated Stanford type B AAD. From 2008 to 2010, a total of 377 consecutive patients with uncomplicated type B AAD were enrolled and then followed up. Clinical data and WBCc on admission were collected. The primary end points were in-hospital death and long-term all-cause death. The in-hospital death rate was 4.2%, and the long-term all-cause mortality rate was 6.9% during a median follow-up of 18.9 months. WBCc on admission was identified as a risk factor for in-hospital death by univariate Cox regression analysis as both a continuous variable and a categorical variable using a cut off of 11.0 × 10 9 cell/L (all P < 0.05). After adjusting for age, sex and other risk factors, elevated admission WBCc was still a significant predictor for in-hospital death as both a continuous variable [hazard ratio (HR): 1.052, 95% CI: 1.024-1.336, P = 0.002] and a categorical variable using a cut off of 11.0 × 10 9 cell/L (HR: 2.056, 95% CI: 1.673-5.253, P = 0.034). No relationship was observed between WBCc on admission and long-term all-cause death. Our results indicate that elevated WBCc upon admission might be used as a predictor for increased risk of in-hospital death in uncomplicated type B AAD. There might be no predictive value of WBCc for the long-term survival of type B AAD.

Analysis of hematopoietic recovery after autologous transplantation as method of quality control for long-term progenitor cell cryopreservation.

PubMed

Pavlů, J; Auner, H W; Szydlo, R M; Sevillano, B; Palani, R; O'Boyle, F; Chaidos, A; Jakob, C; Kanfer, E; MacDonald, D; Milojkovic, D; Rahemtulla, A; Bradshaw, A; Olavarria, E; Apperley, J F; Pello, O M

2017-12-01

Hematopoietic precursor cells (HPC) are able to restore hematopoiesis after high-dose chemotherapy and their cryopreservation is routinely employed prior to the autologous hematopoietic cell transplantation (AHCT). Although previous studies showed feasibility of long-term HPC storage, concerns remain about possible negative effects on their potency. To study the effects of long-term cryopreservation, we compared time to neutrophil and platelet recovery in 50 patients receiving two AHCT for multiple myeloma at least 2 years apart between 2006 and 2016, using HPC obtained from one mobilization and collection attempt before the first transplant. This product was divided into equivalent fractions allowing a minimum of 2 × 10 6 CD34+ cells/kg recipient's weight. One fraction was used for the first transplant after median storage of 60 days (range, 17-165) and another fraction was used after median storage of 1448 days (range, 849-3510) at the second AHCT. Neutrophil recovery occurred at 14 days (median; range, 11-21) after the first and 13 days (10-20) after the second AHCT. Platelets recovered at a median of 16 days after both procedures. Considering other factors, such as disease status, conditioning and HPC dose, this single institution data demonstrated no reduction in the potency of HPC after long-term storage.

Long-term in vivo single-cell tracking reveals the switch of migration patterns in adult-born juxtaglomerular cells of the mouse olfactory bulb

PubMed Central

Liang, Yajie; Li, Kaizhen; Riecken, Kristoffer; Maslyukov, Anatoliy; Gomez-Nicola, Diego; Kovalchuk, Yury; Fehse, Boris; Garaschuk, Olga

2016-01-01

The behavior of adult-born cells can be easily monitored in cell culture or in lower model organisms, but longitudinal observation of individual mammalian adult-born cells in their native microenvironment still proves to be a challenge. Here we have established an approach named optical cell positioning system for long-term in vivo single-cell tracking, which integrates red-green-blue cell labeling with repeated angiography. By combining this approach with in vivo two-photon imaging technique, we characterized the in vivo migration patterns of adult-born neurons in the olfactory bulb. In contrast to the traditional view of mere radial migration of adult-born cells within the bulb, we found that juxtaglomerular cells switch from radial migration to long distance lateral migration upon arrival in their destination layer. This unique long-distance lateral migration has characteristic temporal (stop-and-go) and spatial (migratory, unidirectional or multidirectional) patterns, with a clear cell age-dependent decrease in the migration speed. The active migration of adult-born cells coincides with the time period of initial fate determination and is likely to impact on the integration sites of adult-born cells, their odor responsiveness, as well as their survival rate. PMID:27174051

Thick-tissue bioreactor as a platform for long-term organotypic culture and drug delivery.

PubMed

Markov, Dmitry A; Lu, Jenny Q; Samson, Philip C; Wikswo, John P; McCawley, Lisa J

2012-11-07

We have developed a novel, portable, gravity-fed, microfluidics-based platform suitable for optical interrogation of long-term organotypic cell culture. This system is designed to provide convenient control of cell maintenance, nutrients, and experimental reagent delivery to tissue-like cell densities housed in a transparent, low-volume microenvironment. To demonstrate the ability of our Thick-Tissue Bioreactor (TTB) to provide stable, long-term maintenance of high-density cellular arrays, we observed the morphogenic growth of human mammary epithelial cell lines, MCF-10A and their invasive variants, cultured under three-dimensional (3D) conditions inside our system. Over the course of 21 days, these cells typically develop into hollow "mammospheres" if cultured in standard 3D Matrigel. This complex morphogenic process requires alterations in a variety of cellular functions, including degradation of extracellular matrix that is regulated by cell-produced matrix proteinases. For our "drug" delivery testing and validation experiments we have introduced proteinase inhibitors into the fluid supply system, and we observed both reduced proteinase activity and inhibited cellular morphogenesis. The size inhibition results correlated well with the overall proteinase activities of the tested cells.

Simple surface engineering of polydimethylsiloxane with polydopamine for stabilized mesenchymal stem cell adhesion and multipotency

PubMed Central

Chuah, Yon Jin; Koh, Yi Ting; Lim, Kaiyang; Menon, Nishanth V.; Wu, Yingnan; Kang, Yuejun

2015-01-01

Polydimethylsiloxane (PDMS) has been extensively exploited to study stem cell physiology in the field of mechanobiology and microfluidic chips due to their transparency, low cost and ease of fabrication. However, its intrinsic high hydrophobicity renders a surface incompatible for prolonged cell adhesion and proliferation. Plasma-treated or protein-coated PDMS shows some improvement but these strategies are often short-lived with either cell aggregates formation or cell sheet dissociation. Recently, chemical functionalization of PDMS surfaces has proved to be able to stabilize long-term culture but the chemicals and procedures involved are not user- and eco-friendly. Herein, we aim to tailor greener and biocompatible PDMS surfaces by developing a one-step bio-inspired polydopamine coating strategy to stabilize long-term bone marrow stromal cell culture on PDMS substrates. Characterization of the polydopamine-coated PDMS surfaces has revealed changes in surface wettability and presence of hydroxyl and secondary amines as compared to uncoated surfaces. These changes in PDMS surface profile contribute to the stability in BMSCs adhesion, proliferation and multipotency. This simple methodology can significantly enhance the biocompatibility of PDMS-based microfluidic devices for long-term cell analysis or mechanobiological studies. PMID:26647719

Protein Phosphatase 1-Dependent Transcriptional Programs for Long-Term Memory and Plasticity

ERIC Educational Resources Information Center

Graff, Johannes; Koshibu, Kyoko; Jouvenceau, Anne; Dutar, Patrick; Mansuy, Isabelle M.

2010-01-01

Gene transcription is essential for the establishment and the maintenance of long-term memory (LTM) and for long-lasting forms of synaptic plasticity. The molecular mechanisms that control gene transcription in neuronal cells are complex and recruit multiple signaling pathways in the cytoplasm and the nucleus. Protein kinases (PKs) and…

Red blood cell storage duration and long-term mortality in patients undergoing cardiac intervention: a Danish register study.

PubMed

Dencker, D; Pedersen, F; Engstrøm, T; Schroeder, T V; Lönn, L; Johansson, P I; De Backer, O

2017-08-01

To study the effect of red blood cell (RBC) storage duration on long-term mortality in patients undergoing cardiac intervention. RBCs undergo numerous structural and functional changes during storage. Observational studies have assessed the association between RBC storage duration and patient outcomes with conflicting results. Between January 2006 and December 2014, 82 408 patients underwent coronary angiography. Of these, 1856 patients received one to four RBC units within 30 days after this procedure. Patients were allocated according to length of RBC storage duration: short-term (≤11 days), intermediate (IM)-term (12-23 days) and long-term (≥24 days). The study endpoints were 30-day and long-term all-cause mortality. A total of 4168 RBC units were given to 1856 patients. The mean RBC storage duration was 8.5 ± 2.1, 17.7 ± 3.4 and 29.9 ± 3.4 days in the short-term, IM-term and long-term storage groups, respectively. There was no difference in baseline characteristics between the groups. The long-term storage group received significantly more units (2.4 ± 1.0 units) as compared to the short-term (2.0 ± 1.0 units; P < 0.001) and IM-term storage group (2.2 ± 1.0 units; P < 0.01). In the survival analysis, there was no significant difference in all-cause mortality between the groups (log-rank: 0.509 for 30-days mortality; 0.493 for 5-year mortality). Additional stratified analysis demonstrated no association between RBC storage duration and long-term mortality. This study did not find an association between RBC storage duration and 30-days or long-term mortality in patients undergoing cardiac intervention. © 2017 British Blood Transfusion Society.

Effect of supraphysiological dose of Nandrolone Decanoate on the testis and testosterone concentration in mature and immature male rats: A time course study.

PubMed

Jannatifar, Rahil; Shokri, Saeed; Farrokhi, Ahmad; Nejatbakhsh, Reza

2015-12-01

Most studies on anabolic-androgenic steroids abuse have been done in adult rats, but few data are available to immature. This study was conducted to assay the effect of Nandrolone Decanoate (ND) on the testis and testosterone concentration in male immature rats compare with mature ones in short and long time. 40 mature rats were divided into 4 groups: group A (short term) and group B (long-term) received 10 mg/kg/day ND interaperitoneally for 35 and 70 days, respectively. Group C (control) without any treatment, and group D (vehicle) received dimethyl sulfoxide (DMSO) solution in two periods 35 and 70 days. 40 immature rats were divided into 4 groups same as mature ones. After surgery body weight, testis size, histomorphometry of testis, and serum testosterone level were evaluated. Our results showed that ND decreased the number of Leydig cells in group B (39.9 ±. 919), group A (43.4 ±. 120), and long term (40.6 ±. 299) immature rats, which could result in a reduction of testosterone concentration significantly in all experimental groups except short term mature group. Number of sertoli cells, testis size, and diameter of seminiferous tubules decreased in the long-term immature group. Eventually, the number of sperm was decreased in mature and immature groups, but a severe depletion of sperm was occurred in both mature and immature in long time in comparison to the control group (p< 0.05). This time course study showed that supraphysiological dose of ND may negatively affect the number of Leydig cells, sperm cell, and testosterone concentration of immature rats in the same matter of mature rats. However, the number of sertoli cell, testis size, and seminferous diameter were decreased only in the long immature rats.

iTRAQ-based proteomic analysis reveals the mechanisms of silicon-mediated cadmium tolerance in rice (Oryza sativa) cells.

PubMed

Ma, Jie; Sheng, Huachun; Li, Xiuli; Wang, Lijun

2016-07-01

Silicon (Si) can alleviate cadmium (Cd) stress in rice (Oryza sativa) plants, however, the understanding of the molecular mechanisms at the single-cell level remains limited. To address these questions, we investigated suspension cells of rice cultured in the dark environment in the absence and presence of Si with either short- (12 h) or long-term (5 d) Cd treatments using a combination of isobaric tags for relative and absolute quantitation (iTRAQ), fluorescent staining, and inductively coupled plasma mass spectroscopy (ICP-MS). We identified 100 proteins differentially regulated by Si under the short- or long-term Cd stress. 70% of these proteins were down-regulated, suggesting that Si may improve protein use efficiency by maintaining cells in the normal physiological status. Furthermore, we showed two different mechanisms for Si-mediated Cd tolerance. Under the short-term Cd stress, the Si-modified cell walls inhibited the uptake of Cd ions into cells and consequently reduced the expressions of glycosidase, cell surface non-specific lipid-transfer proteins (nsLTPs), and several stress-related proteins. Under the long-term Cd stress, the amount of Cd in the cytoplasm in Si-accumulating (+Si) cells was decreased by compartmentation of Cd into vacuoles, thus leading to a lower expression of glutathione S-transferases (GST). These results provide protein-level insights into the Si-mediated Cd detoxification in rice single cells. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Magnetic resonance imaging with superparamagnetic iron oxide fails to track the long-term fate of mesenchymal stem cells transplanted into heart.

PubMed

Ma, Ning; Cheng, Huaibing; Lu, Minjie; Liu, Qiong; Chen, Xiuyu; Yin, Gang; Zhu, Hao; Zhang, Lianfeng; Meng, Xianmin; Tang, Yue; Zhao, Shihua

2015-03-12

MRI for in vivo stem cell tracking remains controversial. Here we tested the hypothesis that MRI can track the long-term fate of the superparamagnetic iron oxide (SPIO) nanoparticles labelled mesenchymal stem cells (MSCs) following intramyocardially injection in AMI rats. MSCs (1 × 10(6)) from male rats doubly labeled with SPIO and DAPI were injected 2 weeks after myocardial infarction. The control group received cell-free media injection. In vivo serial MRI was performed at 24 hours before cell delivery (baseline), 3 days, 1, 2, and 4 weeks after cell delivery, respectively. Serial follow-up MRI demonstrated large persistent intramyocardial signal-voids representing SPIO during the follow-up of 4 weeks, and MSCs did not moderate the left ventricular dysfunction. The TUNEL analysis confirmed that MSCs engrafted underwent apoptosis. The histopathological studies revealed that the site of cell injection was infiltrated by inflammatory cells progressively and the iron-positive cells were macrophages identified by CD68 staining, but very few or no DAPI-positive stem cells at 4 weeks after cells transplantation. The presence of engrafted cells was confirmed by real-time PCR, which showed that the amount of Y-chromosome-specific SRY gene was consistent with the results. MRI may not reliably track the long-term fate of SPIO-labeled MSCs engraftment in heart.

Microcapsules engineered to support mesenchymal stem cell (MSC) survival and proliferation enable long-term retention of MSCs in infarcted myocardium.

PubMed

Blocki, Anna; Beyer, Sebastian; Dewavrin, Jean-Yves; Goralczyk, Anna; Wang, Yingting; Peh, Priscilla; Ng, Michael; Moonshi, Shehzahdi S; Vuddagiri, Susmitha; Raghunath, Michael; Martinez, Eliana C; Bhakoo, Kishore K

2015-06-01

The limited efficacy of cardiac cell-based therapy is thought to be due to poor cell retention within the myocardium. Hence, there is an urgent need for biomaterials that aid in long-term cell retention. This study describes the development of injectable microcapsules for the delivery of mesenchymal stem cells (MSCs) into the infarcted cardiac wall. These microcapsules comprise of low concentrations of agarose supplemented with extracellular matrix (ECM) proteins collagen and fibrin. Dextran sulfate, a negatively charged polycarbohydrate, was added to mimic glycosaminoglycans in the ECM. Cell viability assays showed that a combination of all components is necessary to support long-term survival and proliferation of MSCs within microcapsules. Following intramyocardial transplantation, microcapsules degraded slowly in vivo and did not induce a fibrotic foreign body response. Pre-labeling of encapsulated MSCs with iron oxide nanoparticles allowed continued cell-tracking by MRI over several weeks following transplantation into infarcted myocardium. In contrast, MSCs injected as cell suspension were only detectable for two days post transplantation by MRI. Histological analysis confirmed integration of transplanted cells at the infarct site. Therefore, microcapsules proved to be suitable for stem cell delivery into the infarcted myocardium and can overcome current limitations of poor cell retention in cardiac cell-based therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparative cytotoxicity of Al2O3, CeO2, TiO2 and ZnO nanoparticles to human lung cells.

PubMed

Kim, In-Sun; Baek, Miri; Choi, Soo-Jin

2010-05-01

The increased applications of nanoparticles in a wide range of industrial fields raise the concern about their potential toxicity to human. The aim of this study was to assess and compare the toxicity of four different oxide nanoparticles (Al2O3, CeO2, TiO2 and ZnO) to human lung epithelial cells, A549 carcinoma cells and L-132 normal cells, in vitro. We focused on the toxicological effects of the present nanoparticles on cell proliferation, cell viability, membrane integrity and oxidative stress. The long-term cytotoxicity of nanoparticles was also evaluated by employing the clonogenic assay. Among four nanoparticles tested, ZnO exhibited the highest cytotoxicity in terms of cell proliferation, cell viability, membrane integrity and colony formation in both cell lines. Al2O3, CeO2 and TiO2 showed little adverse effects on cell proliferation and cell viability. However, TiO2 induced oxidative stress in a concentration- and time-dependent manner. CeO2 caused membrane damage and inhibited colony formation in long-term, but with different degree depending on cell lines. Al2O3 seems to be less toxic than the other nanoparticles even after long time exposure. These results highlight the need for caution during manufacturing process of nanomaterials as well as further investigation on the toxicity mechanism.

980nm laser for difficult-to-treat basal cell carcinoma

NASA Astrophysics Data System (ADS)

Derjabo, A. D.; Cema, I.; Lihacova, I.; Derjabo, L.

2013-06-01

Begin basal cell carcinoma (BCC) is most common skin cancer over the world. There are around 20 modalities for BCC treatment. Laser surgery is uncommon option. We demonstrate our long term follow up results. Aim: To evaluate long term efficacy of a 980nm diode laser for the difficult-to-treat basal cell carcinoma. Materials and Methods: 167 patients with 173 basal cell carcinoma on the nose were treated with a 980 nm diode laser from May 1999 till May 2005 at Latvian Oncology center. All tumors were morphologically confirmed. 156 patients were followed for more than 5 years. Results: The lowest recurrence rate was observed in cases of superficial BCC, diameter<6mm bet the highest recurrence rate was in cases of infiltrative BCC and nodular recurrent BCC. Conclusions: 980 nm diode laser is useful tool in dermatology with high long term efficacy, good acceptance by the patients and good cosmetics results.

Mesenchymal stem cells from the Wharton's jelly of umbilical cord segments provide stromal support for the maintenance of cord blood hematopoietic stem cells during long-term ex vivo culture.

PubMed

Bakhshi, Tiki; Zabriskie, Ryan C; Bodie, Shamanique; Kidd, Shannon; Ramin, Susan; Paganessi, Laura A; Gregory, Stephanie A; Fung, Henry C; Christopherson, Kent W

2008-12-01

Hematopoietic stem cells (HSCs) are routinely obtained from marrow, mobilized peripheral blood, and umbilical cord blood. Mesenchymal stem cells (MSCs) are traditionally isolated from marrow. Bone marrow-derived MSCs (BM-MSCs) have previously demonstrated their ability to act as a feeder layer in support of ex vivo cord blood expansion. However, the use of BM-MSCs to support the growth, differentiation, and engraftment of cord blood may not be ideal for transplant purposes. Therefore, the potential of MSCs from a novel source, the Wharton's jelly of umbilical cords, to act as stromal support for the long-term culture of cord blood HSC was evaluated. Umbilical cord-derived MSCs (UC-MSCs) were cultured from the Wharton's jelly of umbilical cord segments. The UC-MSCs were then profiled for expression of 12 cell surface receptors and tested for their ability to support cord blood HSCs in a long-term culture-initiating cell (LTC-IC) assay. Upon culture, UC-MSCs express a defined set of cell surface markers (CD29, CD44, CD73, CD90, CD105, CD166, and HLA-A) and lack other markers (CD45, CD34, CD38, CD117, and HLA-DR) similar to BM-MSCs. Like BM-MSCs, UC-MSCs effectively support the growth of CD34+ cord blood cells in LTC-IC assays. These data suggest the potential therapeutic application of Wharton's jelly-derived UC-MSCs to provide stromal support structure for the long-term culture of cord blood HSCs as well as the possibility of cotransplantation of genetically identical, HLA-matched, or unmatched cord blood HSCs and UC-MSCs in the setting of HSC transplantation.

Cytotoxicity evaluation of Curcuma zedoaria (Christm.) Roscoe fluid extract used in oral hygiene products.

PubMed

Fernandes, Joao Paulo Dos Santos; Mello-Moura, Anna Carolina Volpi; Marques, Marcia Martins; Nicoletti, Maria Aparecida

2012-12-01

This in vitro study evaluated the cytotoxic effects of the Curcuma zedoaria (Christm.) Roscoe (popular name: zedoary) fluid extract, as used in preparations for oral hygiene, mostly for anti-septic purposes. The cell viability and cell growth were assessed by Trypan blue dye exclusion assay using the LMF cell line derived from oral mucosa. Cell viability (short-term assay) was measured 0, 6, 12 and 24 h after contact with the fluid extract. Cell growth (long-term assay) was analyzed in 1, 3, 5 and 7 days. The experimental groups were those testing the fluid extract obtained from the zedoary rhizome and the extractor liquid (ethanol 70° GL) in the concentrations of 0.01-0.0001% v/v. Fresh DMEM were used in the control cultures. Short-term assay-all studied cultures maintained stable cell viability; Long-term assay-there was progressive cell growth in all studied cultures. According to the results, the zedoary fluid extract presents low cytotoxicity and probably can be used in the oral hygiene products.

Rahnella sp. strain EK12: Cell surface properties and diesel oil biodegradation after long-term contact with natural surfactants and diesel oil.

PubMed

Smułek, Wojciech; Zdarta, Agata; Guzik, Urszula; Dudzińska-Bajorek, Beata; Kaczorek, Ewa

2015-07-01

The changes in cell surface properties of Rahnella sp. strain EK12 and modifications in genetic material after long-term contact with saponins and rhamnolipids, were investigated. Rhamnolipids caused a decrease of hydrophobicity in liquid cultures compared with saponins. On the other hand, in cultures with rhamnolipids, the addition of diesel oil results in a rapid rise of cell surface hydrophobicity. The similar effect was not so significant in the presence of saponins. For the bacteria grown in the presence of saponins or rhamnolipids, but without diesel oil, the ratio of unsaturated to saturated fatty acids decreased, in comparison to the control culture. The differences observed in hydrophobicity, zeta potential and fatty acids profiles, indicated various mechanisms of an interaction between a surfactant and a bacterial cells. The results have also shown an impact of the long-term contact on changes in genetic material of Rahnella sp. strain EK12 cells. Moreover, the presence of saponins led to significant increase of diesel oil biodegradation. Copyright © 2015 Elsevier GmbH. All rights reserved.

As-prepared MoS2 quantum dot as a facile fluorescent probe for long-term tracing of live cells

NASA Astrophysics Data System (ADS)

Zhou, Kai; Zhang, Yue; Xia, Zhining; Wei, Weili

2016-07-01

Recently, the newly emerged two-dimensional nanomaterials, layered transition metal dichalcogenide (e.g. MoS2) nanosheets, have drawn tremendous attentions due to their extraordinary electronic and optical properties, and MoS2 quantum dots (MoS2 QDs) with lateral sizes less than 10 nm have been found to be highly luminescent. In the present study, a facile approach for large-scale preparation of MoS2 QDs by Na intercalation reaction without using any toxic organic reagents is proposed. MoS2 QDs were carefully characterized by various techniques including transmission electron microscopy, atomic force microscopy, dynamic light scattering, spectroscopy, in vitro cytotoxicology, and capillary electrophoresis. The as-prepared MoS2 QDs were strongly fluorescent, highly photo-stable, low in cytotoxicity, and readily reactive to thiols. These inherent properties of MoS2 QDs make them excellent fluorescent probes for long-term live cell tracing. The results of live cells imaging indicated that MoS2 QD stained cells remained highly fluorescent after long-term culture, and could be easily traced from other co-cultured cell lines.

Lower gingival squamous cell carcinoma with brain metastasis during long-term cetuximab treatment: A case report.

PubMed

Naruse, Tomofumi; Tokuhisa, Mitsuko; Yanamoto, Souichi; Sakamoto, Yuki; Okuyama, Kohei; Tsuchihashi, Hiroki; Umeda, Masahiro

2018-05-01

Long-term cetuximab treatment can lead to acquired resistance, and tumor progression and/or new lesions often occur. The present report describes a case of lower gingival squamous cell carcinoma with brain metastasis during long-term cetuximab treatment in a 60-year-old man, including findings of an immunohistochemical study. The resected primary tumors, biopsy of the lung metastasis before administration of cetuximab, and brain metastasis specimens mediated by cetuximab were immunohistochemically examined. Histologically, the metastatic brain lesion showed hyperkeratinizing tumor cells with deeply stained irregular nuclei with necrotizing tumor cells, and a decrease in cell density was exhibited in part of the tumor nest. Moreover, the brain lesion was less malignant compared with the primary tumor and metastatic lung lesions. Immunohistochemically, the metastatic brain lesions showed low expression of epidermal growth factor receptor (EGFR) and high expression of N-cadherin compared with the primary tumor and metastatic lung lesions. These results suggest that acquired resistance to cetuximab may be associated with low EGFR expression and increased epithelial-to-mesenchymal transition potential.

Long-term protection against SHIV89.6P replication in HIV-1 Tat vaccinated cynomolgus monkeys.

PubMed

Maggiorella, Maria Teresa; Baroncelli, Silvia; Michelini, Zuleika; Fanales-Belasio, Emanuele; Moretti, Sonia; Sernicola, Leonardo; Cara, Andrea; Negri, Donatella R M; Buttò, Stefano; Fiorelli, Valeria; Tripiciano, Antonella; Scoglio, Arianna; Caputo, Antonella; Borsetti, Alessandra; Ridolfi, Barbara; Bona, Roberta; ten Haaft, Peter; Macchia, Iole; Leone, Pasqualina; Pavone-Cossut, Maria Rosaria; Nappi, Filomena; Ciccozzi, Massimo; Heeney, Jonathan; Titti, Fausto; Cafaro, Aurelio; Ensoli, Barbara

2004-09-03

Vaccination with a biologically active Tat protein or tat DNA contained infection with the highly pathogenic SHIV89.6P virus, preventing CD4 T-cell decline and disease onset. Here we show that protection was prolonged, since neither CD4 T-cell decline nor active virus replication was observed in all vaccinated animals that controlled virus replication up to week 104 after the challenge. In contrast, virus persisted and replicated in peripheral blood mononuclear cells and lymph nodes of infected animals, two of which died. Tat-specific antibody, CD4 and CD8 T-cell responses were high and stable only in the animals controlling the infection. In contrast, Gag-specific antibody production and CD4 and CD8 T-cell responses were consistently and persistently positive only in the monkeys that did not control primary virus replication. These results indicate that vaccination with Tat protein or DNA induced long-term memory Tat-specific immune responses and controlled primary infection at its early stages allowing a long-term containment of virus replication and spread in blood and tissues.

Soybean agglutinin binding to corneal endothelial cell surfaces disrupts in situ monolayer integrity and actin organization and interferes with wound repair.

PubMed

Gordon, Sheldon R; Wood, Meredith

2009-03-01

Rat corneal endothelium demonstrates cell-surface soybean agglutinin (SBA) binding during organ-culture or injury. When organ-cultured in medium containing SBA, the endothelial monolayer is disrupted because of cell-cell and cell-matrix alterations. SBA binding disorganizes the circumferential microfilament bundles (CMBs), an effect that is partially prevented by phallacidin preincubation. This disruption is reversible if tissues are returned to standard culture medium. Serum heightens SBA binding, whereas puromycin prevents it. Neither actinomycin D nor alpha-amanitin inhibits SBA binding, suggesting that SBA-binding protein(s) may be post-transcriptionally regulated. During injury-induced cell migration in the presence of SBA, cellular processes are blunted and fail to extend significantly outward. By 72 h post-injury, cells of SBA-treated tissues repopulate the wound but demonstrate little association with neighboring cells. Cells migrating in the presence of N-acetylgalactosamine appear normal but also fail to reassociate with other cells in the jury zone. Immunofluorescent staining for ZO-1 reveals punctuate patterns in cells of control tissues, whereas neither SBA- nor N-acetylgalactosamine-treated tissues exhibit ZO-1 staining. Terminal N-acetylgalactosamine removal fails to affect cell morphology, actin organization, or migration but prevents lectin binding. Our results suggest that SBA binding reflects the synthesis of a stress-induced protein(s) that may play a role in reestablishing cell-cell relationships during monolayer reorganization following injury.

Increased efficiency of gamma-irradiated versus mitomycin C-treated feeder cells for the expansion of normal human cells in long-term cultures.

PubMed

Roy, A; Krzykwa, E; Lemieux, R; Néron, S

2001-12-01

Several normal human cells, such as hematopoietic stem cells, dendritic cells, and B cells, can be cultured in vitro in defined optimal conditions. Several ex vivo culture systems require the use of feeder cells to support the growth of target cells. In such systems, proliferation of feeder cells has to be stopped, so that they can be used as nonreplicating viable support cells. Because feeder cells need to provide one or few active signals, it is important to maintain them in an metabolically active state, allowing continued expression of specific ligands or cytokines. Mitomycin C and gamma-irradiation treatments are commonly used to prepare nonproliferating feeder cells and are usually considered to be equivalent. Normal human B lymphocytes can be expanded in vitro in the presence of feeder cells expressing the CD40 ligand CD154. Here we compared the ability of gamma-irradiation- and mitomycin C-treated feeder cells to support the expansion of normal human B lymphocytes. The results indicate that expansion of B cells during a long-term culture was 100 times more potent using gamma-irradiated feeder cells compared to mitomycin C-treated cells. This difference could be related to a significant reduction in both cellular metabolism and level of CD154 expression observed in mitomycin C-treated feeder cells, but not in gamma-irradiated cells nor in control untreated cells. These results indicate that mitomycin C-treated feeder cells are metabolically altered, and consequently less efficient at maintaining cell expansion in the long-term cell culture system used.

Evidence for mesenchymal-like sub-populations within squamous cell carcinomas possessing chemoresistance and phenotypic plasticity.

PubMed

Basu, D; Nguyen, T-T K; Montone, K T; Zhang, G; Wang, L-P; Diehl, J A; Rustgi, A K; Lee, J T; Weinstein, G S; Herlyn, M

2010-07-22

Variable drug responses among malignant cells within individual tumors may represent a barrier to their eradication using chemotherapy. Carcinoma cells expressing mesenchymal markers resist conventional and epidermal growth factor receptor (EGFR)-targeted chemotherapy. In this study, we evaluated whether mesenchymal-like sub-populations within human squamous cell carcinomas (SCCs) with predominantly epithelial features contribute to overall therapy resistance. We identified a mesenchymal-like subset expressing low E-cadherin (Ecad-lo) and high vimentin within the upper aerodigestive tract SCCs. This subset was both isolated from the cell lines and was identified in xenografts and primary clinical specimens. The Ecad-lo subset contained more low-turnover cells, correlating with resistance to the conventional chemotherapeutic paclitaxel in vitro. Epidermal growth factor induced less stimulation of the mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathways in Ecad-lo cells, which was likely due to lower EGFR expression in this subset and correlated with in vivo resistance to the EGFR-targeted antibody, cetuximab. The Ecad-lo and high E-cadherin subsets were dynamic in phenotype, showing the capacity to repopulate each other from single-cell clones. Taken together, these results provide evidence for a low-turnover, mesenchymal-like sub-population in SCCs with diminished EGFR pathway function and intrinsic resistance to conventional and EGFR-targeted chemotherapies.

Overexpression of IL-7R alpha provides a competitive advantage during early T-cell development.

PubMed

Laouar, Yasmina; Crispe, I Nicholas; Flavell, Richard A

2004-03-15

Critical checkpoints controlling early thymic T-cell development and homeostasis are set by the proper signaling function of the interleukin 7 receptor (IL-7R) and the pre-T-cell antigen receptor. Although alpha beta T-cell development is observed in IL-7- and IL-7R alpha-deficient mice, the number of thymocytes is significantly reduced, implying a role for the IL-7R in controlling the size of the thymic T-cell compartment. Here, we report the overexpression of IL-7R alpha that occurs in the early T-cell compartment from AKR/J mice, animals that are highly susceptible to the spontaneous development of thymoma. Increased IL-7R alpha was revealed by surface staining, and increased IL-7R alpha mRNA was documented by using reverse transcriptase-polymerase chain reaction (RT-PCR). This resulted in increased survival of AKR/J early thymocytes, shown by the decreased frequency of TUNEL(+) (terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate [dUTP]-fluorescein nick end labeling) cells. In an in vivo thymocyte repopulation model, AKR/J thymocytes had a selective advantage over healthy thymocytes. This advantage occurred at early stages of T-cell development. Our findings support the model that overexpression of growth factor receptors can contribute to proliferation and malignancy.

Bone marrow mesenchymal stem cells from patients with aplastic anemia maintain functional and immune properties and do not contribute to the pathogenesis of the disease.

PubMed

Bueno, Clara; Roldan, Mar; Anguita, Eduardo; Romero-Moya, Damia; Martín-Antonio, Beatriz; Rosu-Myles, Michael; del Cañizo, Consuelo; Campos, Francisco; García, Regina; Gómez-Casares, Maite; Fuster, Jose Luis; Jurado, Manuel; Delgado, Mario; Menendez, Pablo

2014-07-01

Aplastic anemia is a life-threatening bone marrow failure disorder characterized by peripheral pancytopenia and marrow hypoplasia. The majority of cases of aplastic anemia remain idiopathic, although hematopoietic stem cell deficiency and impaired immune responses are hallmarks underlying the bone marrow failure in this condition. Mesenchymal stem/stromal cells constitute an essential component of the bone marrow hematopoietic microenvironment because of their immunomodulatory properties and their ability to support hematopoiesis, and they have been involved in the pathogenesis of several hematologic malignancies. We investigated whether bone marrow mesenchymal stem cells contribute, directly or indirectly, to the pathogenesis of aplastic anemia. We found that mesenchymal stem cell cultures can be established from the bone marrow of aplastic anemia patients and display the same phenotype and differentiation potential as their counterparts from normal bone marrow. Mesenchymal stem cells from aplastic anemia patients support the in vitro homeostasis and the in vivo repopulating function of CD34(+) cells, and maintain their immunosuppressive and anti-inflammatory properties. These data demonstrate that bone marrow mesenchymal stem cells from patients with aplastic anemia do not have impaired functional and immunological properties, suggesting that they do not contribute to the pathogenesis of the disease. Copyright© Ferrata Storti Foundation.

Enhanced in vivo selection of bone marrow cells by retroviral-mediated coexpression of mutant O6-methylguanine-DNA-methyltransferase and HOXB4.

PubMed

Milsom, Michael D; Woolford, Lorna B; Margison, Geoffrey P; Humphries, R Keith; Fairbairn, Leslie J

2004-11-01

To attain therapeutic levels of gene-modified hematopoietic stem cells, it may be necessary in the majority of disorders to provide an in vivo selective advantage that facilitates the expansion of their numbers. A popular strategy to achieve in vivo selection has been to employ drug selection while coexpressing a transgene that conveys chemoresistance, such as O6-methylguanine-DNA-methyltransferase (MGMT). An alternate approach is to confer an enhanced proliferative potential upon gene-modified hematopoietic stem cells through the delivery of the homeobox transcription factor HOXB4. By developing a novel tricistronic retroviral vector, we have facilitated the simultaneous coexpression of a mutant version of MGMT and HOXB4 in retrovirally transduced bone marrow. Using an in vivo competitive repopulation assay, we demonstrate that primary bone marrow cells containing this construct show enhanced reconstitution following transplant and improved selection subsequent to chemotherapeutic challenge in comparison to cells expressing either HOXB4 or MGMT alone. This selection advantage was evident even when HOXB4/MGMT-coexpressing cells were infused along with a large excess of unmodified cells. We propose that this selection cassette may facilitate the in vivo expansion of gene-modified hematopoietic stem cells at a level in excess of previous strategies.

In vivo competitive studies between normal and common gamma chain-defective bone marrow cells: implications for gene therapy.

PubMed

Otsu, M; Sugamura, K; Candotti, F

2000-09-20

Corrective gene transfer into hematopoietic stem cells (HSCs) is being investigated as therapy for X-linked severe combined immunodeficiency (XSCID) and it is hoped that selective advantage of gene-corrected HSCs will help in achieving full immune reconstitution after treatment. Lines of evidence from the results of allogeneic bone marrow transplantation in patients with XSCID support this hypothesis that, however, has not been rigorously tested in an experimental system. We studied the competition kinetics between normal and XSCID bone marrow (BM) cells using a murine bone marrow transplantation (BMT) model. For easy chimerism determination, we used genetic marking with retrovirus-mediated expression of the enhanced green fluorescent protein (EGFP). We found that XSCID BM cells were able to compete with normal BM cells for engraftment of myeloid lineages in a dose-dependent manner, whereas we observed selective repopulation of T, B, and NK cells deriving from normal BM cells. This was true despite the evidence of competitive engraftment of XSCID lineage marker-negative/c-Kit-positive (Lin-/c-Kit+) cells in the bone marrow of treated animals. From these results we extrapolate that genetic correction of XSCID HSCs will result in selective advantage of gene-corrected lymphoid lineages with consequent restoration of lymphocyte populations and high probability of clinical benefit.

Decade-Long Safety and Function of Retroviral-Modified Chimeric Antigen Receptor T-cells

PubMed Central

Scholler, John; Brady, Troy L.; Binder-Scholl, Gwendolyn; Hwang, Wei-Ting; Plesa, Gabriela; Hege, Kristen M.; Vogel, Ashley N.; Kalos, Michael; Riley, James L.; Deeks, Steven G.; Mitsuyasu, Ronald T.; Bernstein, Wendy B.; Aronson, Naomi E.; Levine, Bruce L.; Bushman, Frederic D.; June, Carl H.

2015-01-01

The success of adoptive T cell gene transfer for treatment of cancer and HIV is predicated on generating a response that is both durable and safe. Here we report long term results from three clinical trials to evaluate gammaretroviral vector engineered T-cells for HIV. The vector encoded a chimeric antigen receptor (CAR) comprised of CD4 linked to the CD3-ζ signaling chain (CD4ζ). CAR T-cells were detected in 98% of samples tested for at least 11 years post-infusion at frequencies that exceed average T cell levels after most vaccine approaches. The CD4ζ transgene retained expression and function. There was no evidence of vector-induced immortalization of cells as integration site distributions showed no evidence of persistent clonal expansion or enrichment for integration sites near genes implicated in growth control or transformation. The CD4ζ T cells have stable levels of engraftment, with decay half-lives that exceed 16 years, in marked contrast to previous trials testing engineered T cells. These findings indicate that host immunosuppression prior to T cell transfer is not required in order to achieve long term persistence of gene-modified T cells. Further, our results emphasize the safety of T cells modified by retroviral gene transfer in clinical application, as measured in >500 patient years of follow up. Thus, previous safety issues with integrating viral vectors are hematopoietic stem cell or transgene intrinsic, and not a general feature of retroviral vectors. Engineered T cells are a promising form of synthetic biology for long term delivery of protein based therapeutics. These results provide a framework to guide the therapy of a wide spectrum of human diseases. PMID:22553251

Decreased "ineffective erythropoiesis" preserves polycythemia in mice under long-term hypoxia.

PubMed

Harada, Tomonori; Tsuboi, Isao; Hirabayashi, Yukio; Kosaku, Kazuhiro; Naito, Michiko; Hara, Hiroyuki; Inoue, Tohru; Aizawa, Shin

2015-05-01

Hypoxia induces innumerable changes in humans and other animals, including an increase in peripheral red blood cells (polycythemia) caused by the activation of erythropoiesis mediated by increased erythropoietin (EPO) production. However, the elevation of EPO is limited and levels return to normal ranges under normoxia within 5-7 days of exposure to hypoxia, whereas polycythemia continues for as long as hypoxia persists. We investigated erythropoiesis in bone marrow and spleens from mouse models of long-term normobaric hypoxia (10 % O2) to clarify the mechanism of prolonged polycythemia in chronic hypoxia. The numbers of erythroid colony-forming units (CFU-E) in the spleen remarkably increased along with elevated serum EPO levels indicating the activation of erythropoiesis during the first 7 days of hypoxia. After 14 days of hypoxia, the numbers of CFU-E returned to normoxic levels, whereas polycythemia persisted for >140 days. Flow cytometry revealed a prolonged increase in the numbers of TER119-positive cells (erythroid cells derived from pro-erythroblasts through mature erythrocyte stages), especially the TER119 (high) CD71 (high) population, in bone marrow. The numbers of annexin-V-positive cells among the TER119-positive cells particularly declined under chronic hypoxia, suggesting that the numbers of apoptotic cells decrease during erythroid cell maturation. Furthermore, RT-PCR analysis showed that the RNA expression of BMP-4 and stem cell factor that reduces apoptotic changes during erythroid cell proliferation and maturation was increased in bone marrow under hypoxia. These findings indicated that decreased apoptosis of erythroid cells during erythropoiesis contributes to polycythemia in mice during chronic exposure to long-term hypoxia.

Magnetic Beads Enhance Adhesion of NIH 3T3 Fibroblasts: A Proof-of-Principle In Vitro Study for Implant-Mediated Long-Term Drug Delivery to the Inner Ear

PubMed Central

Aliuos, Pooyan; Schulze, Jennifer; Schomaker, Markus; Reuter, Günter; Stolle, Stefan R. O.; Werner, Darja; Ripken, Tammo; Lenarz, Thomas; Warnecke, Athanasia

2016-01-01

Introduction Long-term drug delivery to the inner ear may be achieved by functionalizing cochlear implant (CI) electrodes with cells providing neuroprotective factors. However, effective strategies in order to coat implant surfaces with cells need to be developed. Our vision is to make benefit of electromagnetic field attracting forces generated by CI electrodes to bind BDNF-secreting cells that are labelled with magnetic beads (MB) onto the electrode surfaces. Thus, the effect of MB-labelling on cell viability and BDNF production were investigated. Materials and Methods Murine NIH 3T3 fibroblasts—genetically modified to produce BDNF—were labelled with MB. Results Atomic force and bright field microscopy illustrated the internalization of MB by fibroblasts after 24 h of cultivation. Labelling cells with MB did not expose cytotoxic effects on fibroblasts and allowed adhesion on magnetic surfaces with sufficient BDNF release. Discussion Our data demonstrate a novel approach for mediating enhanced long-term adhesion of BDNF-secreting fibroblasts on model electrode surfaces for cell-based drug delivery applications in vitro and in vivo. This therapeutic strategy, once transferred to cells suitable for clinical application, may allow the biological modifications of CI surfaces with cells releasing neurotrophic or other factors of interest. PMID:26918945

Induction of transplantation tolerance to fully mismatched cardiac allografts by T cell mediated delivery of alloantigen

PubMed Central

Tian, Chaorui; Yuan, Xueli; Jindra, Peter T.; Bagley, Jessamyn; Sayegh, Mohamed H.; Iacomini, John

2010-01-01

Induction of transplantation tolerance has the potential to allow for allograft acceptance without the need for life-long immunosuppression. Here we describe a novel approach that uses delivery of alloantigen by mature T cells to induce tolerance to fully allogeneic cardiac grafts. Adoptive transfer of mature alloantigen-expressing T cells into myeloablatively conditioned mice results in long-term acceptance of fully allogeneic heart transplants without evidence of chronic rejection. Since myeloablative conditioning is clinically undesirable we further demonstrated that adoptive transfer of mature alloantigen-expressing T cells alone into mice receiving non-myeloablative conditioning resulted in long-term acceptance of fully allogeneic heart allografts with minimal evidence of chronic rejection. Mechanistically, tolerance induction involved both deletion of donor-reactive host T cells and the development of regulatory T cells. Thus, delivery of alloantigen by mature T cells induces tolerance to fully allogeneic organ allografts in non-myeloablatively conditioned recipients, representing a novel approach for tolerance induction in transplantation. PMID:20452826

Genetically encoded calcium indicators for studying long-term calcium dynamics during apoptosis.

PubMed

Garcia, M Iveth; Chen, Jessica J; Boehning, Darren

2017-01-01

Intracellular calcium release is essential for regulating almost all cellular functions. Specific spatio-temporal patterns of cytosolic calcium elevations are critical determinants of cell fate in response to pro-apoptotic cellular stressors. As the apoptotic program can take hours or days, measurement of long-term calcium dynamics are essential for understanding the mechanistic role of calcium in apoptotic cell death. Due to the technical limitations of using calcium-sensitive dyes to measure cytosolic calcium little is known about long-term calcium dynamics in living cells after treatment with apoptosis-inducing drugs. Genetically encoded calcium indicators could potentially overcome some of the limitations of calcium-sensitive dyes. Here, we compared the performance of the genetically encoded calcium indicators GCaMP6s and GCaMP6f with the ratiometric dye Fura-2. GCaMP6s performed as well or better than Fura-2 in detecting agonist-induced calcium transients. We then examined the utility of GCaMP6s for continuously measuring apoptotic calcium release over the course of ten hours after treatment with staurosporine. We found that GCaMP6s was suitable for measuring apoptotic calcium release over long time courses and revealed significant heterogeneity in calcium release dynamics in individual cells challenged with staurosporine. Our results suggest GCaMP6s is an excellent indicator for monitoring long-term changes cytosolic calcium during apoptosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Central memory CD4 T cells are associated with incomplete restoration of the CD4 T cell pool after treatment-induced long-term undetectable HIV viraemia.

PubMed

Rallón, Norma; Sempere-Ortells, José M; Soriano, Vincent; Benito, José M

2013-11-01

It is unclear to what extent T cell reconstitution may be possible in HIV-1-infected individuals on continuous successful highly active antiretroviral therapy (HAART). Herein, we analysed distinct phenotypic markers of immune recovery in patients with undetectable viraemia for 8 years, taking as reference untreated patients and healthy controls. Seventy-two subjects were examined: 28 HIV-1+ patients on successful long-term HAART, 24 HIV-1+ untreated viraemic patients and 20 age-matched healthy controls. Analysis of naive and memory CD4 and CD8 T cells was combined with measurements of activation status (expression of CD38) and with thymic function (expression of CD31). Statistical significance was determined by non-parametric tests. After long-term HAART, the majority of parameters were normalized compared with age-matched control values, including T cell activation and thymic function. However, absolute counts of naive and central memory CD4 T cells remained below normal levels. The only parameters significantly associated with CD4 counts at the end of follow-up were the pre-HAART CD4 count ( β ± SD = 0.54 ± 0.16, P = 0.003) and the level of CD4 central memory cells at the end of follow-up (β ± SD = 1.18 ± 0.23, P < 0.0001). Only patients starting HAART with CD4 counts >350 cells/mm(3) reached a complete normalization of CD4 counts. Even after long-term successful HAART, complete CD4 restoration may be attainable only in patients starting therapy with moderately high CD4 counts, prompting early initiation of antiretroviral therapy. Incomplete CD4 restoration may be associated with a defective restoration of central memory CD4 T cells, a cell subset with a pivotal role in T cell homeostasis.

In vitro and in vivo cell invasion and systemic spreading of Mycoplasma agalactiae in the sheep infection model.

PubMed

Hegde, Shivanand; Hegde, Shrilakshmi; Spergser, Joachim; Brunthaler, René; Rosengarten, Renate; Chopra-Dewasthaly, Rohini

2014-11-01

Generally regarded as extracellular pathogens, molecular mechanisms of mycoplasma persistence, chronicity and disease spread are largely unknown. Mycoplasma agalactiae, an economically important pathogen of small ruminants, causes chronic infections that are difficult to eradicate. Animals continue to shed the agent for several months and even years after the initial infection, in spite of long antibiotic treatment. However, little is known about the strategies that M. agalactiae employs to survive and spread within an immunocompetent host to cause chronic disease. Here, we demonstrate for the first time its ability to invade cultured human (HeLa) and ruminant (BEND and BLF) host cells. Presence of intracellular mycoplasmas is clearly substantiated using differential immunofluorescence technique and quantitative gentamicin invasion assays. Internalized M. agalactiae could survive and exit the cells in a viable state to repopulate the extracellular environment after complete removal of extracellular bacteria with gentamicin. Furthermore, an experimental sheep intramammary infection was carried out to evaluate its systemic spread to organs and host niches distant from the site of initial infection. Positive results obtained via PCR, culture and immunohistochemistry, especially the latter depicting the presence of M. agalactiae in the cytoplasm of mammary duct epithelium and macrophages, clearly provide the first formal proof of M. agalactiae's capability to translocate across the mammary epithelium and systemically disseminate to distant inner organs. Altogether, the findings of these in vitro and in vivo studies indicate that M. agalactiae is capable of entering host cells and this might be the strategy that it employs at a population level to ward off the host immune response and antibiotic action, and to disseminate to new and safer niches to later egress and once again proliferate upon the return of favorable conditions to cause persistent chronic infections. Copyright © 2014 The Authors. Published by Elsevier GmbH.. All rights reserved.

“Humanized mice for HIV and AIDS research”

PubMed Central

Garcia, J. Victor

2016-01-01

HIV has a very limited species tropism that prevents the use of most conventional small animal models for AIDS research. The in vivo analysis of HIV/AIDS has benefited extensively from novel chimeric animal models that accurately recapitulate key aspects of the human condition. Specifically, immunodeficient mice that are systemically repopulated with human hematolymphoid cells offer a viable alternative for the study of a multitude of highly relevant aspects of HIV replication, pathogenesis, therapy, transmission, prevention, and eradication. This article summarizes some of the multiple contributions that humanized mouse models of HIV infection have made to the field of AIDS research. These models have proven to be highly informative and hold great potential for accelerating multiple aspects of HIV research in the future. PMID:27447446