Adult subependymal neural precursors, but not differentiated cells, undergo rapid cathodal migration in the presence of direct current electric fields.

PubMed

Babona-Pilipos, Robart; Droujinine, Ilia A; Popovic, Milos R; Morshead, Cindi M

2011-01-01

The existence of neural stem and progenitor cells (together termed neural precursor cells) in the adult mammalian brain has sparked great interest in utilizing these cells for regenerative medicine strategies. Endogenous neural precursors within the adult forebrain subependyma can be activated following injury, resulting in their proliferation and migration toward lesion sites where they differentiate into neural cells. The administration of growth factors and immunomodulatory agents following injury augments this activation and has been shown to result in behavioural functional recovery following stroke. With the goal of enhancing neural precursor migration to facilitate the repair process we report that externally applied direct current electric fields induce rapid and directed cathodal migration of pure populations of undifferentiated adult subependyma-derived neural precursors. Using time-lapse imaging microscopy in vitro we performed an extensive single-cell kinematic analysis demonstrating that this galvanotactic phenomenon is a feature of undifferentiated precursors, and not differentiated phenotypes. Moreover, we have shown that the migratory response of the neural precursors is a direct effect of the electric field and not due to chemotactic gradients. We also identified that epidermal growth factor receptor (EGFR) signaling plays a role in the galvanotactic response as blocking EGFR significantly attenuates the migratory behaviour. These findings suggest direct current electric fields may be implemented in endogenous repair paradigms to promote migration and tissue repair following neurotrauma.

A novel population of local pericyte precursor cells in tumor stroma that require Notch signaling for differentiation.

PubMed

Patenaude, Alexandre; Woerher, Stefan; Umlandt, Patricia; Wong, Fred; Ibrahim, Rawa; Kyle, Alastair; Unger, Sandy; Fuller, Megan; Parker, Jeremy; Minchinton, Andrew; Eaves, Connie J; Karsan, Aly

2015-09-01

Pericytes are perivascular support cells, the origin of which in tumor tissue is not clear. Recently, we identified a Tie1(+) precursor cell that differentiates into vascular smooth muscle, in a Notch-dependent manner. To understand the involvement of Notch in the ontogeny of tumor pericytes we used a novel flow immunophenotyping strategy to define CD146(+)/CD45(-)/CD31(-/lo) pericytes in the tumor stroma. This strategy combined with ex vivo co-culture experiments identified a novel pericyte progenitor cell population defined as Sca1(hi)/CD146(-)/CD45(-)/CD31(-). The differentiation of these progenitor cells was stimulated by co-culture with endothelial cells. Overexpression of the Notch ligand Jagged1 in endothelial cells further stimulated the differentiation of Sca1(hi)/CD146(-)/CD45(-)/CD31(-) cells into pericytes, while inhibition of Notch signaling with a γ-secretase inhibitor reduced this differentiation. However, Notch inhibition specifically in Tie1-expressing cells did not change the abundance of pericytes in tumors, suggesting that the pericyte precursor is distinct from the vascular smooth muscle cell precursor. Transplant experiments showed that the bone marrow contributes minimally to tumor pericytes. Immunophenotyping revealed that Sca1(hi)/CD146(-)/CD45(-)/CD31(-) cells have greater potential to differentiate into pericytes and have increased expression of classic mesenchymal stem cell markers (CD13, CD44, Nt5e and Thy-1) compared to Sca1(-/lo)/CD146(-)/CD45(-)/CD31(-) cells. Our results suggest that a local Sca1(hi)/CD146(-)/CD45(-)/CD31(-) pericyte progenitor resides in the tumor microenvironment and requires Notch signaling for differentiation into mature pericytes. Copyright © 2015 Elsevier Inc. All rights reserved.

Msx genes define a population of mural cell precursors required for head blood vessel maturation.

PubMed

Lopes, Miguel; Goupille, Olivier; Saint Cloment, Cécile; Lallemand, Yvan; Cumano, Ana; Robert, Benoît

2011-07-01

Vessels are primarily formed from an inner endothelial layer that is secondarily covered by mural cells, namely vascular smooth muscle cells (VSMCs) in arteries and veins and pericytes in capillaries and veinules. We previously showed that, in the mouse embryo, Msx1(lacZ) and Msx2(lacZ) are expressed in mural cells and in a few endothelial cells. To unravel the role of Msx genes in vascular development, we have inactivated the two Msx genes specifically in mural cells by combining the Msx1(lacZ), Msx2(lox) and Sm22α-Cre alleles. Optical projection tomography demonstrated abnormal branching of the cephalic vessels in E11.5 mutant embryos. The carotid and vertebral arteries showed an increase in caliber that was related to reduced vascular smooth muscle coverage. Taking advantage of a newly constructed Msx1(CreERT2) allele, we demonstrated by lineage tracing that the primary defect lies in a population of VSMC precursors. The abnormal phenotype that ensues is a consequence of impaired BMP signaling in the VSMC precursors that leads to downregulation of the metalloprotease 2 (Mmp2) and Mmp9 genes, which are essential for cell migration and integration into the mural layer. Improper coverage by VSMCs secondarily leads to incomplete maturation of the endothelial layer. Our results demonstrate that both Msx1 and Msx2 are required for the recruitment of a population of neural crest-derived VSMCs.

Multipurpose Dissociation Cell for Enhanced ETD of Intact Protein Species

PubMed Central

Rose, Christopher M.; Russell, Jason D.; Ledvina, Aaron R.; McAlister, Graeme C.; Westphall, Michael S.; Griep-Raming, Jens; Schwartz, Jae C.; Coon, Joshua J.; Syka, John E.P.

2013-01-01

We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section RF ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion-ion reactions. The approximately two-fold higher quadrupole field frequency of this cell relative to that of the A-QLT, enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell’s longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation. PMID:23609185

Understanding the reconstitution of the B-cell compartment in bone marrow and blood after treatment for B-cell precursor acute lymphoblastic leukaemia.

PubMed

Theunissen, Prisca M J; van den Branden, Anouk; Van Der Sluijs-Gelling, Alita; De Haas, Valerie; Beishuizen, Auke; van Dongen, Jacques J M; Van Der Velden, Vincent H J

2017-07-01

A better understanding of the reconstitution of the B-cell compartment during and after treatment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) will help to assess the immunological status and needs of post-treatment BCP-ALL patients. Using 8-colour flow cytometry and proliferation-assays, we studied the composition and proliferation of both the B-cell precursor (BCP) population in the bone marrow (BM) and mature B-cell population in peripheral blood (PB) during and after BCP-ALL therapy. We found a normal BCP differentiation pattern and a delayed formation of classical CD38 dim -naive mature B-cells, natural effector B-cells and memory B-cells in patients after chemotherapy. This B-cell differentiation/maturation pattern was strikingly similar to that during initial B-cell development in healthy infants. Tissue-resident plasma cells appeared to be partly protected from chemotherapy. Also, we found that the fast recovery of naive mature B-cell numbers after chemotherapy was the result of increased de novo BCP generation, rather than enhanced B-cell proliferation in BM or PB. These results indicate that post-treatment BCP-ALL patients will eventually re-establish a B-cell compartment with a composition and B-cell receptor repertoire similar to that in healthy children. Additionally, the formation of a new memory B-cell compartment suggests that revaccination might be beneficial after BCP-ALL therapy. © 2017 John Wiley & Sons Ltd.

Coculture with endothelial cells reduces the population of cycling LeX neural precursors but increases that of quiescent cells with a side population phenotype

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

Mathieu, Celine; Fouchet, Pierre; Gauthier, Laurent R.

2006-04-01

Neural stem cell proliferation and differentiation are regulated by external cues from their microenvironment. As endothelial cells are closely associated with neural stem cell in brain germinal zones, we investigated whether endothelial cells may interfere with neurogenesis. Neural precursor cells (NPC) from telencephalon of EGFP mouse embryos were cocultured in direct contact with endothelial cells. Endothelial cells did not modify the overall proliferation and apoptosis of neural cells, albeit they transiently delayed spontaneous apoptosis. These effects appeared to be specific to endothelial cells since a decrease in proliferation and a raise in apoptosis were observed in cocultures with fibroblasts. Endothelialmore » cells stimulated the differentiation of NPC into astrocytes and into neurons, whereas they reduced differentiation into oligodendrocytes in comparison to adherent cultures on polyornithine. Determination of NPC clonogenicity and quantification of LeX expression, a marker for NPC, showed that endothelial cells decreased the number of cycling NPC. On the other hand, the presence of endothelial cells increased the number of neural cells having 'side population' phenotype, another marker reported on NPC, which we have shown to contain quiescent cells. Thus, we show that endothelial cells may regulate neurogenesis by acting at different level of NPC differentiation, proliferation and quiescence.« less

Opposing Effects of α2- and β-Adrenergic Receptor Stimulation on Quiescent Neural Precursor Cell Activity and Adult Hippocampal Neurogenesis

PubMed Central

Prosper, Boris W.; Marathe, Swanand; Husain, Basma F. A.; Kernie, Steven G.; Bartlett, Perry F.; Vaidya, Vidita A.

2014-01-01

Norepinephrine regulates latent neural stem cell activity and adult hippocampal neurogenesis, and has an important role in modulating hippocampal functions such as learning, memory and mood. Adult hippocampal neurogenesis is a multi-stage process, spanning from the activation and proliferation of hippocampal stem cells, to their differentiation into neurons. However, the stage-specific effects of noradrenergic receptors in regulating adult hippocampal neurogenesis remain poorly understood. In this study, we used transgenic Nestin-GFP mice and neurosphere assays to show that modulation of α2- and β-adrenergic receptor activity directly affects Nestin-GFP/GFAP-positive precursor cell population albeit in an opposing fashion. While selective stimulation of α2-adrenergic receptors decreases precursor cell activation, proliferation and immature neuron number, stimulation of β-adrenergic receptors activates the quiescent precursor pool and enhances their proliferation in the adult hippocampus. Furthermore, our data indicate no major role for α1-adrenergic receptors, as we did not observe any change in either the activation and proliferation of hippocampal precursors following selective stimulation or blockade of α1-adrenergic receptors. Taken together, our data suggest that under physiological as well as under conditions that lead to enhanced norepinephrine release, the balance between α2- and β-adrenergic receptor activity regulates precursor cell activity and hippocampal neurogenesis. PMID:24922313

Hepatic progenitor populations in embryonic, neonatal, and adult liver.

PubMed

Brill, S; Holst, P; Sigal, S; Zvibel, I; Fiorino, A; Ochs, A; Somasundaran, U; Reid, L M

1993-12-01

Oval cells, small cells with oval-shaped nuclei, are induced to proliferate in the livers of animals treated with carcinogens and are thought to be related to liver stem cells and/or committed liver progenitor cell populations. We have developed protocols for identifying and isolating antigenically related cell populations present in normal tissues using monoclonal antibodies to oval cell antigens and fluorescence-activated cell sorting. We have isolated oval cell-antigen-positive (OCAP) cells from embryonic, neonatal, and adult rat livers and have identified culture conditions permitting their growth in culture. The requirements for growth of the OCAP cells included substrata of type IV collagen mixed with laminin, basal medium with complex lipids and low calcium, specific growth factors (most potently, insulin-like growth factor II and granulocyte-macrophage colony-stimulating factor), and co-cultures of embryonic, liver-specific stroma, strongly suggesting paracrine signaling between hepatic and hemopoietic precursor cells. The growing OCAP cultures proved to be uniformly expressing oval cell markers but were nevertheless a mixture of hepatic and hemopoietic precursor cells. To separate the hepatic and hemopoietic subpopulations of OCAP cells, we surveyed known antibodies and found ones that uniquely identify either hepatic or hemopoietic cells. Several of these antibodies were used in panning procedures and fluorescence-activated cell sorting to eliminate contaminant cell populations, particularly hemopoietic and endothelial cells. Using specific flow cytometric parameters, three cellular subpopulations could be isolated separately that were identified by immunochemistry and molecular hybridization assays as probable: (i) committed progenitors to hepatocytes; (ii) committed progenitors to bile ducts; or (iii) a mixed population of hemopoietic cells that contained a small percentage of hepatic blasts that are possibly pluripotent. The hepatic precursor cells have been characterized using immunochemistry, flow cytometry, and molecular hybridization assays. The hepatic blasts are small (7-10 microns) cells with high nuclear to cytoplasmic ratios and with minimal complexity of the cytoplasm. Cultures of the committed progenitors were found to differentiate into cells with recognizable parenchymal cell fates. We discuss our studies in the context of our model of the liver as stem cell and lineage system and suggest that a slow, unidirectional, terminal differentiation process, paralleling more rapid ones in the skin or gut, occurs at all times in the liver and is thought to vary primarily in kinetics during quiescent versus regenerative states.(ABSTRACT TRUNCATED AT 400 WORDS)

Defining High-Risk Precursor Signaling to Advance Breast Cancer Risk Assessment and Prevention

DTIC Science & Technology

2017-03-01

KEYWORDS: 3. ACCOMPLISHMENTS: Aim 1: Functional analysis of progenitor and stem cells in high-risk tissues. Major Task 1Functional...and stem cells in high-risk tissues. Major Task 1: Quantitation of LP (Luminal Progenitor) and basal stem cell (MASC) populations A. Quantitation of...LP and basal stem cell (MASC) populations We have continued to add patients to the cohorts between months 12 and 24. (This reporting period

Clonal analysis identifies hemogenic endothelium as the source of the blood-endothelial common lineage in the mouse embryo

PubMed Central

Padrón-Barthe, Laura; Temiño, Susana; Villa del Campo, Cristina; Carramolino, Laura; Isern, Joan

2014-01-01

The first blood and endothelial cells of amniote embryos appear in close association in the blood islands of the yolk sac (YS). This association and in vitro lineage analyses have suggested a common origin from mesodermal precursors called hemangioblasts, specified in the primitive streak during gastrulation. Fate mapping and chimera studies, however, failed to provide strong evidence for a common origin in the early mouse YS. Additional in vitro studies suggest instead that mesodermal precursors first generate hemogenic endothelium, which then generate blood cells in a linear sequence. We conducted an in vivo clonal analysis to determine the potential of individual cells in the mouse epiblast, primitive streak, and early YS. We found that early YS blood and endothelial lineages mostly derive from independent epiblast populations, specified before gastrulation. Additionally, a subpopulation of the YS endothelium has hemogenic activity and displays characteristics similar to those found later in the embryonic hemogenic endothelium. Our results show that the earliest blood and endothelial cell populations in the mouse embryo are specified independently, and that hemogenic endothelium first appears in the YS and produces blood precursors with markers related to definitive hematopoiesis. PMID:25139355

CD22 is required for formation of memory B cell precursors within germinal centers.

PubMed

Chappell, Craig P; Draves, Kevin E; Clark, Edward A

2017-01-01

CD22 is a BCR co-receptor that regulates B cell signaling, proliferation and survival and is required for T cell-independent Ab responses. To investigate the role of CD22 during T cell-dependent (TD) Ab responses and memory B cell formation, we analyzed Ag-specific B cell responses generated by wild-type (WT) or CD22-/- B cells following immunization with a TD Ag. CD22-/- B cells mounted normal early Ab responses yet failed to generate either memory B cells or long-lived plasma cells, whereas WT B cells formed both populations. Surprisingly, B cell expansion and germinal center (GC) differentiation were comparable between WT and CD22-/- B cells. CD22-/- B cells, however, were significantly less capable of generating a population of CXCR4hiCD38hi GC B cells, which we propose represent memory B cell precursors within GCs. These results demonstrate a novel role for CD22 during TD humoral responses evident during primary GC formation and underscore that CD22 functions not only during B cell maturation but also during responses to both TD and T cell-independent antigens.

CD22 is required for formation of memory B cell precursors within germinal centers

PubMed Central

Chappell, Craig P.; Draves, Kevin E.

2017-01-01

CD22 is a BCR co-receptor that regulates B cell signaling, proliferation and survival and is required for T cell-independent Ab responses. To investigate the role of CD22 during T cell-dependent (TD) Ab responses and memory B cell formation, we analyzed Ag-specific B cell responses generated by wild-type (WT) or CD22-/- B cells following immunization with a TD Ag. CD22-/- B cells mounted normal early Ab responses yet failed to generate either memory B cells or long-lived plasma cells, whereas WT B cells formed both populations. Surprisingly, B cell expansion and germinal center (GC) differentiation were comparable between WT and CD22-/- B cells. CD22-/- B cells, however, were significantly less capable of generating a population of CXCR4hiCD38hi GC B cells, which we propose represent memory B cell precursors within GCs. These results demonstrate a novel role for CD22 during TD humoral responses evident during primary GC formation and underscore that CD22 functions not only during B cell maturation but also during responses to both TD and T cell-independent antigens. PMID:28346517

Fetal liver contains committed NK progenitors, but is not a site for development of CD34+ cells into T cells.

PubMed

Jaleco, A C; Blom, B; Res, P; Weijer, K; Lanier, L L; Phillips, J H; Spits, H

1997-07-15

The presence of T and NK cells in the human fetal liver and the fact that fetal liver hemopoietic progenitor cells develop into T and NK cells suggest a role for the fetal liver compartment in T and NK cell development. In this work, we show that the capacity of fetal liver progenitors to develop into T cells, in a human/mouse fetal thymic organ culture system, is restricted to an immature subset of CD34+ CD38- cells. No T cell-committed precursors are contained within the more differentiated CD34+ CD38+ population. This conclusion is supported by the observations that no TCR-delta gene rearrangements and no pre-TCR-alpha expression can be detected in this population. However, NK cells were derived from CD34+ CD38- and CD34+ CD38+ fetal liver cells cultured in the presence of IL-15, IL-7, and Flt-3 ligand. Eighty to ninety percent of cells arising from the CD34+ CD38+ population expressed the NK cell-associated markers CD56, CD16, CD94, and NKR-P1A. Several subpopulations of NK cell precursors were identified by differential expression of these receptors. Based on the detection of populations with a similar antigenic profile in freshly isolated fetal liver cells, we propose a model of NK cell differentiation. Collectively, our findings suggest that CD34+ cells differentiate into NK cells, but not into mature T cells, in the human fetal liver.

Identification of a novel intergenic miRNA located between the human DDC and COBL genes with a potential function in cell cycle arrest.

PubMed

Hoballa, Mohamad Hussein; Soltani, Bahram M; Mowla, Seyed Javad; Sheikhpour, Mojgan; Kay, Maryam

2018-07-01

Frequent abnormalities in 7p12 locus in different tumors like lung cancer candidate this region for novel regulatory elements. MiRNAs as novel regulatory elements encoded within the human genome are potentially oncomiRs or miR suppressors. Here, we have used bioinformatics tools to search for the novel miRNAs embedded within human chromosome 7p12. A bona fide stem loop (named mirZa precursor) had the features of producing a real miRNA (named miRZa) which was detected through RT-qPCR following the overexpression of its precursor. Then, endogenous miRZa was detected in human cell lines and tissues and sequenced. Consistent to the bioinformatics prediction, RT-qPCR as well as dual luciferase assay indicated that SMAD3 and IGF1R genes were targeted by miRZa. MiRZa-3p and miRZa-5p were downregulated in lung tumor tissue samples detected by RT-qPCR, and mirZa precursor overexpression in SW480 cells resulted in increased sub-G1 cell population. Overall, here we introduced a novel miRNA which is capable of targeting SMAD3 and IGF1R regulatory genes and increases the cell population in sub-G1 stage.

The rise and fall of long-lived humoral immunity: terminal differentiation of plasma cells in health and disease

PubMed Central

O'Connor, Brian P.; Gleeson, Michael W.; Noelle, Randolph J.; Erickson, Loren D.

2010-01-01

Summary Long-lived humoral immune responses are a hallmark of thymus-dependent immunity. The cellular basis for enduring antibody-mediated immunity is long-lived memory B cells and plasma cells (PCs). Both of these cell populations acquire longevity as a result of antigen-specific, CD40–dependent, cognate interactions with helper T cells within germinal centers (GCs). At the molecular level, defined functional domains of CD40 control the post-GC fate of B cells. PC precursors that emerge from these GC reactions are highly proliferative and terminally differentiate to end-stage cells within the bone marrow (BM). The striking phenotypic similarities between the PC precursors and the putative malignant cell in multiple myeloma (MM) suggests that MM may result from the transformation of PC precursors. Within the domain of autoimmune disease, recent studies have shown that dysregulated migration of PCs to the BM may impact immune homeostasis and the development of lupus. Understanding the processes of normal PC differentiation will provide strategic insights into identifying therapeutic targets for the treatment of differentiated B-cell disorders. PMID:12846808

Deglycosylation of serum vitamin D3-binding protein leads to immunosuppression in cancer patients.

PubMed

Yamamoto, N; Naraparaju, V R; Asbell, S O

1996-06-15

Serum vitamin D3-binding protein (Gc protein) can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor of the macrophage activating factor (MAF). Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high titered MAF, Gc-MAF. When peripheral blood monocytes/macrophages of 52 patients bearing various types of cancer were incubated with 100 pg/ml of GcMAF, the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of patient plasma Gc protein was found to be severely reduced in about 25% of this patient population. About 45% of the patients had moderately reduced MAF precursor activities. Loss of the precursor activity was found to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase detected in the patient's bloodstream. The source of the enzyme appeared to be cancerous cells. Radiation therapy decreased plasma alpha-N-acetylgalactosaminidase activity with concomitant increase of precursor activity. This implies that radiation therapy decreases the number of cancerous cells capable of secreting alpha-N-acetylgalactosaminidase. Both alpha-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in patient bloodstream can serve as diagnostic and prognostic indices.

Cellular Basis of Pineal Gland Development: Emerging Role of Microglia as Phenotype Regulator.

PubMed

Ibañez Rodriguez, María P; Noctor, Stephen C; Muñoz, Estela M

2016-01-01

The adult pineal gland is composed of pinealocytes, astrocytes, microglia, and other interstitial cells that have been described in detail. However, factors that contribute to pineal development have not been fully elucidated, nor have pineal cell lineages been well characterized. We applied systematic double, triple and quadruple labeling of cell-specific markers on prenatal, postnatal and mature rat pineal gland tissue combined with confocal microscopy to provide a comprehensive view of the cellular dynamics and cell lineages that contribute to pineal gland development. The pineal gland begins as an evagination of neuroepithelium in the roof of the third ventricle. The pineal primordium initially consists of radially aligned Pax6+ precursor cells that express vimentin and divide at the ventricular lumen. After the tubular neuroepithelium fuses, the distribution of Pax6+ cells transitions to include rosette-like structures and later, dispersed cells. In the developing gland all dividing cells express Pax6, indicating that Pax6+ precursor cells generate pinealocytes and some interstitial cells. The density of Pax6+ cells decreases across pineal development as a result of cellular differentiation and microglial phagocytosis, but Pax6+ cells remain in the adult gland as a distinct population. Microglial colonization begins after pineal recess formation. Microglial phagocytosis of Pax6+ cells is not common at early stages but increases as microglia colonize the gland. In the postnatal gland microglia affiliate with Tuj1+ nerve fibers, IB4+ blood vessels, and Pax6+ cells. We demonstrate that microglia engulf Pax6+ cells, nerve fibers, and blood vessel-related elements, but not pinealocytes. We conclude that microglia play a role in pineal gland formation and homeostasis by regulating the precursor cell population, remodeling blood vessels and pruning sympathetic nerve fibers.

Cellular Basis of Pineal Gland Development: Emerging Role of Microglia as Phenotype Regulator

PubMed Central

Ibañez Rodriguez, María P.

2016-01-01

The adult pineal gland is composed of pinealocytes, astrocytes, microglia, and other interstitial cells that have been described in detail. However, factors that contribute to pineal development have not been fully elucidated, nor have pineal cell lineages been well characterized. We applied systematic double, triple and quadruple labeling of cell-specific markers on prenatal, postnatal and mature rat pineal gland tissue combined with confocal microscopy to provide a comprehensive view of the cellular dynamics and cell lineages that contribute to pineal gland development. The pineal gland begins as an evagination of neuroepithelium in the roof of the third ventricle. The pineal primordium initially consists of radially aligned Pax6+ precursor cells that express vimentin and divide at the ventricular lumen. After the tubular neuroepithelium fuses, the distribution of Pax6+ cells transitions to include rosette-like structures and later, dispersed cells. In the developing gland all dividing cells express Pax6, indicating that Pax6+ precursor cells generate pinealocytes and some interstitial cells. The density of Pax6+ cells decreases across pineal development as a result of cellular differentiation and microglial phagocytosis, but Pax6+ cells remain in the adult gland as a distinct population. Microglial colonization begins after pineal recess formation. Microglial phagocytosis of Pax6+ cells is not common at early stages but increases as microglia colonize the gland. In the postnatal gland microglia affiliate with Tuj1+ nerve fibers, IB4+ blood vessels, and Pax6+ cells. We demonstrate that microglia engulf Pax6+ cells, nerve fibers, and blood vessel-related elements, but not pinealocytes. We conclude that microglia play a role in pineal gland formation and homeostasis by regulating the precursor cell population, remodeling blood vessels and pruning sympathetic nerve fibers. PMID:27861587

PDGF-responsive progenitors persist in the subventricular zone across the lifespan

PubMed Central

Moore, Lisamarie; Bain, Jennifer M.; Loh, Ji Meng; Levison, Steven W.

2013-01-01

The SVZ (subventricular zone) contains neural stem cells and progenitors of various potentialities. Although initially parsed into A, B, and C cells, this germinal zone is comprised of a significantly more diverse population of cells. Here, we characterized a subset of postnatal PRPs (PDGF-AA-responsive precursors) that express functional PDGFα and β receptors from birth to adulthood. When grown in PDGF-AA, dissociated neonatal rat SVZ cells divided to produce non-adherent clusters of progeny. Unlike the self-renewing EGF/FGF-2-responsive precursors that produce neurospheres, these PRPs failed to self-renew after three passages; therefore, we refer to the colonies they produce as spheroids. Upon differentiation these spheroids could produce neurons, type 1 astrocytes and oligodendrocytes. When maintained in medium supplemented with BMP-4 they also produced type 2 astrocytes. Using lineage tracing methods, it became evident that there were multiple types of PRPs, including a subset that could produce neurons, oligodendrocytes, and type 1 and type 2 astrocytes; thus some of these PRPs represent a unique population of precursors that are quatropotential. Spheroids also could be generated from the newborn neocortex and they had the same potentiality as those from the SVZ. By contrast, the adult neocortex produced less than 20% of the numbers of spheroids than the adult SVZ and spheroids from the adult neocortex only differentiated into glial cells. Interestingly, SVZ spheroid producing capacity diminished only slightly from birth to adulthood. Altogether these data demonstrate that there are PRPs that persist in the SVZ that includes a unique population of quatropotential PRPs. PMID:24367913

Molecular Features of Neural Stem Cells Enable their Enrichment Using Pharmacological Inhibitors of Survival-Promoting Kinases

PubMed Central

Brazel, Christine Y.; Alaythan, Abdulaziz A.; Felling, Ryan J.; Calderon, Frances; Levison, Steven W.

2013-01-01

Isolating a pure population of neural stem cells (NSCs) has been difficult since no exclusive surface markers have been identified for panning or FACS purification. Moreover, additional refinements for maintaining NSCs in culture are required, since NSCs generate a variety of neural precursors (NPs) as they proliferate. Here, we demonstrate that postnatal rat NPs express low levels of pro-apoptotic molecules and resist PI3K and ERK1/2 inhibition as compared to late oligodendrocyte progenitors. Furthermore, maintaining SVZ precursors in LY294002 and PD98059, inhibitors of PI3K and ERK1/2 signaling, eliminated lineage-restricted precursors as revealed by enrichment for Nestin+/SOX-2+ cells. The cells that survived formed neurospheres and 89% of these neurospheres were tripotential, generating neurons, astrocytes and oligodendrocytes. Without this enrichment step, less than 50% of the NPs were Nestin+/SOX-2+ and 42% of the neurospheres were tripotential. Additionally, neurospheres enriched using this procedure produced 3-times more secondary neurospheres, supporting the conclusion that this procedure enriches for NSCs. A number of genes that enhance survival were more highly expressed in neurospheres compared to late oligodendrocyte progenitors. Altogether, these studies demonstrate that primitive neural precursors can be enriched using a relatively simple and inexpensive means that will facilitate cell replacement strategies using stem cells as well as other studies whose goal is to reveal the fundamental properties of primitive neural precursors. PMID:24032666

Genetic modification of hematopoietic stem cells: recent advances in the gene therapy of inherited diseases.

PubMed

Bueren, Juan A; Guenechea, Guillermo; Casado, José A; Lamana, María Luisa; Segovia, José C

2003-01-01

Hematopoietic stem cells constitute a rare population of precursor cells with remarkable properties for being used as targets in gene therapy protocols. The last years have been particularly productive both in the fields of gene therapy and stem cell biology. Results from ongoing clinical trials have shown the first unquestionable clinical benefits of immunodeficient patients transplanted with genetically modified autologous stem cells. On the other hand, severe side effects in a few patients treated with gene therapy have also been reported, indicating the usefulness of further improving the vectors currently used in gene therapy clinical trials. In the field of stem cell biology, evidence showing the plastic potential of adult hematopoietic stem cells and data indicating the multipotency of adult mesenchymal precursor cells have been presented. Also, the generation of embryonic stem cells by means of nuclear transfer techniques has appeared as a new methodology with direct implications in gene therapy.

p57KIP2 regulates radial glia and intermediate precursor cell cycle dynamics and lower layer neurogenesis in developing cerebral cortex

PubMed Central

Mairet-Coello, Georges; Tury, Anna; Van Buskirk, Elise; Robinson, Kelsey; Genestine, Matthieu; DiCicco-Bloom, Emanuel

2012-01-01

During cerebral cortex development, precise control of precursor cell cycle length and cell cycle exit is required for balanced precursor pool expansion and layer-specific neurogenesis. Here, we defined the roles of cyclin-dependent kinase inhibitor (CKI) p57KIP2, an important regulator of G1 phase, using deletion mutant mice. Mutant mice displayed macroencephaly associated with cortical hyperplasia during late embryogenesis and postnatal development. Embryonically, proliferation of radial glial cells (RGC) and intermediate precursors (IPC) was increased, expanding both populations, with greater effect on IPCs. Furthermore, cell cycle re-entry was increased during early corticogenesis, whereas cell cycle exit was augmented at middle stage. Consequently, neurogenesis was reduced early, whereas it was enhanced during later development. In agreement, the timetable of early neurogenesis, indicated by birthdating analysis, was delayed. Cell cycle dynamics analyses in mutants indicated that p57KIP2 regulates cell cycle length in both RGCs and IPCs. By contrast, related CKI p27KIP1 controlled IPC proliferation exclusively. Furthermore, p57KIP2 deficiency markedly increased RGC and IPC divisions at E14.5, whereas p27KIP1 increased IPC proliferation at E16.5. Consequently, loss of p57KIP2 increased primarily layer 5-6 neuron production, whereas loss of p27KIP1 increased neurons specifically in layers 2-5. In conclusion, our observations suggest that p57KIP2 and p27KIP1 control neuronal output for distinct cortical layers by regulating different stages of precursor proliferation, and support a model in which IPCs contribute to both lower and upper layer neuron generation. PMID:22223678

Base Composition Differences between Avian Myeloblastosis Virus Transfer RNA and Transfer RNA Isolated from Host Cells

PubMed Central

Randerath, Kurt; Rosenthal, Leonard J.; Zamecnik, Paul C.

1971-01-01

Using a novel chemical tritium derivative method, we have determined the base composition of 4S RNA isolated from an RNA tumor virus, the avian myeloblastosis virus, and from normal and neoplastic host cells. Extensive differences were detected, particularly with respect to the amount of methylated bases in the viral RNA. The viral 4S RNA, which fulfills the criteria for designation as transfer RNA, appears to be derived from a precursor pool that is different from the precursor population of host-cell 4S RNA. These results are discussed in regard to the possible relationship between transfer RNA of avian mycoblastosis virus and cellular transfer RNA. Images PMID:4332019

USSR Report, Life Sciences Biomedical and Behavioral Sciences.

DTIC Science & Technology

1984-03-14

demonstrated that infection with tick-borne encephalitis (TBE) virus or Langat virus leads to the appearance of two populations of autoreactive T...cells formed in mice ^fected wiA the TBE or Langat viruses exert a protective effect by preventing the maturation of the ACTL precursor cells Into

A complex between contactin-1 and the protein tyrosine phosphatase PTPRZ controls the development of oligodendrocyte precursor cells

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

Lamprianou, Smaragda; Chatzopoulou, Elli; Thomas, Jean-Léon

The six members of the contactin (CNTN) family of neural cell adhesion molecules are involved in the formation and maintenance of the central nervous system (CNS) and have been linked to mental retardation and neuropsychiatric disorders such as autism. Five of the six CNTNs bind to the homologous receptor protein tyrosine phosphatases gamma (PTPRG) and zeta (PTPRZ), but the biological roles of these interactions remain unclear. We report here the cocrystal structure of the carbonic anhydrase-like domain of PTPRZ bound to tandem Ig repeats of CNTN1 and combine these structural data with binding assays to show that PTPRZ binds specificallymore » to CNTN1 expressed at the surface of oligodendrocyte precursor cells. Furthermore, analyses of glial cell populations in wild-type and PTPRZ-deficient mice show that the binding of PTPRZ to CNTN1 expressed at the surface of oligodendrocyte precursor cells inhibits their proliferation and promotes their development into mature oligodendrocytes. Overall, these results implicate the PTPRZ/CNTN1 complex as a previously unknown modulator of oligodendrogenesis.« less

Signaling Networks among Stem Cell Precursors, Transit-Amplifying Progenitors, and their Niche in Developing Hair Follicles.

PubMed

Rezza, Amélie; Wang, Zichen; Sennett, Rachel; Qiao, Wenlian; Wang, Dongmei; Heitman, Nicholas; Mok, Ka Wai; Clavel, Carlos; Yi, Rui; Zandstra, Peter; Ma'ayan, Avi; Rendl, Michael

2016-03-29

The hair follicle (HF) is a complex miniorgan that serves as an ideal model system to study stem cell (SC) interactions with the niche during growth and regeneration. Dermal papilla (DP) cells are required for SC activation during the adult hair cycle, but signal exchange between niche and SC precursors/transit-amplifying cell (TAC) progenitors that regulates HF morphogenetic growth is largely unknown. Here we use six transgenic reporters to isolate 14 major skin and HF cell populations. With next-generation RNA sequencing, we characterize their transcriptomes and define unique molecular signatures. SC precursors, TACs, and the DP niche express a plethora of ligands and receptors. Signaling interaction network analysis reveals a bird's-eye view of pathways implicated in epithelial-mesenchymal interactions. Using a systematic tissue-wide approach, this work provides a comprehensive platform, linked to an interactive online database, to identify and further explore the SC/TAC/niche crosstalk regulating HF growth. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Pax-3 expression in segmental mesoderm marks early stages in myogenic cell specification.

PubMed

Williams, B A; Ordahl, C P

1994-04-01

Specification of the myogenic lineage begins prior to gastrulation and culminates in the emergence of determined myogenic precursor cells from the somites. The myoD family (MDF) of transcriptional activators controls late step(s) in myogenic specification that are closely followed by terminal muscle differentiation. Genes expressed in myogenic specification at stages earlier than MDFs are unknown. The Pax-3 gene is expressed in all the cells of the caudal segmental plate, the early mesoderm compartment that contains the precursors of skeletal muscle. As somites form from the segmental plate and mature, Pax-3 expression is progressively modulated. Beginning at the time of segmentation, Pax-3 becomes repressed in the ventral half of the somite, leaving Pax-3 expression only in the dermomyotome. Subsequently, differential modulation of Pax-3 expression levels delineates the medial and lateral halves of the dermomyotome, which contain precursors of axial (back) muscle and limb muscle, respectively. Pax-3 expression is then repressed as dermomyotome-derived cells activate MDFs. Quail-chick chimera and ablation experiments confirmed that the migratory precursors of limb muscle continue to express Pax-3 during migration. Since limb muscle precursors do not activate MDFs until 2 days after they leave the somite, Pax-3 represents the first molecular marker for this migratory cell population. A null mutation of the mouse Pax-3 gene, Splotch, produces major disruptions in early limb muscle development (Franz, T., Kothary, R., Surani, M. A. H., Halata, Z. and Grim, M. (1993) Anat. Embryol. 187, 153-160; Goulding, M., Lumsden, A. and Paquette, A. (1994) Development 120, 957-971). We conclude, therefore, that Pax-3 gene expression in the paraxial mesoderm marks earlier stages in myogenic specification than MDFs and plays a crucial role in the specification and/or migration of limb myogenic precursors.

Generation of avian cells resembling osteoclasts from mononuclear phagocytes

NASA Technical Reports Server (NTRS)

Alvarez, J. I.; Teitelbaum, S. L.; Blair, H. C.; Greenfield, E. M.; Athanasou, N. A.; Ross, F. P.

1991-01-01

Several lines of indirect evidence suggest that a monocyte family precursor gives rise to the osteoclast, although this hypothesis is controversial. Starting with a uniform population of nonspecific esterase positive, tartrate-sensitive, acid phosphatase-producing, mannose receptor-bearing mononuclear cells, prepared from dispersed marrow of calcium-deprived laying hens by cell density separation and selective cellular adherence, we generated multinucleated cells in vitro. When cultured with devitalized bone, these cells show, by electron microscopy, the characteristic osteoclast morphology in that they are mitochondria-rich, multinucleated, and, most importantly, develop characteristic ruffled membranes at the matrix attachment site. Moreover, as documented by scanning electron microscopy, these cells pit bone slices in a manner identical to freshly isolated osteoclasts. In addition, isoenzymes of acid phosphatase from generated osteoclasts, separated by 7.5% polyacrylamide gel electrophoresis at pH 4, are identical to those of mature osteoclasts in migration pattern and tartrate resistance, although the precursor cells from which the osteoclasts are generated produce an entirely different isoenzyme, which is tartrate-sensitive and migrates less rapidly at pH 4. The fused cells also exhibit a cAMP response to prostaglandin E2. Therefore, osteoclast-like cells can be derived by in vitro culture of a marrow-derived monocyte cell population.

Regionally distinct responses of microglia and glial progenitor cells to whole brain irradiation in adult and aging rats.

PubMed

Hua, Kun; Schindler, Matthew K; McQuail, Joseph A; Forbes, M Elizabeth; Riddle, David R

2012-01-01

Radiation therapy has proven efficacy for treating brain tumors and metastases. Higher doses and larger treatment fields increase the probability of eliminating neoplasms and preventing reoccurrence, but dose and field are limited by damage to normal tissues. Normal tissue injury is greatest during development and in populations of proliferating cells but also occurs in adults and older individuals and in non-proliferative cell populations. To better understand radiation-induced normal tissue injury and how it may be affected by aging, we exposed young adult, middle-aged, and old rats to 10 Gy of whole brain irradiation and assessed in gray- and white matter the responses of microglia, the primary cellular mediators of radiation-induced neuroinflammation, and oligodendrocyte precursor cells, the largest population of proliferating cells in the adult brain. We found that aging and/or irradiation caused only a few microglia to transition to the classically "activated" phenotype, e.g., enlarged cell body, few processes, and markers of phagocytosis, that is seen following more damaging neural insults. Microglial changes in response to aging and irradiation were relatively modest and three markers of reactivity - morphology, proliferation, and expression of the lysosomal marker CD68- were regulated largely independently within individual cells. Proliferation of oligodendrocyte precursors did not appear to be altered during normal aging but increased following irradiation. The impacts of irradiation and aging on both microglia and oligodendrocyte precursors were heterogeneous between white- and gray matter and among regions of gray matter, indicating that there are regional regulators of the neural response to brain irradiation. By several measures, the CA3 region of the hippocampus appeared to be differentially sensitive to effects of aging and irradiation. The changes assessed here likely contribute to injury following inflammatory challenges like brain irradiation and represent important end-points for analysis in studies of therapeutic strategies to protect patients from neural dysfunction.

Lin- CD34hi CD117int/hi FcεRI+ cells in human blood constitute a rare population of mast cell progenitors.

PubMed

Dahlin, Joakim S; Malinovschi, Andrei; Öhrvik, Helena; Sandelin, Martin; Janson, Christer; Alving, Kjell; Hallgren, Jenny

2016-01-28

Mast cells are rare tissue-resident immune cells that are involved in allergic reactions, and their numbers are increased in the lungs of asthmatics. Murine lung mast cells arise from committed bone marrow-derived progenitors that enter the blood circulation, migrate through the pulmonary endothelium, and mature in the tissue. In humans, mast cells can be cultured from multipotent CD34(+) progenitor cells. However, a population of distinct precursor cells that give rise to mast cells has remained undiscovered. To our knowledge, this is the first report of human lineage-negative (Lin(-)) CD34(hi) CD117(int/hi) FcεRI(+) progenitor cells, which represented only 0.0053% of the isolated blood cells in healthy individuals. These cells expressed integrin β7 and developed a mast cell-like phenotype, although with a slow cell division capacity in vitro. Isolated Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells had an immature mast cell-like appearance and expressed high levels of many mast cell-related genes as compared with human blood basophils in whole-transcriptome microarray analyses. Furthermore, serglycin, tryptase, and carboxypeptidase A messenger RNA transcripts were detected by quantitative reverse transcription-polymerase chain reaction. Altogether, we propose that the Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells are closely related to human tissue mast cells and likely constitute an immediate precursor population, which can give rise to predominantly mast cells. Furthermore, asthmatics with reduced lung function had a higher frequency of Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood mast cell progenitors than asthmatics with normal lung function. © 2016 by The American Society of Hematology.

Embryonic Heart Progenitors and Cardiogenesis

PubMed Central

Brade, Thomas; Pane, Luna S.; Moretti, Alessandra; Chien, Kenneth R.; Laugwitz, Karl-Ludwig

2013-01-01

The mammalian heart is a highly specialized organ, comprised of many different cell types arising from distinct embryonic progenitor populations during cardiogenesis. Three precursor populations have been identified to contribute to different myocytic and nonmyocytic cell lineages of the heart: cardiogenic mesoderm cells (CMC), the proepicardium (PE), and cardiac neural crest cells (CNCCs). This review will focus on molecular cues necessary for proper induction, expansion, and lineage-specific differentiation of these progenitor populations during cardiac development in vivo. Moreover, we will briefly discuss how the knowledge gained on embryonic heart progenitor biology can be used to develop novel therapeutic strategies for the management of congenital heart disease as well as for improvement of cardiac function in ischemic heart disease. PMID:24086063

A Subpopulation of Smooth Muscle Cells, Derived from Melanocyte-Competent Precursors, Prevents Patent Ductus Arteriosus

PubMed Central

Puig, Isabel; Champeval, Delphine; Kumasaka, Mayuko; Belloir, Elodie; Bonaventure, Jacky; Mark, Manuel; Yamamoto, Hiroaki; Taketo, Mark M.; Choquet, Philippe; Etchevers, Heather C.; Beermann, Friedrich; Delmas, Véronique; Monassier, Laurent; Larue, Lionel

2013-01-01

Background Patent ductus arteriosus is a life-threatening condition frequent in premature newborns but also present in some term infants. Current mouse models of this malformation generally lead to perinatal death, not reproducing the full phenotypic spectrum in humans, in whom genetic inheritance appears complex. The ductus arteriosus (DA), a temporary fetal vessel that bypasses the lungs by shunting the aortic arch to the pulmonary artery, is constituted by smooth muscle cells of distinct origins (SMC1 and SMC2) and many fewer melanocytes. To understand novel mechanisms preventing DA closure at birth, we evaluated the importance of cell fate specification in SMC that form the DA during embryonic development. Upon specific Tyr::Cre-driven activation of Wnt/β-catenin signaling at the time of cell fate specification, melanocytes replaced the SMC2 population of the DA, suggesting that SMC2 and melanocytes have a common precursor. The number of SMC1 in the DA remained similar to that in controls, but insufficient to allow full DA closure at birth. Thus, there was no cellular compensation by SMC1 for the loss of SMC2. Mice in which only melanocytes were genetically ablated after specification from their potential common precursor with SMC2, demonstrated that differentiated melanocytes themselves do not affect DA closure. Loss of the SMC2 population, independent of the presence of melanocytes, is therefore a cause of patent ductus arteriosus and premature death in the first months of life. Our results indicate that patent ductus arteriosus can result from the insufficient differentiation, proliferation, or contractility of a specific smooth muscle subpopulation that shares a common neural crest precursor with cardiovascular melanocytes. PMID:23382837

CCR7 Plays No Appreciable Role in Trafficking of Central Memory CD4 T Cells to Lymph Nodes

PubMed Central

Lugt, Bryan Vander; Tubo, Noah J.; Nizza, Suzanne T.; Boes, Marianne; Malissen, Bernard; Fuhlbrigge, Robert C.; Kupper, Thomas S.; Campbell, James J.

2013-01-01

CCR7−/− mice exhibit profound anomalies in LN and spleen architecture, which complicates the study of CCR7-mediated T cell trafficking in vivo. To circumvent this problem, we established in vivo models in which WT and CCR7−/− populations coexist within mice possessing normal lymphoid organs, and must compete for developmental niches within the tissues of these mice. Under the conditions we have created in vivo, we find the entry of memory CD4 T cells into LN from the blood to be independent of CCR7. Thus, the central memory CD4 T cells that traffic though LN, which are often defined by their expression of CCR7, do not appear to gain any competitive homing advantage by expressing this receptor. Furthermore, in contrast to cutaneous DC populations, we found that CCR7 deficiency had no appreciable effect on the exit of CD4 T cells from inflamed skin. Finally, we found that WT and CCR7−/− precursors were equally represented within the major thymic subpopulations, despite previous findings that CCR7 plays a role in seeding the thymus from bone marrow-derived T cell precursors. PMID:23935190

GM-CSF: An Immune Modulatory Cytokine that can Suppress Autoimmunity

PubMed Central

Bhattacharya, Palash; Thiruppathi, Muthusamy; Elshabrawy, Hatem A.; Alharshawi, Khaled; Kumar, Prabhakaran; Prabhakar, Bellur S.

2015-01-01

GM-CSF was originally identified as a colony stimulating factor (CSF) because of its ability to induce granulocyte and macrophage populations from precursor cells. Multiple studies have demonstrated that GM-CSF is also an immune-modulatory cytokine, capable of affecting not only the phenotype of myeloid lineage cells, but also T-cell activation through various myeloid intermediaries. This property has been implicated in the sustenance of several autoimmune diseases like arthritis and multiple sclerosis. In contrast, several studies using animal models have shown that GM-CSF is also capable of suppressing many autoimmune diseases like Crohn's disease, Type-1 diabetes, Myasthenia gravis and experimental autoimmune thyroiditis. Knockout mouse studies have suggested that the role of GM-CSF in maintaining granulocyte and macrophage populations in the physiological steady state is largely redundant. Instead, its immune-modulatory role plays a significant role in the development or resolution of autoimmune diseases. This is mediated either through the differentiation of precursor cells into specialized non-steady state granulocytes, macrophages and dendritic cells, or through the modulation of the phenotype of mature myeloid cells. Thus, outside of myelopoiesis, GM-CSF has a profound role in regulating the immune response and maintaining immunological tolerance. PMID:26113402

Location and cellular stages of NK cell development

PubMed Central

Yu, Jianhua; Freud, Aharon G.; Caligiuri, Michael A

2013-01-01

The identification of distinct tissue-specific natural killer (NK) cell populations that apparently mature from local precursor populations has brought new insight into the diversity and developmental regulation of this important lymphoid subset. NK cells provide a necessary link between the early (innate) and late (adaptive) immune responses to infection. Gaining a better understanding of the processes that govern NK cell development should allow us to better harness NK cell functions in multiple clinical settings as well as to gain further insight into how these cells undergo malignant transformation. In this review, we summarize recent advances in understanding sites and cellular stages of NK cell development in humans and mice. PMID:24055329

Ptf1a determines horizontal and amacrine cell fates during mouse retinal development.

PubMed

Fujitani, Yoshio; Fujitani, Shuko; Luo, Huijun; Qiu, Feng; Burlison, Jared; Long, Qiaoming; Kawaguchi, Yoshiya; Edlund, Helena; MacDonald, Raymond J; Furukawa, Takahisa; Fujikado, Takashi; Magnuson, Mark A; Xiang, Mengqing; Wright, Christopher V E

2006-11-01

The vertebrate neural retina comprises six classes of neurons and one class of glial cells, all derived from a population of multipotent progenitors. There is little information on the molecular mechanisms governing the specification of cell type identity from multipotent progenitors in the developing retina. We report that Ptf1a, a basic-helix-loop-helix (bHLH) transcription factor, is transiently expressed by post-mitotic precursors in the developing mouse retina. Recombination-based lineage tracing analysis in vivo revealed that Ptf1a expression marks retinal precursors with competence to exclusively produce horizontal and amacrine neurons. Inactivation of Ptf1a leads to a fate-switch in these precursors that causes them to adopt a ganglion cell fate. This mis-specification of neurons results in a complete loss of horizontal cells, a profound decrease of amacrine cells and an increase in ganglion cells. Furthermore, we identify Ptf1a as a primary downstream target for Foxn4, a forkhead transcription factor involved in the genesis of horizontal and amacrine neurons. These data, together with the previous findings on Foxn4, provide a model in which the Foxn4-Ptf1a pathway plays a central role in directing the differentiation of retinal progenitors towards horizontal and amacrine cell fates.

Microfluidic co-culture platform for investigating osteocyte-osteoclast signalling during fluid shear stress mechanostimulation.

PubMed

Middleton, K; Al-Dujaili, S; Mei, X; Günther, A; You, L

2017-07-05

Bone cells exist in a complex environment where they are constantly exposed to numerous dynamic biochemical and mechanical stimuli. These stimuli regulate bone cells that are involved in various bone disorders, such as osteoporosis. Knowledge of how these stimuli affect bone cells have been utilised to develop various treatments, such as pharmaceuticals, hormone therapy, and exercise. To investigate the role that bone loading has on these disorders in vitro, bone cell mechanotransduction studies are typically performed using parallel plate flow chambers (PPFC). However, these chambers do not allow for dynamic cellular interactions among different cell populations to be investigated. We present a microfluidic approach that exposes different cell populations, which are located at physiologically relevant distances within adjacent channels, to different levels of fluid shear stress, and promotes cell-cell communication between the different channels. We employed this microfluidic system to assess mechanically regulated osteocyte-osteoclast communication. Osteoclast precursors (RAW264.7 cells) responded to cytokine gradients (e.g., RANKL, OPG, PGE-2) developed by both mechanically stimulated (fOCY) and unstimulated (nOCY) osteocyte-like MLO-Y4 cells simultaneously. Specifically, we observed increased osteoclast precursor cell densities and osteoclast differentiation towards nOCY. We also used this system to show an increased mechanoresponse of osteocytes when in co-culture with osteoclasts. We envision broad applicability of the presented approach for microfluidic perfusion co-culture of multiple cell types in the presence of fluid flow stimulation, and as a tool to investigate osteocyte mechanotransduction, as well as bone metastasis extravasation. This system could also be applied to any multi-cell population cross-talk studies that are typically performed using PPFCs (e.g. endothelial cells, smooth muscle cells, and fibroblasts). Copyright © 2017 Elsevier Ltd. All rights reserved.

New melanogenesis and photobiological processes in activation and proliferation of precursor melanocytes after UV-exposure: ultrastructural differentiation of precursor melanocytes from Langerhans cells

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

Jimbow, K.; Uesugi, T.

1982-02-01

Photobiological processes involving new melanogenesis after exposure to ultraviolet (UV) light were experimentally studied in C57 black adult mice by histochemistry, cytochemistry, and autoradiography. The trunk and the plantar region of the foot, where no functioning melanocytes were present before exposure, were exposed to UV-A for 14 consecutive days. Both regions revealed a basically similar pattern for new melanogenesis which involved an activation of precursor melanocytes. Essentially all of ''indeterminate'' cells appeared to be precursor melanocytes, the fine structure of which could be differentiated even from poorly developed Langerhans cells. New melanogenesis was manifested by 4 stages of cellular andmore » subcellular reactions of these cells as indicated by histochemistry of dihydroxyphenylalanine (dopa) and autoradiography of thymidine incorporation: (a) an initial lag in the activation of precursor melanocytes with development of Golgi cisternae and rough endoplasmic reticulum followed by formation of unmelanized melanosomes (day 0 to 2); (b) synthesis of active tyrosinase accumulated in Golgi cisternae and vesicles with subsequent formation of melanized melanosomes in these cells (day 3 to 5); (c) mitotic proliferation of many of these activated cells, followed by an exponential increase of new melanocytes (day 6 to 7); and (d) melanosome transfer with differentiation of 10 nm filaments and arborization of dendrites, but without any significant change in the melanocyte population (day 8 to 14). The melanosome transfer was, however, not obvious until after 7 days of exposure. The size of newly synthesized melanosomes was similar to that of tail skin where native melanocytes were present before exposure.« less

SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.

PubMed

Dubois, Nicole C; Craft, April M; Sharma, Parveen; Elliott, David A; Stanley, Edouard G; Elefanty, Andrew G; Gramolini, Anthony; Keller, Gordon

2011-10-23

To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiac precursors and cardiomyocytes from hESC/hiPSC differentiation cultures, yielding populations of up to 98% cardiac troponin T-positive cells. When plated in culture, SIRPA-positive cells were contracting and could be maintained over extended periods of time. These findings provide a simple method for isolating populations of cardiomyocytes from human pluripotent stem cell cultures, and thereby establish a readily adaptable technology for generating large numbers of enriched cardiomyocytes for therapeutic applications.

A morphological and electrophysiological study on the postnatal development of oligodendrocyte precursor cells in the rat brain.

PubMed

Chen, Peng-hui; Cai, Wen-qin; Wang, Li-yan; Deng, Qi-yue

2008-12-03

A widespread population of cells in CNS is identified by specific expression of the NG2 chondroitin sulphate proteoglycan and named as oligodendrocyte precursor cell (OPC). OPCs may possess stem cell-like characteristics, including multipotentiality in vitro and in vivo. It was proposed that OPCs in the CNS parenchyma comprise a unique population of glia, distinct from oligodendrocytes and astrocytes. This study confirmed that NG2 immunoreactive OPCs were continuously distributed in cerebral cortex and hippocampus during different postnatal developmental stages. These cells rapidly increased in number over the postnatal 7 days and migrate extensively to populate with abundant processes both in developing cortex and hippocampus. The morphology of OPCs exhibited extremely complex changes with the distribution of long distance primary process gradually increased from neonatal to adult CNS. Immunohistochemical studies showed that OPCs exhibited the morphological properties that can be distinguished from astrocytes. The electrophysiological properties showed that OPCs expressed a small amount of inward Na(+) currents which was distinguished from Na(+) currents in neurons owing to their lower Na-to-K conductance ratio and higher command voltage step depolarized maximum Na(+) current amplitude. These observations suggest that OPCs can be identified as the third type of macroglia because of their distribution in the CNS, the morphological development in process diversity and the electrophysiological difference from astrocyte.

Large Population-Based Study Reveals Disparities in Myeloma Precursor Disease | Center for Cancer Research

Cancer.gov

Multiple myeloma (MM) is a cancer of plasma cells, which are antibody-producing white blood cells. Patients with MM have a characteristic excess of monoclonal antibodies, so called M proteins, in their serum, urine, or both and plasma cell infiltration into their bone marrow at multiple sites. African Americans are more than twice as likely as whites to develop MM, but the

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

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

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

2013-11-01

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

Flow cytometric cell cycle analysis of muscle precursor cells cultured within 3D scaffolds in a perfusion bioreactor.

PubMed

Flaibani, Marina; Luni, Camilla; Sbalchiero, Elisa; Elvassore, Nicola

2009-01-01

It has been widely demonstrated that perfusion bioreactors improve in vitro three-dimensional (3D) cultures in terms of high cell density and uniformity of cell distribution; however, the studies reported in literature were primarily based on qualitative analysis (histology, immunofluorescent staining) or on quantitative data averaged on the whole population (DNA assay, PCR). Studies on the behavior, in terms of cell cycle, of a cell population growing in 3D scaffolds in static or dynamic conditions are still absent. In this work, a perfusion bioreactor suitable to culture C(2)C(12) muscle precursor cells within 3D porous collagen scaffolds was designed and developed and a method based on flowcytometric analyses for analyzing the cell cycle in the cell population was established. Cells were extracted by enzymatic digestion of the collagen scaffolds after 4, 7, and 10 days of culture, and flow cytometric live/dead and cell cycle analyses were performed with Propidium Iodide. A live/dead assay was used for validating the method for cell extraction and staining. Moreover, to investigate spatial heterogeneity of the cell population under perfusion conditions, two stacked scaffolds in the 3D domain, of which only the upstream layer was seeded, were analyzed separately. All results were compared with those obtained from static 3D cultures. The live/dead assay revealed the presence of less than 20% of dead cells, which did not affect the cell cycle analysis. Cell cycle analyses highlighted the increment of cell fractions in proliferating phases (S/G(2)/M) owing to medium perfusion in long-term cultures. After 7-10 days, the percentage of proliferating cells was 8-12% for dynamic cultures and 3-5% for the static controls. A higher fraction of proliferating cells was detected in the downstream scaffold. From a general perspective, this method provided data with a small standard deviation and detected the differences between static and dynamic cultures and between upper and lower scaffolds. Our methodology can be extended to other cell types to investigate the influence of 3D culture conditions on the expression of other relevant cell markers.

Neural stem cell apoptosis after low-methylmercury exposures in postnatal hippocampus produce persistent cell loss and adolescent memory deficits.

PubMed

Sokolowski, Katie; Obiorah, Maryann; Robinson, Kelsey; McCandlish, Elizabeth; Buckley, Brian; DiCicco-Bloom, Emanuel

2013-12-01

The developing brain is particularly sensitive to exposures to environmental contaminants. In contrast to the adult, the developing brain contains large numbers of dividing neuronal precursors, suggesting that they may be vulnerable targets. The postnatal day 7 (P7) rat hippocampus has populations of both mature neurons in the CA1-3 region as well as neural stem cells (NSC) in the dentate gyrus (DG) hilus, which actively produce new neurons that migrate to the granule cell layer (GCL). Using this well-characterized NSC population, we examined the impact of low levels of methylmercury (MeHg) on proliferation, neurogenesis, and subsequent adolescent learning and memory behavior. Assessing a range of exposures, we found that a single subcutaneous injection of 0.6 µg/g MeHg in P7 rats induced caspase activation in proliferating NSC of the hilus and GCL. This acute NSC death had lasting impact on the DG at P21, reducing cell numbers in the hilus by 22% and the GCL by 27%, as well as reductions in neural precursor proliferation by 25%. In contrast, non-proliferative CA1-3 pyramidal neuron cell number was unchanged. Furthermore, animals exposed to P7 MeHg exhibited an adolescent spatial memory deficit as assessed by Morris water maze. These results suggest that environmentally relevant levels of MeHg exposure may decrease NSC populations and, despite ongoing neurogenesis, the brain may not restore the hippocampal cell deficits, which may contribute to hippocampal-dependent memory deficits during adolescence. Copyright © 2013 Wiley Periodicals, Inc.

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

PubMed

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

2017-12-15

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

Auditory cortex interneuron development requires cadherins operating hair-cell mechanoelectrical transduction.

PubMed

Libé-Philippot, Baptiste; Michel, Vincent; Boutet de Monvel, Jacques; Le Gal, Sébastien; Dupont, Typhaine; Avan, Paul; Métin, Christine; Michalski, Nicolas; Petit, Christine

2017-07-25

Many genetic forms of congenital deafness affect the sound reception antenna of cochlear sensory cells, the hair bundle. The resulting sensory deprivation jeopardizes auditory cortex (AC) maturation. Early prosthetic intervention should revive this process. Nevertheless, this view assumes that no intrinsic AC deficits coexist with the cochlear ones, a possibility as yet unexplored. We show here that many GABAergic interneurons, from their generation in the medial ganglionic eminence up to their settlement in the AC, express two cadherin-related (cdhr) proteins, cdhr23 and cdhr15, that form the hair bundle tip links gating the mechanoelectrical transduction channels. Mutant mice lacking either protein showed a major decrease in the number of parvalbumin interneurons specifically in the AC, and displayed audiogenic reflex seizures. Cdhr15 - and Cdhr23 -expressing interneuron precursors in Cdhr23 -/- and Cdhr15 -/- mouse embryos, respectively, failed to enter the embryonic cortex and were scattered throughout the subpallium, consistent with the cell polarity abnormalities we observed in vitro. In the absence of adhesion G protein-coupled receptor V1 (adgrv1), another hair bundle link protein, the entry of Cdhr23 - and Cdhr15 -expressing interneuron precursors into the embryonic cortex was also impaired. Our results demonstrate that a population of newborn interneurons is endowed with specific cdhr proteins necessary for these cells to reach the developing AC. We suggest that an "early adhesion code" targets populations of interneuron precursors to restricted neocortical regions belonging to the same functional area. These findings open up new perspectives for auditory rehabilitation and cortical therapies in patients.

Muscle satellite cell heterogeneity and self-renewal

PubMed Central

Motohashi, Norio; Asakura, Atsushi

2014-01-01

Adult skeletal muscle possesses extraordinary regeneration capacities. After muscle injury or exercise, large numbers of newly formed muscle fibers are generated within a week as a result of expansion and differentiation of a self-renewing pool of muscle stem cells termed muscle satellite cells. Normally, satellite cells are mitotically quiescent and reside beneath the basal lamina of muscle fibers. Upon regeneration, satellite cells are activated, and give rise to daughter myogenic precursor cells. After several rounds of proliferation, these myogenic precursor cells contribute to the formation of new muscle fibers. During cell division, a minor population of myogenic precursor cells returns to quiescent satellite cells as a self-renewal process. Currently, accumulating evidence has revealed the essential roles of satellite cells in muscle regeneration and the regulatory mechanisms, while it still remains to be elucidated how satellite cell self-renewal is molecularly regulated and how satellite cells are important in aging and diseased muscle. The number of satellite cells is decreased due to the changing niche during ageing, resulting in attenuation of muscle regeneration capacity. Additionally, in Duchenne muscular dystrophy (DMD) patients, the loss of satellite cell regenerative capacity and decreased satellite cell number due to continuous needs for satellite cells lead to progressive muscle weakness with chronic degeneration. Thus, it is necessary to replenish muscle satellite cells continuously. This review outlines recent findings regarding satellite cell heterogeneity, asymmetric division and molecular mechanisms in satellite cell self-renewal which is crucial for maintenance of satellite cells as a muscle stem cell pool throughout life. In addition, we discuss roles in the stem cell niche for satellite cell maintenance, as well as related cell therapies for approaching treatment of DMD. PMID:25364710

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.

Endogenous stem cell proliferation induced by intravenous hedgehog agonist administration after contusion in the adult rat spinal cord.

PubMed

Bambakidis, Nicholas C; Horn, Eric M; Nakaji, Peter; Theodore, Nicholas; Bless, Elizabeth; Dellovade, Tammy; Ma, Chiyuan; Wang, Xukui; Preul, Mark C; Coons, Stephen W; Spetzler, Robert F; Sonntag, Volker K H

2009-02-01

Sonic hedgehog (Shh) is a glycoprotein molecule that upregulates the transcription factor Gli1. The Shh protein plays a critical role in the proliferation of endogenous neural precursor cells when directly injected into the spinal cord after a spinal cord injury in adult rodents. Small-molecule agonists of the hedgehog (Hh) pathway were used in an attempt to reproduce these findings through intravenous administration. The expression of Gli1 was measured in rat spinal cord after the intravenous administration of an Hh agonist. Ten adult rats received a moderate contusion and were treated with either an Hh agonist (10 mg/kg, intravenously) or vehicle (5 rodents per group) 1 hour and 4 days after injury. The rats were killed 5 days postinjury. Tissue samples were immediately placed in fixative. Samples were immunohistochemically stained for neural precursor cells, and these cells were counted. Systemic dosing with an Hh agonist significantly upregulated Gli1 expression in the spinal cord (p < 0.005). After spinal contusion, animals treated with the Hh agonist had significantly more nestin-positive neural precursor cells around the rim of the lesion cavity than in vehicle-treated controls (means +/- SDs, 46.9 +/- 12.9 vs 20.9 +/- 8.3 cells/hpf, respectively, p < 0.005). There was no significant difference in the area of white matter injury between the groups. An intravenous Hh agonist at doses that upregulate spinal cord Gli1 transcription also increases the population of neural precursor cells after spinal cord injury in adult rats. These data support previous findings based on injections of Shh protein directly into the spinal cord.

Increasing magnetite contents of polymeric magnetic particles dramatically improves labeling of neural stem cell transplant populations.

PubMed

Adams, Christopher F; Rai, Ahmad; Sneddon, Gregor; Yiu, Humphrey H P; Polyak, Boris; Chari, Divya M

2015-01-01

Safe and efficient delivery of therapeutic cells to sites of injury/disease in the central nervous system is a key goal for the translation of clinical cell transplantation therapies. Recently, 'magnetic cell localization strategies' have emerged as a promising and safe approach for targeted delivery of magnetic particle (MP) labeled stem cells to pathology sites. For neuroregenerative applications, this approach is limited by the lack of available neurocompatible MPs, and low cell labeling achieved in neural stem/precursor populations. We demonstrate that high magnetite content, self-sedimenting polymeric MPs [unfunctionalized poly(lactic acid) coated, without a transfecting component] achieve efficient labeling (≥90%) of primary neural stem cells (NSCs)-a 'hard-to-label' transplant population of major clinical relevance. Our protocols showed high safety with respect to key stem cell regenerative parameters. Critically, labeled cells were effectively localized in an in vitro flow system by magnetic force highlighting the translational potential of the methods used. Copyright © 2015 Elsevier Inc. All rights reserved.

Building the mammalian testis: origins, differentiation, and assembly of the component cell populations

PubMed Central

Svingen, Terje; Koopman, Peter

2013-01-01

Development of testes in the mammalian embryo requires the formation and assembly of several cell types that allow these organs to achieve their roles in male reproduction and endocrine regulation. Testis development is unusual in that several cell types such as Sertoli, Leydig, and spermatogonial cells arise from bipotential precursors present in the precursor tissue, the genital ridge. These cell types do not differentiate independently but depend on signals from Sertoli cells that differentiate under the influence of transcription factors SRY and SOX9. While these steps are becoming better understood, the origins and roles of many testicular cell types and structures—including peritubular myoid cells, the tunica albuginea, the arterial and venous blood vasculature, lymphatic vessels, macrophages, and nerve cells—have remained unclear. This review synthesizes current knowledge of how the architecture of the testis unfolds and highlights the questions that remain to be explored, thus providing a roadmap for future studies that may help illuminate the causes of XY disorders of sex development, infertility, and testicular cancers. PMID:24240231

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.

Laboratory management of cervical intraepithelial neoplasia: proposing a new paradigm.

PubMed

Herfs, Michael; Crum, Christopher P

2013-03-01

Since the discovery of human papillomavirus (HPV) type 16 in early 80s, the link between HPV and cervical cancer has been established with certainty, a function of the discovery and cloning of a range of HPV types associated with both cancer precursors (cervical intraepithelial neoplasia or CIN) and carcinomas and extensive epidemiologic, clinical, pathologic, and experimental data. These accumulated results have culminated in new paradigms of cancer prevention through screening and triage. Despite this, the management of women with CIN is still suboptimal and the overtreatment of these conditions still occurs, largely due to the lack of clarity regarding which precancerous lesions are most likely to progress in grade. Recently, a discrete population of cuboidal cells was discovered at the cervical squamocolumnar junction, the anatomic site where the large majority of HPV-related (pre)neoplastic lesions develop. These cells seem to be embryonic in nature and participate both in benign metaplasias and the initial phase of precancer development. This review summarizes the historical evolution of precursor management, assesses the potential role of this and other discoveries in segregating lower from higher-risk precursors, and examines their potential impact on the management of women with real or potential cervical cancer precursors.

Cell size control and a cell-intrinsic maturation program in proliferating oligodendrocyte precursor cells.

PubMed

Gao, F B; Raff, M

1997-09-22

We have used clonal analysis and time-lapse video recording to study the proliferative behavior of purified oligodendrocyte precursor cells isolated from the perinatal rat optic nerve growing in serum-free cultures. First, we show that the cell cycle time of precursor cells decreases with increasing concentrations of PDGF, the main mitogen for these cells, suggesting that PDGF levels may regulate the cell cycle time during development. Second, we show that precursor cells isolated from embryonic day 18 (E18) nerves differ from precursor cells isolated from postnatal day 7 (P7) or P14 nerves in a number of ways: they have a simpler morphology, and they divide faster and longer before they stop dividing and differentiate into postmitotic oligodendrocytes. Third, we show that purified E18 precursor cells proliferating in culture progressively change their properties to resemble postnatal cells, suggesting that progressive maturation is an intrinsic property of the precursors. Finally, we show that precursor cells, especially mature ones, sometimes divide unequally, such that one daughter cell is larger than the other; in each of these cases the larger daughter cell divides well before the smaller one, suggesting that the precursor cells, just like single-celled eucaryotes, have to reach a threshold size before they can divide. These and other findings raise the possibility that such stochastic unequal divisions, rather than the stochastic events occurring in G1 proposed by "transition probability" models, may explain the random variability of cell cycle times seen within clonal cell lines in culture.

Cell Size Control and a Cell-intrinsic Maturation Program in Proliferating Oligodendrocyte Precursor Cells

PubMed Central

Gao, Fen-Biao; Raff, Martin

1997-01-01

We have used clonal analysis and time-lapse video recording to study the proliferative behavior of purified oligodendrocyte precursor cells isolated from the perinatal rat optic nerve growing in serum-free cultures. First, we show that the cell cycle time of precursor cells decreases with increasing concentrations of PDGF, the main mitogen for these cells, suggesting that PDGF levels may regulate the cell cycle time during development. Second, we show that precursor cells isolated from embryonic day 18 (E18) nerves differ from precursor cells isolated from postnatal day 7 (P7) or P14 nerves in a number of ways: they have a simpler morphology, and they divide faster and longer before they stop dividing and differentiate into postmitotic oligodendrocytes. Third, we show that purified E18 precursor cells proliferating in culture progressively change their properties to resemble postnatal cells, suggesting that progressive maturation is an intrinsic property of the precursors. Finally, we show that precursor cells, especially mature ones, sometimes divide unequally, such that one daughter cell is larger than the other; in each of these cases the larger daughter cell divides well before the smaller one, suggesting that the precursor cells, just like single-celled eucaryotes, have to reach a threshold size before they can divide. These and other findings raise the possibility that such stochastic unequal divisions, rather than the stochastic events occurring in G1 proposed by “transition probability” models, may explain the random variability of cell cycle times seen within clonal cell lines in culture. PMID:9298991

C-kit+ cells isolated from developing kidneys are a novel population of stem cells with regenerative potential

PubMed Central

Rangel, Erika B; Gomes, Samirah A; Dulce, Raul A; Premer, Courtney; Rodrigues, Claudia O; Kanashiro-Takeuchi, Rosemeire M; Oskouei, Behzad; Carvalho, Decio A; Ruiz, Phillip; Reiser, Jochen; Hare, Joshua M

2013-01-01

The presence of tissue specific precursor cells is an emerging concept in organ formation and tissue homeostasis. Several progenitors are described in the kidneys. However, their identity as a true stem cell remains elusive. Here, we identify a neonatal kidney-derived c-kit+ cell population that fulfills all of the criteria as a stem cell. These cells were found in the thick ascending limb of Henle's loop and exhibited clonogenicity, self-renewal, and multipotentiality with differentiation capacity into mesoderm and ectoderm progeny. Additionally, c-kit+ cells formed spheres in nonadherent conditions when plated at clonal density and expressed markers of stem cells, progenitors, and differentiated cells. Ex-vivo expanded c-kit+ cells integrated into several compartments of the kidney, including tubules, vessels, and glomeruli, and contributed to functional and morphological improvement of the kidney following acute ischemia-reperfusion injury in rats. Together these findings document a novel neonatal rat kidney c-kit+ stem cell population that can be isolated, expanded, cloned, differentiated, and employed for kidney repair following acute kidney injury. These cells have important biological and therapeutic implications. PMID:23733311

Long-lived self-renewing bone marrow-derived macrophages displace embryo-derived cells to inhabit adult serous cavities

PubMed Central

Bain, Calum C.; Hawley, Catherine A.; Garner, Hannah; Scott, Charlotte L.; Schridde, Anika; Steers, Nicholas J.; Mack, Matthias; Joshi, Anagha; Guilliams, Martin; Mowat, Allan Mc I.; Geissmann, Frederic; Jenkins, Stephen J.

2016-01-01

Peritoneal macrophages are one of the most studied macrophage populations in the body, yet the composition, developmental origin and mechanisms governing the maintenance of this compartment are controversial. Here we show resident F4/80hiGATA6+ macrophages are long-lived, undergo non-stochastic self-renewal and retain cells of embryonic origin for at least 4 months in mice. However, Ly6C+ monocytes constitutively enter the peritoneal cavity in a CCR2-dependent manner, where they mature into short-lived F4/80loMHCII+ cells that act, in part, as precursors of F4/80hiGATA6+ macrophages. Notably, monocyte-derived F4/80hi macrophages eventually displace the embryonic population with age in a process that is highly gender dependent and not due to proliferative exhaustion of the incumbent embryonic population, despite the greater proliferative activity of newly recruited cells. Furthermore, although monocyte-derived cells acquire key characteristics of the embryonic population, expression of Tim4 was impaired, leading to cumulative changes in the population with age. PMID:27292029

Evidence of heterogeneity within bovine satellite cells isolated from young and adult animals.

PubMed

Li, J; Gonzalez, J M; Walker, D K; Hersom, M J; Ealy, A D; Johnson, S E

2011-06-01

Satellite cells are a heterogeneous population of myogenic precursors responsible for muscle growth and repair in mammals. The objectives of the experiment were to examine the growth rates and degree of heterogeneity within bovine satellite cells (BSC) isolated from young and adult animals. The BSC were harvested from the semimembranosus of young (4.3 ± 0.5 d) and adult (estimated 24 to 27 mo) cattle and cultured en masse. Young animal BSC re-enter the cell cycle sooner and reach maximal 5-ethynyl-2'-deoxyuridine (EdU) incorporation earlier (P < 0.05) than adult contemporaries. Adult BSC contain fewer (P < 0.05) MyoD and myogenin immunopositive nuclei than BSC isolated from young animals after 3, 4, and 5 d in culture. These results indicate that BSC from young animals activate, proliferate, and differentiate sooner than isolates from adult animals. Lineage heterogeneity within BSC was examined using antibodies specific for Pax7 and Myf5, lineage markers of satellite cells, and myoblasts. Immunocytochemistry revealed the majority of Pax7-expressing BSC also express Myf5; a minor population (~5%) fails to exhibit Myf5 immunoreactivity. The percentage of Pax7:Myf5 BSC from young animals decreases sooner (P < 0.05) in culture than adult BSC, indicating a more rapid rate of muscle fiber formation. A subpopulation immunopositive for Myf5 only was identified in both ages of BSC isolates. The growth kinetics and heterogeneity of young BSC was further evaluated by clonal analysis. Single cell clones were established and analyzed after 10 d. Colonies segregated into 2 groups based upon population doubling time. Immunostaining of the slow-growing colonies (population doubling time ≥ 3 d) revealed that a portion exhibited asymmetric distribution of the lineage markers Pax7 and Myf5, similar to self-renewable mouse muscle stem cells. In summary, these results offer insight into the heterogeneity of BSC and provide evidence for subtle differences between rodent and bovine myogenic precursors.

Processing, distribution, and function of VGF, a neuronal and endocrine peptide precursor.

PubMed

Levi, Andrea; Ferri, Gian-Luca; Watson, Elizabeth; Possenti, Roberta; Salton, Stephen R J

2004-08-01

1. The vgf gene encodes a neuropeptide precursor with a restricted pattern of expression that is limited to a subset of neurons in the central and peripheral nervous systems and to specific populations of endocrine cells in the adenohypophysis, adrenal medulla, gastrointestinal tract, and pancreas. In responsive neurons, vgf transcription is upregulated by neurotrophins. the basis for the original identification of VGF as nerve growth factor- (NGF) inducible in PC12 cells (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985). 2. In this review, we shall summarize data concerning the transcriptional regulation of vgf in vitro, the structural organization of the vgf promoter as well as the transcription factors which regulate its activity. 3. On the basis of in situ hybridization and immunohistochemical studies, the in vivo tissue-specific expression of VGF during differentiation and in the adult will be summarized. 4. Parallel biochemical data will be reviewed, addressing the proteolytical processing of the pro-VGF precursor within the secretory compartment of neuroendocrine cells. 5. Finally, analysis of the phenotype of VGF knockout mice will be discussed, implying a nonredundant role of VGF products in the regulation of energy storage and expenditure.

Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties

PubMed Central

Gudjonsson, Thorarinn; Villadsen, René; Nielsen, Helga Lind; Rønnov-Jessen, Lone; Bissell, Mina J.; Petersen, Ole William

2002-01-01

The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting, we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC+) and epithelial-specific antigen (ESA+) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC−/ESA+). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins, claudin-1 and occludin, and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures, the MUC+/ESA+ epithelial cell line was luminal epithelial restricted in its differentiation repertoire, the suprabasal-derived MUC−/ESA+ epithelial cell line was able to generate itself as well as MUC+/ESA+ epithelial cells and Thy-1+/α-smooth muscle actin+ (ASMA+) myoepithelial cells. The MUC−/ESA+ epithelial cell line further differed from the MUC+/ESA+ epithelial cell line by the expression of keratin K19, a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane, the MUC+/ESA+ epithelial cell line formed acinus-like spheres. In contrast, the MUC−/ESA+ epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by inoculating the extracellular matrix-embedded cells subcutaneously in nude mice. Thus, MUC−/ESA+ epithelial cells within the luminal epithelial lineage may function as precursor cells of terminal duct lobular units in the human breast. PMID:11914275

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

Identification of early B cell precursors (stage 1 and 2 hematogones) in the peripheral blood.

PubMed

Kurzer, Jason H; Weinberg, Olga K

2018-05-25

Differentiating malignant B-lymphoblasts from early benign B cell precursors (hematogones) is a vital component of the diagnosis of B-lymphoblastic leukaemia. It has been previously reported that only late-stage B cell precursors circulate in the peripheral blood. Consequently, flow cytometric detection of cells with immunophenotypic findings similar to earlier stage precursors in the peripheral blood justifiably raises concern for involvement by B-lymphoblastic leukaemia. We report here, however, that benign early B cell precursors can indeed be detected in the peripheral blood, thus complicating the interpretation of flow cytometric findings derived from these sample types. A retrospective search of our collective databases identified 13 cases containing circulating early stage B cell precursors. The patients ranged in age from 15 days to 85 years old. All positive cases demonstrated that the earlier B cell precursors were associated with later stage precursors, a finding that could help differentiate these cells from B-lymphoblastic leukaemia. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Single-cell RNA-seq and computational analysis using temporal mixture modelling resolves Th1/Tfh fate bifurcation in malaria

PubMed Central

Lönnberg, Tapio; Svensson, Valentine; James, Kylie R.; Fernandez-Ruiz, Daniel; Sebina, Ismail; Montandon, Ruddy; Soon, Megan S. F.; Fogg, Lily G.; Nair, Arya Sheela; Liligeto, Urijah; Stubbington, Michael J. T.; Ly, Lam-Ha; Bagger, Frederik Otzen; Zwiessele, Max; Lawrence, Neil D.; Souza-Fonseca-Guimaraes, Fernando; Bunn, Patrick T.; Engwerda, Christian R.; Heath, William R.; Billker, Oliver; Stegle, Oliver; Haque, Ashraful; Teichmann, Sarah A.

2017-01-01

Differentiation of naïve CD4+ T cells into functionally distinct T helper subsets is crucial for the orchestration of immune responses. Due to extensive heterogeneity and multiple overlapping transcriptional programs in differentiating T cell populations, this process has remained a challenge for systematic dissection in vivo. By using single-cell transcriptomics and computational analysis using a temporal mixtures of Gaussian processes model, termed GPfates, we reconstructed the developmental trajectories of Th1 and Tfh cells during blood-stage Plasmodium infection in mice. By tracking clonality using endogenous TCR sequences, we first demonstrated that Th1/Tfh bifurcation had occurred at both population and single-clone levels. Next, we identified genes whose expression was associated with Th1 or Tfh fates, and demonstrated a T-cell intrinsic role for Galectin-1 in supporting a Th1 differentiation. We also revealed the close molecular relationship between Th1 and IL-10-producing Tr1 cells in this infection. Th1 and Tfh fates emerged from a highly proliferative precursor that upregulated aerobic glycolysis and accelerated cell cycling as cytokine expression began. Dynamic gene expression of chemokine receptors around bifurcation predicted roles for cell-cell in driving Th1/Tfh fates. In particular, we found that precursor Th cells were coached towards a Th1 but not a Tfh fate by inflammatory monocytes. Thus, by integrating genomic and computational approaches, our study has provided two unique resources, a database www.PlasmoTH.org, which facilitates discovery of novel factors controlling Th1/Tfh fate commitment, and more generally, GPfates, a modelling framework for characterizing cell differentiation towards multiple fates. PMID:28345074

Maturation of Human Embryonic Stem Cell–Derived Pancreatic Progenitors Into Functional Islets Capable of Treating Pre-existing Diabetes in Mice

PubMed Central

Rezania, Alireza; Bruin, Jennifer E.; Riedel, Michael J.; Mojibian, Majid; Asadi, Ali; Xu, Jean; Gauvin, Rebecca; Narayan, Kavitha; Karanu, Francis; O’Neil, John J.; Ao, Ziliang; Warnock, Garth L.

2012-01-01

Diabetes is a chronic debilitating disease that results from insufficient production of insulin from pancreatic β-cells. Islet cell replacement can effectively treat diabetes but is currently severely limited by the reliance upon cadaveric donor tissue. We have developed a protocol to efficiently differentiate commercially available human embryonic stem cells (hESCs) in vitro into a highly enriched PDX1+ pancreatic progenitor cell population that further develops in vivo to mature pancreatic endocrine cells. Immature pancreatic precursor cells were transplanted into immunodeficient mice with streptozotocin-induced diabetes, and glycemia was initially controlled with exogenous insulin. As graft-derived insulin levels increased over time, diabetic mice were weaned from exogenous insulin and human C-peptide secretion was eventually regulated by meal and glucose challenges. Similar differentiation of pancreatic precursor cells was observed after transplant in immunodeficient rats. Throughout the in vivo maturation period hESC-derived endocrine cells exhibited gene and protein expression profiles that were remarkably similar to the developing human fetal pancreas. Our findings support the feasibility of using differentiated hESCs as an alternative to cadaveric islets for treating patients with diabetes. PMID:22740171

IMMUNOLOGIC MEMORY CELLS OF BONE MARROW ORIGIN

PubMed Central

Miller, Harold C.; Cudkowicz, Gustavo

1972-01-01

Individual immunocompetent precursor cells of (C57BL/10 x C3H)F1 mouse marrow generate, on transplantation, three to five times more antibody-forming cells localized in recipient spleens during secondary than during primary immune responses. The increased burst size is immunologically specific since antigens of horse and chicken erythrocytes and of Salmonella typhimurium do not cause this effect in marrow cells responsive to sheep red blood cells. Both sensitized and nonsensitized precursors require the helper function of thymus-derived cells and antigen for the final steps of differentiation and maturation. The burst size of primed precursor cells is the same after cooperative interactions with virgin or educated helper cells of thymic origin. The greater potential of these marrow precursors may be attributable to self-replication and migration before differentiation into antibody-forming descendants. In fact, the progeny cells of primed precursor units are distributed among a multiplicity of foci, whereas those of nonimmune precursors are clustered into one focus. The described properties of specifically primed marrow precursors are those underlying immunologic memory. It remains to be established whether memory cells are induced or selected by antigens and whether the thymus plays a role in this process. PMID:4553850

TET2 mutations in B cells of patients affected by angioimmunoblastic T-cell lymphoma.

PubMed

Schwartz, Friederike H; Cai, Qian; Fellmann, Eva; Hartmann, Sylvia; Mäyränpää, Mikko I; Karjalainen-Lindsberg, Marja-Liisa; Sundström, Christer; Scholtysik, René; Hansmann, Martin-Leo; Küppers, Ralf

2017-06-01

Angioimmunoblastic T-cell lymphomas (AITLs) frequently carry mutations in the TET2 and IDH2 genes. TET2 mutations represent early genetic lesions as they had already been detected in haematopoietic precursor cells of AITL patients. We show by analysis of whole-tissue sections and microdissected PD1 + cells that the frequency of TET2-mutated AITL is presumably even higher than reported (12/13 cases in our collection; 92%). In two-thirds of informative AITLs (6/9), a fraction of B cells was also TET2-mutated. Investigation of four AITLs by TET2 and IGHV gene sequencing of single microdissected B cells showed that between 10% and 60% of polyclonal B cells in AITL lymph nodes harboured the identical TET2 mutations of the respective T-cell lymphoma clone. Thus, TET2-mutated haematopoietic precursor cells in AITL patients not only give rise to the T-cell lymphoma but also generate a large population of mutated mature B cells. Future studies will show whether this is a reason why AITL patients frequently also develop B-cell lymphomas. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pluripotent stem cell-derived natural killer cells for cancer therapy

PubMed Central

Knorr, David A.; Kaufman, Dan S.

2010-01-01

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide an accessible, genetically tractable and homogenous starting cell populations to efficiently study human blood cell development. These cell populations provide platforms to develop new cell-based therapies to treat both malignant and non-malignant hematological diseases. Our group has previously demonstrated the ability of hESC-derived hematopoietic precursors to produce functional natural killer (NK) cells as well as an explanation of the underlying mechanism responsible for inefficient development of T and B cells from hESCs. hESCs and iPSCs, which can be reliably engineered in vitro, provide an important new model system to study human lymphocyte development and produce enhanced cell-based therapies with potential to serve as a “universal” source of anti-tumor lymphocytes for novel clinical therapies. This review will focus on the application of hESC-derived NK cells with currently used and novel therapeutics for clinical trials, current barriers to translation, and future applications through genetic engineering approaches. PMID:20801411

[The role of endothelial cells and endothelial precursor cells in angiogenesis].

PubMed

Poreba, Małgorzata; Usnarska-Zubkiewicz, Lidia; Kuliczkowski, Kazimierz

2006-01-01

Endothelium plays a key role in maintenance of vascular homeostasis in human organism. According to new data endothelial cells and hematopoietic cells have a common precursor in prenatal life--a hemangioblast, which explains the fact of sharing the same determinants on the surface of both type of cells. Circulating endothelial precursors were identified in adults and this suggests that hemangioblasts may be present not only during embriogenesis. In some clinical situations the increased numbers of endothelial cells and endothelial precursors were noted, and especially in patients with neoplastic diseases, which is probably the result of increased angiogenesis. Endothelial precursors are thought to be the promice for therapeutic purposes in future--to increase local angiogenesis.

SomethiNG 2 talk about-Transcriptional regulation in embryonic and adult oligodendrocyte precursors.

PubMed

Küspert, Melanie; Wegner, Michael

2016-05-01

Glial cells that express the chondroitin sulfate proteoglycan NG2 represent an inherently heterogeneous population. These so-called NG2-glia are present during development and in the adult CNS, where they are referred to as embryonic oligodendrocyte precursors and adult NG2-glia, respectively. They give rise to myelinating oligodendrocytes at all times of life. Over the years much has been learnt about the transcriptional network in embryonic oligodendrocyte precursors, and several transcription factors from the HLH, HMG-domain, zinc finger and homeodomain protein families have been identified as main constituents. Much less is known about the corresponding network in adult NG2-glia. Here we summarize and discuss current knowledge on functions of each of these transcription factor families in NG2-glia, and where possible compare transcriptional regulation in embryonic oligodendrocyte precursors and adult NG2-glia. This article is part of a Special Issue entitled SI:NG2-glia (Invited only). Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed

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

2004-12-06

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

Phenytoin enhances the phosphorylation of epidermal growth factor receptor and fibroblast growth factor receptor in the subventricular zone and promotes the proliferation of neural precursor cells and oligodendrocyte differentiation.

PubMed

Galvez-Contreras, Alma Y; Gonzalez-Castaneda, Rocio E; Campos-Ordonez, Tania; Luquin, Sonia; Gonzalez-Perez, Oscar

2016-01-01

Phenytoin is a widely used antiepileptic drug that induces cell proliferation in several tissues, such as heart, bone, skin, oral mucosa and neural precursors. Some of these effects are mediated via fibroblast growth factor receptor (FGFR) and epidermal growth factor receptor (EGFR). These receptors are strongly expressed in the adult ventricular-subventricular zone (V-SVZ), the main neurogenic niche in the adult brain. The aim of this study was to determine the cell lineage and cell fate of V-SVZ neural progenitors expanded by phenytoin, as well as the effects of this drug on EGFR/FGFR phosphorylation. Male BALB/C mice received 10 mg/kg phenytoin by oral cannula for 30 days. We analysed the proliferation of V-SVZ neural progenitors by immunohistochemistry and western blot. Our findings indicate that phenytoin enhanced twofold the phosphorylation of EGFR and FGFR in the V-SVZ, increased the number of bromodeoxyuridine (BrdU)+/Sox2+ and BrdU+/doublecortin+ cells in the V-SVZ, and expanded the population of Olig2-expressing cells around the lateral ventricles. After phenytoin removal, a large number of BrdU+/Receptor interacting protein (RIP)+ cells were observed in the olfactory bulb. In conclusion, phenytoin enhanced the phosphorylation of FGFR and EGFR, and promoted the expression of neural precursor markers in the V-SVZ. In parallel, the number of oligodendrocytes increased significantly after phenytoin removal. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Skin-derived neural precursors competitively generate functional myelin in adult demyelinated mice

PubMed Central

Mozafari, Sabah; Laterza, Cecilia; Roussel, Delphine; Bachelin, Corinne; Marteyn, Antoine; Deboux, Cyrille; Martino, Gianvito; Evercooren, Anne Baron-Van

2015-01-01

Induced pluripotent stem cell–derived (iPS-derived) neural precursor cells may represent the ideal autologous cell source for cell-based therapy to promote remyelination and neuroprotection in myelin diseases. So far, the therapeutic potential of reprogrammed cells has been evaluated in neonatal demyelinating models. However, the repair efficacy and safety of these cells has not been well addressed in the demyelinated adult CNS, which has decreased cell plasticity and scarring. Moreover, it is not clear if these induced pluripotent–derived cells have the same reparative capacity as physiologically committed CNS-derived precursors. Here, we performed a side-by-side comparison of CNS-derived and skin-derived neural precursors in culture and following engraftment in murine models of adult spinal cord demyelination. Grafted induced neural precursors exhibited a high capacity for survival, safe integration, migration, and timely differentiation into mature bona fide oligodendrocytes. Moreover, grafted skin–derived neural precursors generated compact myelin around host axons and restored nodes of Ranvier and conduction velocity as efficiently as CNS-derived precursors while outcompeting endogenous cells. Together, these results provide important insights into the biology of reprogrammed cells in adult demyelinating conditions and support use of these cells for regenerative biomedicine of myelin diseases that affect the adult CNS. PMID:26301815

Immature MEF2C-dysregulated T-cell leukemia patients have an early T-cell precursor acute lymphoblastic leukemia gene signature and typically have non-rearranged T-cell receptors

PubMed Central

Zuurbier, Linda; Gutierrez, Alejandro; Mullighan, Charles G.; Canté-Barrett, Kirsten; Gevaert, A. Olivier; de Rooi, Johan; Li, Yunlei; Smits, Willem K.; Buijs-Gladdines, Jessica G.C.A.M.; Sonneveld, Edwin; Look, A. Thomas; Horstmann, Martin; Pieters, Rob; Meijerink, Jules P.P.

2014-01-01

Three distinct immature T-cell acute lymphoblastic leukemia entities have been described including cases that express an early T-cell precursor immunophenotype or expression profile, immature MEF2C-dysregulated T-cell acute lymphoblastic leukemia cluster cases based on gene expression analysis (immature cluster) and cases that retain non-rearranged TRG@ loci. Early T-cell precursor acute lymphoblastic leukemia cases exclusively overlap with immature cluster samples based on the expression of early T-cell precursor acute lymphoblastic leukemia signature genes, indicating that both are featuring a single disease entity. Patients lacking TRG@ rearrangements represent only 40% of immature cluster cases, but no further evidence was found to suggest that cases with absence of bi-allelic TRG@ deletions reflect a distinct and even more immature disease entity. Immature cluster/early T-cell precursor acute lymphoblastic leukemia cases are strongly enriched for genes expressed in hematopoietic stem cells as well as genes expressed in normal early thymocyte progenitor or double negative-2A T-cell subsets. Identification of early T-cell precursor acute lymphoblastic leukemia cases solely by defined immunophenotypic criteria strongly underestimates the number of cases that have a corresponding gene signature. However, early T-cell precursor acute lymphoblastic leukemia samples correlate best with a CD1 negative, CD4 and CD8 double negative immunophenotype with expression of CD34 and/or myeloid markers CD13 or CD33. Unlike various other studies, immature cluster/early T-cell precursor acute lymphoblastic leukemia patients treated on the COALL-97 protocol did not have an overall inferior outcome, and demonstrated equal sensitivity levels to most conventional therapeutic drugs compared to other pediatric T-cell acute lymphoblastic leukemia patients. PMID:23975177

Defining the Diverse Cell Populations Contributing to Lignification in Arabidopsis Stems.

PubMed

Smith, Rebecca A; Schuetz, Mathias; Karlen, Steven D; Bird, David; Tokunaga, Naohito; Sato, Yasushi; Mansfield, Shawn D; Ralph, John; Samuels, A Lacey

2017-06-01

Many land plants evolved tall and sturdy growth habits due to specialized cells with thick lignified cell walls: tracheary elements that function in water transport and fibers that function in structural support. The objective of this study was to define how and when diverse cell populations contribute lignin precursors, monolignols, to secondary cell walls during lignification of the Arabidopsis ( Arabidopsis thaliana ) inflorescence stem. Previous work demonstrated that, when lignin biosynthesis is suppressed in fiber and tracheary element cells with thickened walls, fibers become lignin-depleted while vascular bundles still lignify, suggesting that nonlignifying neighboring xylem cells are contributing to lignification. In this work, we dissect the contributions of different cell types, specifically xylary parenchyma and fiber cells, to lignification of the stem using cell-type-specific promoters to either knock down an essential monolignol biosynthetic gene or to introduce novel monolignol conjugates. Analysis of either reductions in lignin in knockdown lines, or the addition of novel monolignol conjugates, directly identifies the xylary parenchyma and fiber cell populations that contribute to the stem lignification and the developmental timing at which each contribution is most important. © 2017 American Society of Plant Biologists. All Rights Reserved.

Cancer cell death induced by the intracellular self-assembly of an enzyme-responsive supramolecular gelator.

PubMed

Tanaka, Akiko; Fukuoka, Yuki; Morimoto, Yuka; Honjo, Takafumi; Koda, Daisuke; Goto, Masahiro; Maruyama, Tatsuo

2015-01-21

We report cancer cell death initiated by the intracellular molecular self-assembly of a peptide lipid, which was derived from a gelator precursor. The gelator precursor was designed to form nanofibers via molecular self-assembly, after cleavage by a cancer-related enzyme (matrix metalloproteinase-7, MMP-7), leading to hydrogelation. The gelator precursor exhibited remarkable cytotoxicity to five different cancer cell lines, while the precursor exhibited low cytotoxicity to normal cells. Cancer cells secrete excessive amounts of MMP-7, which converted the precursor into a supramolecular gelator prior to its uptake by the cells. Once inside the cells, the supramolecular gelator formed a gel via molecular self-assembly, exerting vital stress on the cancer cells. The present study thus describes a new drug where molecular self-assembly acts as the mechanism of cytotoxicity.

Endogenous oncogenic Nras mutation promotes aberrant GM-CSF signaling in granulocytic/monocytic precursors in a murine model of chronic myelomonocytic leukemia.

PubMed

Wang, Jinyong; Liu, Yangang; Li, Zeyang; Du, Juan; Ryu, Myung-Jeom; Taylor, Philip R; Fleming, Mark D; Young, Ken H; Pitot, Henry; Zhang, Jing

2010-12-23

Oncogenic NRAS mutations are frequently identified in myeloid diseases involving monocyte lineage. However, its role in the genesis of these diseases remains elusive. We report a mouse bone marrow transplantation model harboring an oncogenic G12D mutation in the Nras locus. Approximately 95% of recipient mice develop a myeloproliferative disease resembling the myeloproliferative variant of chronic myelomonocytic leukemia (CMML), with a prolonged latency and acquisition of multiple genetic alterations, including uniparental disomy of oncogenic Nras allele. Based on single-cell profiling of phospho-proteins, a novel population of CMML cells is identified to display aberrant granulocyte-macrophage colony stimulating factor (GM-CSF) signaling in both the extracellular signal-regulated kinase (ERK) 1/2 and signal transducer and activator of transcription 5 (Stat5) pathways. This abnormal signaling is acquired during CMML development. Further study suggests that aberrant Ras/ERK signaling leads to expansion of granulocytic/monocytic precursors, which are highly responsive to GM-CSF. Hyperactivation of Stat5 in CMML cells is mainly through expansion of these precursors rather than up-regulation of surface expression of GM-CSF receptors. Our results provide insights into the aberrant cytokine signaling in oncogenic NRAS-associated myeloid diseases.

Clonal nature of spontaneously immortalized 3T3 cells.

PubMed

Rittling, S R

1996-11-25

Mouse embryo fibroblasts (MEFs), when plated at appropriate densities, proliferate vigorously for several passages, and then the growth rate of the culture slows considerably. If the cells are plated at a high enough density and continuously passed, the cultures will eventually overcome this "crisis" period and resume rapid growth. Here, we have addressed the question of what the changes are that cells undergo in overcoming the growth restraints of crisis. Primary MEF cells were infected with a retrovirus which confers G418 resistance and selected in G418. The resultant pre-crisis population comprised cells which each contained a retrovirus integrated at a unique genomic location. These cells were then passed according to the 3T3 protocol until immortal, rapidly growing cells emerged. The integration pattern of the retrovirus in the immortal population was examined. In two independent experiments, the immortal population of cells grown in the presence of G418 comprised two independent clones of cells, with additional clones undetectable at the level of detection of the assays used. The integration pattern was also examined in parallel infected cultures grown in the absence of selection. In one experiment the unselected immortal population contained the same labeled clone that appeared in the sister infected culture, indicating that an immortal precursor was present in the precrisis population. These results are consistent with the idea that a mutation is responsible for the immortal phenotype.

CD200/BTLA deletions in pediatric precursor B-cell acute lymphoblastic leukemia treated according to the EORTC-CLG 58951 protocol

PubMed Central

Ghazavi, Farzaneh; Clappier, Emmanuelle; Lammens, Tim; Suciu, Stefan; Caye, Aurélie; Zegrari, Samira; Bakkus, Marleen; Grardel, Nathalie; Benoit, Yves; Bertrand, Yves; Minckes, Odile; Costa, Vitor; Ferster, Alina; Mazingue, Françoise; Plat, Geneviève; Plouvier, Emmanuel; Poirée, Marilyne; Uyttebroeck, Anne; van der Werff-ten Bosch, Jutte; Yakouben, Karima; Helsmoortel, Hetty; Meul, Magali; Van Roy, Nadine; Philippé, Jan; Speleman, Frank; Cavé, Hélène; Van Vlierberghe, Pieter; De Moerloose, Barbara

2015-01-01

DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% ± 1.2% for patients with deletions versus 83.5% ± 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23–3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P<0.0001), but were also identified in patients who did not have any genetic abnormality that is currently used for risk stratification. Within the latter population of patients, the presence of CD200/BTLA deletions was associated with inferior event-free survival and overall survival. Moreover, the multivariate Cox model indicated that these deletions had independent prognostic impact on event-free survival when adjusting for conventional risk criteria. All together, these findings further underscore the rationale for copy number profiling as an important tool for risk stratification in human B-cell precursor acute lymphoblastic leukemia. This trial was registered at www.ClinicalTrials.gov as #NCT00003728. PMID:26137961

CD200/BTLA deletions in pediatric precursor B-cell acute lymphoblastic leukemia treated according to the EORTC-CLG 58951 protocol.

PubMed

Ghazavi, Farzaneh; Clappier, Emmanuelle; Lammens, Tim; Suciu, Stefan; Caye, Aurélie; Zegrari, Samira; Bakkus, Marleen; Grardel, Nathalie; Benoit, Yves; Bertrand, Yves; Minckes, Odile; Costa, Vitor; Ferster, Alina; Mazingue, Françoise; Plat, Geneviève; Plouvier, Emmanuel; Poirée, Marilyne; Uyttebroeck, Anne; van der Werff-Ten Bosch, Jutte; Yakouben, Karima; Helsmoortel, Hetty; Meul, Magali; Van Roy, Nadine; Philippé, Jan; Speleman, Frank; Cavé, Hélène; Van Vlierberghe, Pieter; De Moerloose, Barbara

2015-10-01

DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% ± 1.2% for patients with deletions versus 83.5% ± 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23-3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P<0.0001), but were also identified in patients who did not have any genetic abnormality that is currently used for risk stratification. Within the latter population of patients, the presence of CD200/BTLA deletions was associated with inferior event-free survival and overall survival. Moreover, the multivariate Cox model indicated that these deletions had independent prognostic impact on event-free survival when adjusting for conventional risk criteria. All together, these findings further underscore the rationale for copy number profiling as an important tool for risk stratification in human B-cell precursor acute lymphoblastic leukemia. This trial was registered at www.ClinicalTrials.gov as #NCT00003728. Copyright© Ferrata Storti Foundation.

Sonic hedgehog acts cell-autonomously on muscle precursor cells to generate limb muscle diversity

PubMed Central

Anderson, Claire; Williams, Victoria C.; Moyon, Benjamin; Daubas, Philippe; Tajbakhsh, Shahragim; Buckingham, Margaret E.; Shiroishi, Toshihiko; Hughes, Simon M.; Borycki, Anne-Gaëlle

2012-01-01

How muscle diversity is generated in the vertebrate body is poorly understood. In the limb, dorsal and ventral muscle masses constitute the first myogenic diversification, as each gives rise to distinct muscles. Myogenesis initiates after muscle precursor cells (MPCs) have migrated from the somites to the limb bud and populated the prospective muscle masses. Here, we show that Sonic hedgehog (Shh) from the zone of polarizing activity (ZPA) drives myogenesis specifically within the ventral muscle mass. Shh directly induces ventral MPCs to initiate Myf5 transcription and myogenesis through essential Gli-binding sites located in the Myf5 limb enhancer. In the absence of Shh signaling, myogenesis is delayed, MPCs fail to migrate distally, and ventral paw muscles fail to form. Thus, Shh production in the limb ZPA is essential for the spatiotemporal control of myogenesis and coordinates muscle and skeletal development by acting directly to regulate the formation of specific ventral muscles. PMID:22987640

Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

DOEpatents

Dolbeare, Frank A.; Gray, Joe W.

1986-01-01

A method for the simultaneous flow cytometric measurement of the total DNA content and the level of DNA synthesis in normal and malignant cells is disclosed. The sensitivity of the method allows a study of cell cycle traverse rates for large scale cell populations as well as single cell measurements. A DNA stain such as propidium iodide is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as bromodeoxyuridine (BrdU) is used as a probe for the measurement of BrdU uptake by the cells as a measure of DNA synthesis.

Development of B cells expressing surface immunoglobulin molecules that lack V(D)J-encoded determinants in the avian embryo bursa of Fabricius

PubMed Central

Sayegh, Camil E.; Demaries, Sandra L.; Iacampo, Sandra; Ratcliffe, Michael J. H.

1999-01-01

Immunoglobulin gene rearrangement in avian B cell precursors generates surface Ig receptors of limited diversity. It has been proposed that specificities encoded by these receptors play a critical role in B lineage development by recognizing endogenous ligands within the bursa of Fabricius. To address this issue directly we have introduced a truncated surface IgM, lacking variable region domains, into developing B precursors by retroviral gene transfer in vivo. Cells expressing this truncated receptor lack endogenous surface IgM, and the low level of endogenous Ig rearrangements that have occurred within this population of cells has not been selected for having a productive reading frame. Such cells proliferate rapidly within bursal epithelial buds of normal morphology. In addition, despite reduced levels of endogenous light chain rearrangement, those light chain rearrangements that have occurred have undergone variable region diversification by gene conversion. Therefore, although surface expression of an Ig receptor is required for bursal colonization and the induction of gene conversion, the specificity encoded by the prediversified receptor is irrelevant and, consequently, there is no obligate ligand for V(D)J-encoded determinants of prediversified avian cell surface IgM receptor. PMID:10485907

Preosteoblast production 55 hours after a 12.5-day spaceflight on Cosmos 1887

NASA Technical Reports Server (NTRS)

Garetto, L. P.; Gonsalves, M. R.; Morey, E. R.; Durnova, G.; Roberts, W. E.; Morey-Holton, E. (Principal Investigator)

1990-01-01

The influence of 12.5 days of spaceflight and a 55 h stressful recovery period (at 1 g) on fibroblastlike osteoblast precursor cells was assessed in the periodontal ligament (PDL) of rats that were 91 days old at launch. Nuclear morphometry was used as a marker for precursor cell differentiation in 3 microns sections cut in the midsagittal plane from the maxillary first molar. According to nuclear volume, cells were classified as preosteoblasts (C + D cells, greater than or equal to 120 microns 3) and less differentiated progenitor cells (A + A' cells, 40-79 microns 3). Compared with synchronous controls (simulated flight conditions), the 55 h postflight recovery period at 1 g resulted in a 40% decrease in the A + A' cell population, a 42% increase in the C + D cells, and a 39% increase in the number of PDL fibroblastlike cells near the bone surface. These results are consistent with a postflight osteogenic response in PDL. This recovery response occurred despite physiological stress in the flight animals that resulted in a highly significant (P less than or equal to 0.001) increase in adrenal weight. The data suggest that after spaceflight there is a strong and rapid recovery mechanism for osteoblast differentiation that is not suppressed by physiological stress.

Postnatal reduction of BDNF regulates the developmental remodeling of taste bud innervation

PubMed Central

Huang, Tao; Ma, Liqun; Krimm, Robin F

2015-01-01

The refinement of innervation is a common developmental mechanism that serves to increase the specificity of connections following initial innervation. In the peripheral gustatory system, the extent to which innervation is refined and how refinement might be regulated is unclear. The initial innervation of taste buds is controlled by brain-derived neurotrophic factor (BDNF). Following initial innervation, taste receptor cells are added and become newly innervated. The connections between the taste receptor cells and nerve fibers are likely to be specific in order to retain peripheral coding mechanisms. Here, we explored the possibility that the down-regulation of BDNF regulates the refinement of taste bud innervation during postnatal development. An analysis of BDNF expression in BdnflacZ/+ mice and real-time reverse transcription polymerase chain reaction (RT-PCR) revealed that BDNF was down-regulated between postnatal day (P) 5 and P10. This reduction in BDNF expression was due to a loss of precursor/progenitor cells that express BDNF, while the expression of BDNF in the subpopulations of taste receptor cells did not change. Gustatory innervation, which was identified by P2X3 immunohistochemistry, was lost around the perimeter where most progenitor/precursor cells are located. In addition, the density of innervation in the taste bud was reduced between P5 and P10, because taste buds increase in size without increasing innervation. This reduction of innervation density was blocked by the overexpression of BDNF in the precursor/progenitor population of taste bud cells. Together these findings indicate that the process of BDNF restriction to a subpopulation of taste receptor cells between P5 and P10, results in a refinement of gustatory innervation. We speculate that this refinement results in an increased specificity of connections between neurons and taste receptor cells during development. PMID:26164656

Primordial Germ Cells in Mice

PubMed Central

Saitou, Mitinori; Yamaji, Masashi

2012-01-01

Germ cell development creates totipotency through genetic as well as epigenetic regulation of the genome function. Primordial germ cells (PGCs) are the first germ cell population established during development and are immediate precursors for both the oocytes and spermatogonia. We here summarize recent findings regarding the mechanism of PGC development in mice. We focus on the transcriptional and signaling mechanism for PGC specification, potential pluripotency, and epigenetic reprogramming in PGCs and strategies for the reconstitution of germ cell development using pluripotent stem cells in culture. Continued studies on germ cell development may lead to the generation of totipotency in vitro, which should have a profound influence on biological science as well as on medicine. PMID:23125014

Cytokines and the Inception of CD8 T Cell Responses

PubMed Central

Cox, Maureen A.; Harrington, Laurie E.; Zajac, Allan J.

2011-01-01

The activation and differentiation of CD8 T cells is a necessary first step that endows these cells with the phenotypic and functional properties required for the control of intracellular pathogens. The induction of the CD8 T cell responses typically results in the development of a massive overall population of effector cells, comprised of both highly functional but short-lived terminally differentiated cells, as well as a smaller subset of precursors that are predisposed to survive and transition into the memory T cell pool. In this article we discuss how inflammatory cytokines and IL-2 bias the initial response towards short-lived effector generation and also highlight the potential counterbalancing role of IL-21. PMID:21371940

Molecular and Functional Characterization of Lymphoid Progenitor Subsets Reveals a Bipartite Architecture of Human Lymphopoiesis.

PubMed

Alhaj Hussen, Kutaiba; Vu Manh, Thien-Phong; Guimiot, Fabien; Nelson, Elisabeth; Chabaane, Emna; Delord, Marc; Barbier, Maxime; Berthault, Claire; Dulphy, Nicolas; Alberdi, Antonio José; Burlen-Defranoux, Odile; Socié, Gerard; Bories, Jean Christophe; Larghero, Jerôme; Vanneaux, Valérie; Verhoeyen, Els; Wirth, Thierry; Dalod, Marc; Gluckman, Jean Claude; Cumano, Ana; Canque, Bruno

2017-10-17

The classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127 - and CD127 + early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127 - and CD127 + ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127 - ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127 + ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis. Copyright © 2017 Elsevier Inc. All rights reserved.

HMC-1 human mast cells synthesize neurotensin (NT) precursor, secrete bioactive NT-like peptide(s) and express NT receptor NTS1.

PubMed

Cochrane, David E; Carraway, Robert E; Harrington, Kimberly; Laudano, Melissa; Rawlings, Stephen; Feldberg, Ross S

2011-12-01

To determine if mast cells synthesize the inflammatory peptide, neurotensin (NT), secrete immunoreactive and bioactive NT, and express the NT receptor NTS1. HMC-1 cells, pleural mast cells from Sprague-Dawley rats, LAD2 mast cells, and human cord blood mast cells were used. HMC-1 cells were stimulated with NT, C48/80, mastoparan, or PGE(2). For changes in cutaneous vascular permeability, anesthetized rats were injected intravenously with Evans Blue dye and intradermally with saline, NT, histamine, diphenhydramine, and C48/80. RT-PCR was used to identify RNA transcripts. Histamine was measured by fluorometric assay. In vivo cutaneous vascular permeability assays, radio-immunoassays for NT, Western blotting for the NT precursor protein and NTS1 protein from HMC-1 cells and tissues from rats were used. Immunohistochemistry was used to identify NT precursor-like proteins in HMC-1 mast cells. HMC-1 cells express mRNAs for NT precursor, PC5A processing enzyme and NTS1 receptor. Human cord blood mast cells and LAD2 mast cells express mRNA transcripts for NT precursor and NTS1. Western blotting showed NT precursor and NTS1 receptor in HMC1. Rat tissues with high numbers of mast cells contained NT precursor proteins. NT-like peptides from HMC-1 displayed NT-like bioactivity. HMC-1 mast cells synthesize and secrete immunoreactive and bioactive NT-like peptide(s) and express the NT receptor, suggesting that NT from mast cells might serve autocrine and paracrine roles.

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

PubMed Central

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

2008-01-01

Background Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. Results In normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA). Conclusion Our study, comparing surface antigen expression of ALDH+/CD34+, ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow, clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia. PMID:18510759

Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell-cell recognition and fusion.

PubMed

Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D; Schulz, Stefan; Fleißner, André

2016-10-18

Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell-cell communication and fusion in the fungus Neurospora crassa Genetically identical germinating spores of this fungus undergo cell-cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell-cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion.

Transplantation of oligodendrocyte precursors and sonic hedgehog results in improved function and white matter sparing in the spinal cords of adult rats after contusion.

PubMed

Bambakidis, Nicholas C; Miller, Robert H

2004-01-01

A substantial cause of neurological disability in spinal cord injury is oligodendrocyte death leading to demyelination and axonal degeneration. Rescuing oligodendrocytes and preserving myelin is expected to result in significant improvement in functional outcome after spinal cord injury. Although previous investigators have used cellular transplantation of xenografted pluripotent embryonic stem cells and observed improved functional outcome, these transplants have required steroid administration and only a minority of these cells develop into oligodendrocytes. The objective of the present study was to determine whether allografts of oligodendrocyte precursors transplanted into an area of incomplete spinal cord contusion would improve behavioral and electrophysiological measures of spinal cord function. Additional treatment incorporated the use of the glycoprotein molecule Sonic hedgehog (Shh), which has been shown to play a critical role in oligodendroglial development and induce proliferation of endogenous neural precursors after spinal cord injury. Laboratory study. Moderate spinal cord contusion injury was produced in 39 adult rats at T9-T10. Ten animals died during the course of the study. Nine rats served as contusion controls (Group 1). Six rats were treated with oligodendrocyte precursor transplantation 5 days after injury (Group 2). The transplanted cells were isolated from newborn rat pups using immunopanning techniques. Another eight rats received an injection of recombinant Shh along with the oligodendrocyte precursors (Group 3), while six more rats were treated with Shh alone (Group 4). Eight additional rats received only T9 laminectomies to serve as noninjured controls (Group 0). Animals were followed for 28 days. After an initial complete hindlimb paralysis, rats of all groups receiving a contusive injury recovered substantial function within 1 week. By 28 days, rats in Groups 2 and 3 scored 4.7 and 5.8 points better on the Basso, Beattie, Bresnahan (BBB) open field locomotor score than rats in group 1 (Groups 2 and 3=18.2 and 19.4 points, respectively, after 28 days vs. Group 1=13.6 points; p=.015). Rats in Group 4 scored no better than those in Group 1 (BBB=16.4). Motor evoked potential (MEP) recordings revealed a strong trend towards significant improvement in latency measurements in all treatment groups compared with controls at 28 days, although three animals in Group 1 and two animals in Group 3 were not recordable. Histological examination demonstrated significantly more spared white matter in the same groups that correlated with the improvements in BBB scores and MEP latencies. Immunohistochemical analysis showed the survival, proliferation and migration of the transplanted cells, as well as the induction of proliferating endogenous neural precursor cells in animals treated with Shh. These findings suggest that the transplantation of oligodendrocyte precursors may improve axonal conduction and spinal cord function in the injured spinal cord. The benefits seem more pronounced with the addition of Shh, and the addition of Shh alone results in the proliferation of an endogenous population of neural precursor cells.

[Analysis of the changes of stromal precursor cell numbers in the thymus and the spleen of animals of different age groups].

PubMed

Lebedinskaia, O V; Gorskaia, Iu F; Shuklina, E Iu; Latsinik, N V; Nesterenko, V G

2005-01-01

The objective of this study was to analyze the species differences in the numbers of stromal precursor cell (CFU-f), their cloning efficiency (CFE-0 and their dynamics in different organs during aging, using the mathematical gradient decrease method. Age changes of CFU-f numbers and of their CFE-f were studied in the thymus and the spleen of mice and guinea pigs. The study was performed using CFU-f cloning in monolayer cultures. CFU-f numbers and CFE-f were found to decrease with aging both in the thymus and the spleen of mice and guinea pigs. However these changes were different in each species and were variable in different organs of the animals of the same species, which, probably was associated with the physiological characteristics and aging peculiarities of the animals of different species and with the functional role of organs studied. The process of reduction was more significant in the thymus of guinea pigs and mice - the numbers of CFU-f were decreased 75- and 12-fold, respectively. Since it is known that the population of CFU-f in the thymus and the spleen includes inducible osteogenic precursor cells, the data obtained indicate the possibility of a reduction in numbers of this category ofstromal precursors, that could be one of the reasons of osteoporosis of aging. The application of a mathematical analysis using the gradient decrease method allows to predict the time-course of age changes and to evaluate the dynamics of CFU-f numbers and of CFE-f in association with organism aging.

Cell cycle progression is an essential regulatory component of phospholipid metabolism and membrane homeostasis

PubMed Central

Sanchez-Alvarez, Miguel; Zhang, Qifeng; Finger, Fabian; Wakelam, Michael J. O.; Bakal, Chris

2015-01-01

We show that phospholipid anabolism does not occur uniformly during the metazoan cell cycle. Transition to S-phase is required for optimal mobilization of lipid precursors, synthesis of specific phospholipid species and endoplasmic reticulum (ER) homeostasis. Average changes observed in whole-cell phospholipid composition, and total ER lipid content, upon stimulation of cell growth can be explained by the cell cycle distribution of the population. TORC1 promotes phospholipid anabolism by slowing S/G2 progression. The cell cycle stage-specific nature of lipid biogenesis is dependent on p53. We propose that coupling lipid metabolism to cell cycle progression is a means by which cells have evolved to coordinate proliferation with cell and organelle growth. PMID:26333836

Cell cycle progression is an essential regulatory component of phospholipid metabolism and membrane homeostasis.

PubMed

Sanchez-Alvarez, Miguel; Zhang, Qifeng; Finger, Fabian; Wakelam, Michael J O; Bakal, Chris

2015-09-01

We show that phospholipid anabolism does not occur uniformly during the metazoan cell cycle. Transition to S-phase is required for optimal mobilization of lipid precursors, synthesis of specific phospholipid species and endoplasmic reticulum (ER) homeostasis. Average changes observed in whole-cell phospholipid composition, and total ER lipid content, upon stimulation of cell growth can be explained by the cell cycle distribution of the population. TORC1 promotes phospholipid anabolism by slowing S/G2 progression. The cell cycle stage-specific nature of lipid biogenesis is dependent on p53. We propose that coupling lipid metabolism to cell cycle progression is a means by which cells have evolved to coordinate proliferation with cell and organelle growth. © 2015 The Authors.

Neuronal cell fate specification in Drosophila.

PubMed

Jan, Y N; Jan, L Y

1994-02-01

Recent work indicates that the Drosophila nervous system develops in a progressive process of cell fate specification. Expression of specific proneural genes in clusters of cells (the proneural clusters) in the cellular blastoderm endows these cells with the potential to form certain types of neural precursors. Intercellular interactions that involve both proneural genes and neurogenic genes then allow the neural precursors to be singled out from the proneural clusters. Expression of neural precursor genes in all neural precursors is likely to account for the universal aspects of neuronal differentiation, such as axonal outgrowth. Selective expression of certain neuronal-type selector genes further specifies the type of neuron(s) that a neural precursor will produce.

Analysis of antigen-specific B-cell memory directly ex vivo.

PubMed

McHeyzer-Williams, Louise J; McHeyzer-Williams, Michael G

2004-01-01

Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.

Development of the intrinsic and extrinsic innervation of the gut.

PubMed

Uesaka, Toshihiro; Young, Heather M; Pachnis, Vassilis; Enomoto, Hideki

2016-09-15

The gastrointestinal (GI) tract is innervated by intrinsic enteric neurons and by extrinsic efferent and afferent nerves. The enteric (intrinsic) nervous system (ENS) in most regions of the gut consists of two main ganglionated layers; myenteric and submucosal ganglia, containing numerous types of enteric neurons and glial cells. Axons arising from the ENS and from extrinsic neurons innervate most layers of the gut wall and regulate many gut functions. The majority of ENS cells are derived from vagal neural crest cells (NCCs), which proliferate, colonize the entire gut, and first populate the myenteric region. After gut colonization by vagal NCCs, the extrinsic nerve fibers reach the GI tract, and Schwann cell precursors (SCPs) enter the gut along the extrinsic nerves. Furthermore, a subpopulation of cells in myenteric ganglia undergoes a radial (inward) migration to form the submucosal plexus, and the intrinsic and extrinsic innervation to the mucosal region develops. Here, we focus on recent progress in understanding the developmental processes that occur after the gut is colonized by vagal ENS precursors, and provide an up-to-date overview of molecular mechanisms regulating the development of the intrinsic and extrinsic innervation of the GI tract. Copyright © 2016 Elsevier Inc. All rights reserved.

Regulation of endogenous neural stem/progenitor cells for neural repair—factors that promote neurogenesis and gliogenesis in the normal and damaged brain

PubMed Central

Christie, Kimberly J.; Turnley, Ann M.

2012-01-01

Neural stem/precursor cells in the adult brain reside in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus. These cells primarily generate neuroblasts that normally migrate to the olfactory bulb (OB) and the dentate granule cell layer respectively. Following brain damage, such as traumatic brain injury, ischemic stroke or in degenerative disease models, neural precursor cells from the SVZ in particular, can migrate from their normal route along the rostral migratory stream (RMS) to the site of neural damage. This neural precursor cell response to neural damage is mediated by release of endogenous factors, including cytokines and chemokines produced by the inflammatory response at the injury site, and by the production of growth and neurotrophic factors. Endogenous hippocampal neurogenesis is frequently also directly or indirectly affected by neural damage. Administration of a variety of factors that regulate different aspects of neural stem/precursor biology often leads to improved functional motor and/or behavioral outcomes. Such factors can target neural stem/precursor proliferation, survival, migration and differentiation into appropriate neuronal or glial lineages. Newborn cells also need to subsequently survive and functionally integrate into extant neural circuitry, which may be the major bottleneck to the current therapeutic potential of neural stem/precursor cells. This review will cover the effects of a range of intrinsic and extrinsic factors that regulate neural stem/precursor cell functions. In particular it focuses on factors that may be harnessed to enhance the endogenous neural stem/precursor cell response to neural damage, highlighting those that have already shown evidence of preclinical effectiveness and discussing others that warrant further preclinical investigation. PMID:23346046

Reactive oxygen species are required for zoledronic acid-induced apoptosis in osteoclast precursors and mature osteoclast-like cells

PubMed Central

Tai, Ta-Wei; Chen, Ching-Yu; Su, Fong-Chin; Tu, Yuan-Kun; Tsai, Tsung-Ting; Lin, Chiou-Feng; Jou, I.-Ming

2017-01-01

Inhibiting osteoclasts and osteoclast precursors to reduce bone resorption is an important strategy to treat osteoclast-related diseases, such as osteoporosis, inflammatory bone loss, and malignant bone metastasis. However, the mechanism by which apoptosis is induced in the osteoclasts and their precursors are not completely understood. Here, we used nitrogen-containing bisphosphonate zoledronic acid (ZA) to induce cell apoptosis in human and murine osteoclast precursors and mature osteoclast-like cells. Caspase-3-mediated cell apoptosis occurred following the ZA (100 μM) treatment. Reactive oxygen species (ROS) were also generated in a time-dependent manner. Following knock-down of the p47phox expression, which is required for ROS activation, or co-treatment with the ROS inhibitor, N-acetyl-L-cysteine, ZA-induced apoptosis was significantly suppressed in both osteoclast precursors and mature osteoclast-like cells. The ROS-activated mitogen-activated protein kinases pathways did not trigger cell apoptosis. However, a ROS-regulated Mcl-1 decrease simultaneously with glycogen synthase kinase (GSK)-3β promoted cell apoptosis. These findings show that ZA induces apoptosis in osteoclast precursors and mature osteoclast-like cells by triggering ROS- and GSK-3β-mediated Mcl-1 down-regulation. PMID:28281643

Thin film solar cells by selenization sulfurization using diethyl selenium as a selenium precursor

DOEpatents

Dhere, Neelkanth G.; Kadam, Ankur A.

2009-12-15

A method of forming a CIGSS absorber layer includes the steps of providing a metal precursor, and selenizing the metal precursor using diethyl selenium to form a selenized metal precursor layer (CIGSS absorber layer). A high efficiency solar cell includes a CIGSS absorber layer formed by a process including selenizing a metal precursor using diethyl selenium to form the CIGSS absorber layer.

Functional heterogeneity of side population cells in skeletal muscle

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

Uezumi, Akiyoshi; Ojima, Koichi; Fukada, So-ichiro

2006-03-17

Skeletal muscle regeneration has been exclusively attributed to myogenic precursors, satellite cells. A stem cell-rich fraction referred to as side population (SP) cells also resides in skeletal muscle, but its roles in muscle regeneration remain unclear. We found that muscle SP cells could be subdivided into three sub-fractions using CD31 and CD45 markers. The majority of SP cells in normal non-regenerating muscle expressed CD31 and had endothelial characteristics. However, CD31{sup -}CD45{sup -} SP cells, which are a minor subpopulation in normal muscle, actively proliferated upon muscle injury and expressed not only several regulatory genes for muscle regeneration but also somemore » mesenchymal lineage markers. CD31{sup -}CD45{sup -} SP cells showed the greatest myogenic potential among three SP sub-fractions, but indeed revealed mesenchymal potentials in vitro. These SP cells preferentially differentiated into myofibers after intramuscular transplantation in vivo. Our results revealed the heterogeneity of muscle SP cells and suggest that CD31{sup -}CD45{sup -} SP cells participate in muscle regeneration.« less

The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function

PubMed Central

Isern, Joan; García-García, Andrés; Martín, Ana M; Arranz, Lorena; Martín-Pérez, Daniel; Torroja, Carlos; Sánchez-Cabo, Fátima; Méndez-Ferrer, Simón

2014-01-01

Mesenchymal stem cells (MSCs) and osteolineage cells contribute to the hematopoietic stem cell (HSC) niche in the bone marrow of long bones. However, their developmental relationships remain unclear. In this study, we demonstrate that different MSC populations in the developing marrow of long bones have distinct functions. Proliferative mesoderm-derived nestin− MSCs participate in fetal skeletogenesis and lose MSC activity soon after birth. In contrast, quiescent neural crest-derived nestin+ cells preserve MSC activity, but do not generate fetal chondrocytes. Instead, they differentiate into HSC niche-forming MSCs, helping to establish the HSC niche by secreting Cxcl12. Perineural migration of these cells to the bone marrow requires the ErbB3 receptor. The neonatal Nestin-GFP+ Pdgfrα− cell population also contains Schwann cell precursors, but does not comprise mature Schwann cells. Thus, in the developing bone marrow HSC niche-forming MSCs share a common origin with sympathetic peripheral neurons and glial cells, and ontogenically distinct MSCs have non-overlapping functions in endochondrogenesis and HSC niche formation. DOI: http://dx.doi.org/10.7554/eLife.03696.001 PMID:25255216

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

PubMed Central

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

2004-01-01

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

Transcriptional diversity during lineage commitment of human blood progenitors.

PubMed

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

2014-09-26

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

1,25-Dihydroxyvitamin D3 and 22-oxa-1,25-dihydroxyvitamin D3 in vivo nuclear receptor binding in developing bone during endochondral and intramembranous ossification.

PubMed

Stumpf, W E; Koike, N; Hayakawa, N; Tokuda, K; Nishimiya, K; Tsuchiya, Y; Hirate, J; Okazaki, A; Kumaki, K

1994-09-01

Target cells for 3H-labeled 1 alpha, 25(OH)2 vitamin D3 [1,25(OH)2D3, vitamin D] and its analog 3H-labeled 22-oxa-1 alpha, 25(OH)2 vitamin D3 (OCT) have been identified during endochondral and intramembranous ossification in developing, undecalcified, unembedded bone, using thaw-mount autoradiography. Two-day-old neonatal rats were injected with [3H]1,25(OH)2D3 or [3H]OCT; after 2 h leg, spine, and head were frozen and sectioned. In the epiphyseal-metaphyseal region specific nuclear concentrations of [3H]1,25(OH)2D3 and [3H]OCT were observed in identical cell populations, being low in cells of the articular and resting zone, intermediate in the proliferating zone, and highest in hypertrophic chondrocytes and in osteoblasts and precursor cells. In the primary spongiosa intertrabecular spaces there were a large number of cells with nuclear labeling--probably osteoblasts and precursor cells. In contrast, in the secondary spongiosa intertrabecular spaces, apparent blood-forming cells were mostly unlabeled. Osteoblasts along bone spicules and compact bone in long bones, vertebrae, and head also showed strong nuclear labeling, as did cells of the periosteum. These data suggest that 1,25(OH)2D3 and OCT regulate development, differentiation, and activities of chondrocytes and osteoblasts, including differentiation of resting chondrocytes into proliferating and hypertrophic chondrocytes that involve "chondroclastic" enlargement of lacunae and "trans-differentiation" of surviving hypertrophic chondrocytes; differentiation of stroma cells into osteoblasts; and in periosteum and other regions of intramembranous ossification differentiation of precursor cells and osteoblasts. Nuclear receptor binding and their selective and hierarchical distribution during cell differentiation appear to correspond to multiple genomic effects toward growth, regeneration and repair. The findings indicate a physiological significance and therapeutic potential of 1,25(OH)2D3 and in particular of its less hypercalcemic analog OCT.

Microparticle Shedding from Neural Progenitor Cells and Vascular Compartment Cells Is Increased in Ischemic Stroke.

PubMed

Chiva-Blanch, Gemma; Suades, Rosa; Crespo, Javier; Peña, Esther; Padró, Teresa; Jiménez-Xarrié, Elena; Martí-Fàbregas, Joan; Badimon, Lina

2016-01-01

Ischemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke. Forty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3-7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells) were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls. Compared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions. Stroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process after stroke. Larger cerebral lesions associate with deeper vessel injury affecting vascular smooth muscle cells.

Microparticle Shedding from Neural Progenitor Cells and Vascular Compartment Cells Is Increased in Ischemic Stroke

PubMed Central

Chiva-Blanch, Gemma; Suades, Rosa; Crespo, Javier; Peña, Esther; Padró, Teresa; Jiménez-Xarrié, Elena; Martí-Fàbregas, Joan; Badimon, Lina

2016-01-01

Purpose Ischemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke. Methods Forty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3–7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells) were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls. Results Compared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions. Conclusions Stroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process after stroke. Larger cerebral lesions associate with deeper vessel injury affecting vascular smooth muscle cells. PMID:26815842

Cryopreservation of GABAergic Neuronal Precursors for Cell-Based Therapy

PubMed Central

2017-01-01

Cryopreservation protocols are essential for stem cells storage in order to apply them in the clinic. Here we describe a new standardized cryopreservation protocol for GABAergic neural precursors derived from the medial glanglionic eminence (MGE), a promising source of GABAergic neuronal progenitors for cell therapy against interneuron-related pathologies. We used 10% Me2SO as cryoprotectant and assessed the effects of cell culture amplification and cellular organization, as in toto explants, neurospheres, or individualized cells, on post-thaw cell viability and retrieval. We confirmed that in toto cryopreservation of MGE explants is an optimal preservation system to keep intact the interneuron precursor properties for cell transplantation, together with a high cell viability (>80%) and yield (>70%). Post-thaw proliferation and self-renewal of the cryopreserved precursors were tested in vitro. In addition, their migration capacity, acquisition of mature neuronal morphology, and potency to differentiate into multiple interneuron subtypes were also confirmed in vivo after transplantation. The results show that the cryopreserved precursor features remained intact and were similar to those immediately transplanted after their dissection from the MGE. We hope this protocol will facilitate the generation of biobanks to obtain a permanent and reliable source of GABAergic precursors for clinical application in cell-based therapies against interneuronopathies. PMID:28122047

Differentiation of vascular smooth muscle cells from local precursors during embryonic and adult arteriogenesis requires Notch signaling

PubMed Central

Chang, Linda; Noseda, Michela; Higginson, Michelle; Ly, Michelle; Patenaude, Alexandre; Fuller, Megan; Kyle, Alastair H.; Minchinton, Andrew I.; Puri, Mira C.; Dumont, Daniel J.; Karsan, Aly

2012-01-01

Vascular smooth muscle cells (VSMC) have been suggested to arise from various developmental sources during embryogenesis, depending on the vascular bed. However, evidence also points to a common subpopulation of vascular progenitor cells predisposed to VSMC fate in the embryo. In the present study, we use binary transgenic reporter mice to identify a Tie1+CD31dimvascular endothelial (VE)-cadherin−CD45− precursor that gives rise to VSMC in vivo in all vascular beds examined. This precursor does not represent a mature endothelial cell, because a VE-cadherin promoter-driven reporter shows no expression in VSMC during murine development. Blockade of Notch signaling in the Tie1+ precursor cell, but not the VE-cadherin+ endothelial cell, decreases VSMC investment of developing arteries, leading to localized hemorrhage in the embryo at the time of vascular maturation. However, Notch signaling is not required in the Tie1+ precursor after establishment of a stable artery. Thus, Notch activity is required in the differentiation of a Tie1+ local precursor to VSMC in a spatiotemporal fashion across all vascular beds. PMID:22509029

Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

DOEpatents

Dolbeare, Frank A.; Gray, Joe W.

1988-01-01

A method for the simultaneous flow cytometric measurement of the total DNA content and the level of DNA synthesis in normal and malignant cells is disclosed. The sensitivity of the method allows a study of cell cycle traverse rates for large scale cell populations as well as single cell measurements. A DNA stain such as propidium iodide or Hoechst 33258 is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as halodeoxy-uridine (HdU), more specifically bromodeoxyuridine (BrdU) is used as a probe for the measurement of HdU or BrdU uptake by the cells as a measure of DNA synthesis.

Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell–cell recognition and fusion

PubMed Central

Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D.; Schulz, Stefan; Fleißner, André

2016-01-01

Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell–cell communication and fusion in the fungus Neurospora crassa. Genetically identical germinating spores of this fungus undergo cell–cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell–cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion. PMID:27708165

Ramifying feedback networks, cross-scale interactions, and emergent quasi individuals in Conway's game of Life.

PubMed

Gotts, Nicholas M

2009-01-01

Small patterns of state 1 cells on an infinite, otherwise empty array of Conway's game of Life can produce sets of growing structures resembling in significant ways a population of spatially situated individuals in a nonuniform, highly structured environment. Ramifying feedback networks and cross-scale interactions play a central role in the emergence and subsequent dynamics of the quasi population. The implications are discussed: It is proposed that analogous networks and interactions may have been precursors to natural selection in the real world.

[Isolation and Characterization of Multipotent Precursor Cells from Murine Adipose Tissue using a Clinically Approved Cell Separation System].

PubMed

Krug, C; Beer, A; Saller, M M; Aszodi, A; Holzbach, T; Giunta, R E; Volkmer, E

2016-04-01

Recent studies underscored the clinical potential of adipose-derived multipotent stem-/precursor cells (ASPCs). One of the main hurdles en route to clinical application was to isolate cells without having to perform expansion cultures outside the OR. A new generation of clinically approved, commercially available cell separation systems claims to provide ASPCs ready for application without further expansion cultures. However, it is unclear if the new systems yield sufficient cells of adequate quality for the use in autologous murine models. The aim of this study was to isolate and characterize adipose-derived precursor cells taken from the inguinal fat pat of wistar rats using InGeneron's clinically approved ARC™-cell separation system. We isolated cells from the inguinal fat pad of 3 male Wistar rats according to the manufacturer's protocol. In order to reduce the influence of the atmospheric oxygen on the multipotent precursor cells, one half of the cell suspension was cultivated under hypoxia (2% O2) simulating physiological conditions for ASPCs. As a control, the other half of the cells were cultivated under normoxia (21% O2). Cell surface markers CD90, CD29, CD45 and CD11b/c were analyzed by FACS, and osteogenic and adipogenic differentiation of the ASPCs was performed. Finally, cellular growth characteristics were assessed by evaluation of the cumulative population doublings and CFU assay, and metabolic activity was evaluated by WST-1 assay. Processing time was 90 (± 12) min. 1 g of adipose tissue yielded approximately 60 000 plastic adhering cells. Both groups showed a high expression of the mesenchymal stem cell markers CD90 and CD29 while they were negative for the leucocyte markers CD45 and CD11b/c. A strong osteogenic differentiation and a sufficient adipogenic differentiation potential was proven for all ASPCs. Under hypoxia, ASPCs showed increased proliferation characteristics and CFU efficiency as well as a significantly increased metabolic activity. This study showed that sufficient multipotent ASPCs of appropriate quality can be isolated from the inguinal fat pad of Wistar rats using the ARC™-cell separation system. As shown in previous studies, cultivation of cells under hypoxic conditions increased their stemness. Our findings will enable future studies that focus on autologous transplantation of ASPCs in a rat model, which most closely resembles a possible clinical application. © Georg Thieme Verlag KG Stuttgart · New York.

Host responses in tissue repair and fibrosis.

PubMed

Duffield, Jeremy S; Lupher, Mark; Thannickal, Victor J; Wynn, Thomas A

2013-01-24

Myofibroblasts accumulate in the spaces between organ structures and produce extracellular matrix (ECM) proteins, including collagen I. They are the primary "effector" cells in tissue remodeling and fibrosis. Previously, leukocyte progenitors termed fibrocytes and myofibroblasts generated from epithelial cells through epithelial-to-mesenchymal transition (EMT) were considered the primary sources of ECM-producing myofibroblasts in injured tissues. However, genetic fate mapping experiments suggest that mesenchyme-derived cells, known as resident fibroblasts, and pericytes are the primary precursors of scar-forming myofibroblasts, whereas epithelial cells, endothelial cells, and myeloid leukocytes contribute to fibrogenesis predominantly by producing key fibrogenic cytokines and by promoting cell-to-cell communication. Numerous cytokines derived from T cells, macrophages, and other myeloid cell populations are important drivers of myofibroblast differentiation. Monocyte-derived cell populations are key regulators of the fibrotic process: They act as a brake on the processes driving fibrogenesis, and they dismantle and degrade established fibrosis. We discuss the origins, modes of activation, and fate of myofibroblasts in various important fibrotic diseases and describe how manipulation of macrophage activation could help ameliorate fibrosis.

Defining the cellular precursors to human breast cancer

PubMed Central

Keller, Patricia J.; Arendt, Lisa M.; Skibinski, Adam; Logvinenko, Tanya; Klebba, Ina; Dong, Shumin; Smith, Avi E.; Prat, Aleix; Perou, Charles M.; Gilmore, Hannah; Schnitt, Stuart; Naber, Stephen P.; Garlick, Jonathan A.; Kuperwasser, Charlotte

2012-01-01

Human breast cancers are broadly classified based on their gene-expression profiles into luminal- and basal-type tumors. These two major tumor subtypes express markers corresponding to the major differentiation states of epithelial cells in the breast: luminal (EpCAM+) and basal/myoepithelial (CD10+). However, there are also rare types of breast cancers, such as metaplastic carcinomas, where tumor cells exhibit features of alternate cell types that no longer resemble breast epithelium. Until now, it has been difficult to identify the cell type(s) in the human breast that gives rise to these various forms of breast cancer. Here we report that transformation of EpCAM+ epithelial cells results in the formation of common forms of human breast cancer, including estrogen receptor-positive and estrogen receptor-negative tumors with luminal and basal-like characteristics, respectively, whereas transformation of CD10+ cells results in the development of rare metaplastic tumors reminiscent of the claudin-low subtype. We also demonstrate the existence of CD10+ breast cells with metaplastic traits that can give rise to skin and epidermal tissues. Furthermore, we show that the development of metaplastic breast cancer is attributable, in part, to the transformation of these metaplastic breast epithelial cells. These findings identify normal cellular precursors to human breast cancers and reveal the existence of a population of cells with epidermal progenitor activity within adult human breast tissues. PMID:21940501

Clonal population of adult stem cells: life span and differentiation potential.

PubMed

Seruya, Mitchel; Shah, Anup; Pedrotty, Dawn; du Laney, Tracey; Melgiri, Ryan; McKee, J Andrew; Young, Henry E; Niklason, Laura E

2004-01-01

Adult stem cells derived from bone marrow, connective tissue, and solid organs can exhibit a range of differentiation potentials. Some controversy exists regarding the classification of mesenchymal stem cells as bona fide stem cells, which is in part derived from the limited ability to propagate true clonal populations of precursor cells. We isolated putative mesenchymal stem cells from the connective tissue of an adult rat (rMSC), and generated clonal populations via three rounds of dilutional cloning. The replicative potential of the clonal rMSC line far exceeded Hayflick's limit of 50-70 population doublings. The high capacity for self-renewal in vitro correlated with telomerase activity, as demonstrated by telomerase repeat amplification protocol (TRAP) assay. Exposure to nonspecific differentiation culture medium revealed multilineage differentiation potential of rMSC clones. Immunostaining confirmed the appearance of mesodermal phenotypes, including adipocytes possessing lipid-rich vacuoles, chondrocytes depositing pericellular type II collagen, and skeletal myoblasts expressing MyoD1. Importantly, the spectrum of differentiation capability was sustained through repeated passaging. Furthermore, serum-free conditions that led to high-efficiency smooth muscle differentiation were identified. rMSCs plated on collagen IV-coated surfaces and exposed to transforming growth factor-beta1 (TGF-beta1) differentiated into a homogeneous population expressing alpha-actin and calponin. Hence, clonogenic analysis confirmed the presence of a putative MSC population derived from the connective tissue of rat skeletal muscle. The ability to differentiate into a smooth muscle cell (SMC) phenotype, combined with a high proliferative capacity, make such a connective tissue-derived MSC population ideal for applications in vascular tissue construction.

Loss of T cell precursors after spaceflight and exposure to vector-averaged gravity

NASA Technical Reports Server (NTRS)

Woods, Chris C.; Banks, Krista E.; Gruener, Raphael; DeLuca, Dominick

2003-01-01

Using fetal thymus organ culture (FTOC), we examined the effects of spaceflight and vector-averaged gravity on T cell development. Under both conditions, the development of T cells was significantly attenuated. Exposure to spaceflight for 16 days resulted in a loss of precursors for CD4+, CD8+, and CD4+CD8+ T cells in a rat/mouse xenogeneic co-culture. A significant decrease in the same precursor cells, as well as a decrease in CD4-CD8- T cell precursors, was also observed in a murine C57BL/6 FTOC after rotation in a clinostat to produce a vector-averaged microgravity-like environment. The block in T cell development appeared to occur between the pre-T cell and CD4+CD8+ T cell stage. These data indicate that gravity plays a decisive role in the development of T cells.

Secondary immunization generates clonally related antigen-specific plasma cells and memory B cells.

PubMed

Frölich, Daniela; Giesecke, Claudia; Mei, Henrik E; Reiter, Karin; Daridon, Capucine; Lipsky, Peter E; Dörner, Thomas

2010-09-01

Rechallenge with T cell-dependent Ags induces memory B cells to re-enter germinal centers (GCs) and undergo further expansion and differentiation into plasma cells (PCs) and secondary memory B cells. It is currently not known whether the expanded population of memory B cells and PCs generated in secondary GCs are clonally related, nor has the extent of proliferation and somatic hypermutation of their precursors been delineated. In this study, after secondary tetanus toxoid (TT) immunization, TT-specific PCs increased 17- to 80-fold on days 6-7, whereas TT-specific memory B cells peaked (delayed) on day 14 with a 2- to 22-fold increase. Molecular analyses of V(H)DJ(H) rearrangements of individual cells revealed no major differences of gene usage and CDR3 length between TT-specific PCs and memory B cells, and both contained extensive evidence of somatic hypermutation with a pattern consistent with GC reactions. This analysis identified clonally related TT-specific memory B cells and PCs. Within clusters of clonally related cells, sequences shared a number of mutations but also could contain additional base pair changes. The data indicate that although following secondary immunization PCs can derive from memory B cells without further somatic hypermutation, in some circumstances, likely within GC reactions, asymmetric mutation can occur. These results suggest that after the fate decision to differentiate into secondary memory B cells or PCs, some committed precursors continue to proliferate and mutate their V(H) genes.

Isolation of Oct4-Expressing Extraembryonic Endoderm Precursor Cell Lines

PubMed Central

Debeb, Bisrat G.; Galat, Vasiliy; Epple-Farmer, Jessica; Iannaccone, Steve; Woodward, Wendy A.; Bader, Michael; Iannaccone, Philip; Binas, Bert

2009-01-01

Background The extraembryonic endoderm (ExEn) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4. Methodology/Principal Findings Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines (“XEN-P cell lines”), which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo. Conclusions/Significance Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity, (ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation. PMID:19784378

Anodes for alkaline electrolysis

DOEpatents

Soloveichik, Grigorii Lev [Latham, NY

2011-02-01

A method of making an anode for alkaline electrolysis cells includes adsorption of precursor material on a carbonaceous material, conversion of the precursor material to hydroxide form and conversion of precursor material from hydroxide form to oxy-hydroxide form within the alkaline electrolysis cell.

Identification, characterization and isolation of a common progenitor for osteoclasts, macrophages and dendritic cells from murine bone marrow and periphery

PubMed Central

Jacome-Galarza, Christian E.; Lee, Sun-Kyeong; Lorenzo, Joseph A.; LeonardoAguila, Hector

2012-01-01

Osteoclasts are specialized bone resorbing cells that derive from monocyte precursors. We have identified three populations of cells with high osteoclastogenic potential in murine bone marrow, which expressed the phenotype: B220−CD3−CD11b−/low CD115+ and either CD117hi, CD117intermediate or CD117low. We have evaluated these populations for their ability to also generate macrophages and dendritic cells. At a single cell level, the population expressing higher CD117 levels was able to generate bone-resorbing osteoclasts, phagocytic macrophages and antigen-presenting dendritic cells in vitro with efficiencies of over 90 percent, indicating that there exists a common developmental pathway for these cell types. Cells with osteoclastogenic potential also exist in blood and peripheral hematopoietic organs. Their functional meaning and/or their relationship with bone marrow progenitors is not well established. Hence, we characterized murine peripheral cell populations for their ability to form osteoclasts, macrophages and dendritic cells in vitro. The spleen and peripheral blood monocyte progenitors share phenotypic markers with bone marrow progenitors, but differ in their expression of CD11b, which was low in bone marrow but high in periphery. We propose that circulating monocyte progenitors are derived from a common bone marrow osteoclasts/macrophage/dendritic cell progenitor (OcMDC), which we have now characterized at a clonal level. However, the lineage relationship between the bone marrow and peripheral monocyte progenitors has yet to be defined. PMID:23165930

The planarian nanos-like gene Smednos is expressed in germline and eye precursor cells during development and regeneration.

PubMed

Handberg-Thorsager, Mette; Saló, Emili

2007-05-01

Planarians are highly regenerative organisms with the ability to remake all their cell types, including the germ cells. The germ cells have been suggested to arise from totipotent neoblasts through epigenetic mechanisms. Nanos is a zinc-finger protein with a widely conserved role in the maintenance of germ cell identity. In this work, we describe the expression of a planarian nanos-like gene Smednos in two kinds of precursor cells namely, primordial germ cells and eye precursor cells, during both development and regeneration of the planarian Schmidtea mediterranea. In sexual planarians, Smednos is expressed in presumptive male primordial germ cells of embryos from stage 8 of embryogenesis and throughout development of the male gonads and in the female primordial germ cells of the ovary. Thus, upon hatching, juvenile planarians do possess primordial germ cells. In the asexual strain, Smednos is expressed in presumptive male and female primordial germ cells. During regeneration, Smednos expression is maintained in the primordial germ cells, and new clusters of Smednos-positive cells appear in the regenerated tissue. Remarkably, during the final stages of development (stage 8 of embryogenesis) and during regeneration of the planarian eye, Smednos is expressed in cells surrounding the differentiating eye cells, possibly corresponding to eye precursor cells. Our results suggest that similar genetic mechanisms might be used to control the differentiation of precursor cells during development and regeneration in planarians.

The alveolar macrophage.

PubMed

Bowden, D H

1984-04-01

The pulmonary macrophagic system is critical to the defense of the lung, keeping the alveoli clean and sterile and responding on demand with an adaptive outpouring of new cells into the air sacs. Under basal conditions alveolar macrophages, in common with other mononuclear phagocytes, are derived from the bone marrow. A population of macrophage precursors within the pulmonary interstitium provides a reserve pool capable of proliferation and delivery of phagocytes in response to unusually heavy loads of inhaled particles. This reserve system also produces macrophages when monocytic precursors in the bone marrow are depleted by diseases such as leukemia. The alveolar macrophage is destined to ingest particulate matter and to be eliminated along the mucociliary pathway; clearance by lymphatics is of minor importance and macrophages probably do not recross the alveolar epithelium to reach the pulmonary interstitial compartment. Although the protective role of the macrophage is dominant, this cell may participate, directly or indirectly, in the genesis of two major groups of chronic pulmonary disease, interstitial fibrosis and emphysema. Such inappropriate responses involve interactions with fibroblastic cells and tissue injury initiated by proteases secreted by the macrophage.

Fibrillar α-Synuclein and Huntingtin Exon 1 Assemblies Are Toxic to the Cells

PubMed Central

Pieri, Laura; Madiona, Karine; Bousset, Luc; Melki, Ronald

2012-01-01

The aggregation of alpha-synuclein (α-syn) and huntingtin (htt) into fibrillar assemblies in nerve and glial cells is a molecular hallmark of Parkinson's and Huntington's diseases. Within the aggregation process, prefibrillar and fibrillar oligomeric species form. Prefibrillar assemblies rather than fibrils are nowadays considered cytotoxic. However, recent reports describing spreading of fibrillar assemblies from one cell to another, in cell cultures, animal models, and brains of grafted patients suggest a critical role for fibrillar assemblies in pathogenesis. Here we compare the cytotoxic effect of defined and comparable particle concentrations of on-assembly pathway oligomeric and fibrillar α-syn and Htt fragment corresponding to the first exon of the protein (HttEx1). We show that homogeneous populations of α-syn and HttEx1 fibrils, rather than their precursor on-assembly pathway oligomers, are highly toxic to cultured cells and induce apoptotic cell death. We document the reasons that make fibrils toxic. We show that α-syn and HttEx1 fibrils bind and permeabilize lipid vesicles. We also show that fibrils binding to the plasma membrane in cultured cells alter Ca2+ homeostasis. Overall, our data indicate that fibrillar α-syn and HttEx1, rather than their precursor oligomers, are highly cytotoxic, the toxicity being associated to their ability to bind and permeabilize the cell membranes. PMID:22735540

Thymus development in Macaca fascicularis (Cynomolgus monkey): an approach for toxicology and embryology.

PubMed

Buse, Eberhard; Habermann, Gunnar; Vogel, Friedhelm

2006-05-01

Thymus development was studied in the cynomolgus monkey from day 35 of gestation (gd 35) to the stage of advanced involution in a 21-year-old monkey. Special emphasis was placed on thymus cell generation and cellular pattern formation. At gd 35, the epithelial bud of the thymus was visible in a sagittal position at the level of the thoracic aperture. At gd 50, first lymphocyte-like cells and few Human Leukocyte Antigen-D Region (HLA-DR) immunoreactive cells appeared. The cortico-medullary differentiation, Hassall's body precursors and faint immunoreactivity for T-lymphocytes (CD 3-positive) were detected from gd 60 onwards. First macrophages (CD 68 positive) were apparent at day 70, first CD 20 immunoreactive cells (B-lymphocyte-like cells) at gd 85, and natural killer cells (M1014 immunoreactive) at gd 100. At gd 100 all evaluated cell populations present in the adult cynomolgus monkey thymus were in place, whereas no B- and T-cell precursors or (CD 34 and CD 117, respectively) dendritic cells (CD 35 positive cells) were present. All these immunopositive cells persisted, partly with changing distribution patterns, until the advanced age of 21 years with the exception of natural killer cells, which were present only until adult ages (evaluation at 4-7 years). The rationale of this study was to analyse thymic development in the cynomolgus monkey and to evaluate the relevance of the development of thymus in non-human primate as a model for corresponding human targeted toxicological research.

Concanavalin A-induced and spontaneous suppressor cell activities in peripheral blood lymphocytes and spleen cells from gastric cancer patients.

PubMed

Toge, T; Hamamoto, S; Itagaki, E; Yajima, K; Tanada, M; Nakane, H; Kohno, H; Nakanishi, K; Hattori, T

1983-11-01

In 173 gastric cancer patients, activities of Concanavalin-A-induced suppressor cells (Con-AS) and spontaneous suppressor cells (SpS) in peripheral blood lymphocytes (PBL), splenic vein lymphocytes (SVL), and spleen cells (SCs) were investigated. Suppressions by Con-AS in PBL were significantly effective in patients of Stages III and IV, while suppressions by SpS were effective in patients with recurrent tumors. Thus, in PBLs of cancer patients, suppressor precursors, which are considered to be activated in vitro by Concanavalin-A, seemed to appear with the advances of the disease, and SpS activities, which could be already activated in vivo, seemed to increase in the terminal stage. In SCs, increased activities of Con-AS, but normal activities of SpS, were observed, and these suppressor-cell populations consisted of glass nonadherent cells. Suppressor activities of SCs would be due to suppressor T-cells, not to other types of cells. Furthermore, Con-AS existed in the medium-sized lymphocytes, which were fractionated on the basis of cell size, while SpS in the large-sized lymphocytes. A higher proportion of T-cells, bearing Fc receptors for IgG, was observed in the larger-sized lymphocyte fractions. Cell numbers in the large-sized lymphocyte fraction tended to increase with the advances of tumors. From these results, it is suggested that higher presence of suppressor precursors and the increase of SpS activities may occur in cancer patients, depending on the tumor advancing.

Bystander hyperactivation of preimmune CD8+ T cells in chronic HCV patients

PubMed Central

Alanio, Cécile; Nicoli, Francesco; Sultanik, Philippe; Flecken, Tobias; Perot, Brieuc; Duffy, Darragh; Bianchi, Elisabetta; Lim, Annick; Clave, Emmanuel; van Buuren, Marit M; Schnuriger, Aurélie; Johnsson, Kerstin; Boussier, Jeremy; Garbarg-Chenon, Antoine; Bousquet, Laurence; Mottez, Estelle; Schumacher, Ton N; Toubert, Antoine; Appay, Victor; Heshmati, Farhad; Thimme, Robert; Pol, Stanislas; Mallet, Vincent; Albert, Matthew L

2015-01-01

Chronic infection perturbs immune homeostasis. While prior studies have reported dysregulation of effector and memory cells, little is known about the effects on naïve T cell populations. We performed a cross-sectional study of chronic hepatitis C (cHCV) patients using tetramer-associated magnetic enrichment to study antigen-specific inexperienced CD8+ T cells (i.e., tumor or unrelated virus-specific populations in tumor-free and sero-negative individuals). cHCV showed normal precursor frequencies, but increased proportions of memory-phenotype inexperienced cells, as compared to healthy donors or cured HCV patients. These observations could be explained by low surface expression of CD5, a negative regulator of TCR signaling. Accordingly, we demonstrated TCR hyperactivation and generation of potent CD8+ T cell responses from the altered T cell repertoire of cHCV patients. In sum, we provide the first evidence that naïve CD8+ T cells are dysregulated during cHCV infection, and establish a new mechanism of immune perturbation secondary to chronic infection. DOI: http://dx.doi.org/10.7554/eLife.07916.001 PMID:26568315

Biosynthesis and processing of platelet GPIIb-IIIa in human megakaryocytes.

PubMed

Duperray, A; Berthier, R; Chagnon, E; Ryckewaert, J J; Ginsberg, M; Plow, E; Marguerie, G

1987-06-01

Platelet membrane glycoprotein IIb-IIIa forms a calcium-dependent heterodimer and constitutes the fibrinogen receptor on stimulated platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIIa is a single chain protein. These proteins are synthesized in the bone marrow by megakaryocytes, but the study of their synthesis has been hampered by the difficulty in obtaining enriched population of megakaryocytes in large numbers. To examine the biosynthesis and processing of GPIIb-IIIa, purified human megakaryocytes were isolated from liquid cultures of cryopreserved leukocytes stem cell concentrates from patients with chronic myelogenous leukemia. Immunoprecipitation of [35S]methionine pulse-chase-labeled cell extracts by antibodies specific for the alpha or beta subunits of GPIIb indicated that GPIIb was derived from a precursor of Mr 130,000 that contains the alpha and beta subunits. This precursor was converted to GPIIb with a half-life of 4-5 h. No precursor form of GPIIIa was detected. The glycosylation of GPIIb-IIIa was examined in megakaryocytes by metabolic labeling in the presence of tunicamycin, monensin, or treatment with endoglycosidase H. The polypeptide backbones of the GPIIb and the GPIIIa have molecular masses of 120 and 90 kD, respectively. High-mannose oligosaccharides are added to these polypeptide backbones co-translationally. The GPIIb precursor is then processed with conversion of high-mannose to complex type carbohydrates yielding the mature subunits GPIIb alpha (Mr 116,000) and GPIIb beta (Mr 25,000). No posttranslational processing of GPIIIa was detected.

Nanoscale liposomal formulation of a SYK P-site inhibitor against B-precursor leukemia

PubMed Central

Qazi, Sanjive; Cely, Ingrid; Sahin, Kazim; Shahidzadeh, Anoush; Ozercan, Ibrahim; Yin, Qian; Gaynon, Paul; Termuhlen, Amanda; Cheng, Jianjun

2013-01-01

We report preclinical proof of principle for effective treatment of B-precursor acute lymphoblastic leukemia (ALL) by targeting the spleen tyrosine kinase (SYK)–dependent antiapoptotic blast cell survival machinery with a unique nanoscale pharmaceutical composition. This nanoscale liposomal formulation (NLF) contains the pentapeptide mimic 1,4-Bis (9-O dihydroquinidinyl) phthalazine/hydroquinidine 1,4-phathalazinediyl diether (C61) as the first and only selective inhibitor of the substrate binding P-site of SYK. The C61 NLF exhibited a very favorable pharmacokinetic and safety profile in mice, induced apoptosis in primary B-precursor ALL blast cells taken directly from patients as well as in vivo clonogenic ALL xenograft cells, destroyed the in vivo clonogenic fraction of ALL blast cells, and, at nontoxic dose levels, exhibited potent in vivo antileukemic activity against patient-derived ALL cells in xenograft models of aggressive B-precursor ALL. Our findings establish SYK as an attractive molecular target for therapy of B-precursor ALL. Further development of the C61 NLF may provide the foundation for therapeutic innovation against therapy-refractory B-precursor ALL. PMID:23568490

Podocytes populate cellular crescents in a murine model of inflammatory glomerulonephritis.

PubMed

Moeller, Marcus J; Soofi, Abdulsalaam; Hartmann, Inge; Le Hir, Michel; Wiggins, Roger; Kriz, Wilhelm; Holzman, Lawrence B

2004-01-01

Cellular crescents are a defining histologic finding in many forms of inflammatory glomerulonephritis. Despite numerous studies, the origin of glomerular crescents remains unresolved. A genetic cell lineage-mapping study with a novel transgenic mouse model was performed to investigate whether visceral glomerular epithelial cells, termed podocytes, are precursors of cells that populate cellular crescents. The podocyte-specific 2.5P-Cre mouse line was crossed with the ROSA26 reporter line, resulting in irreversible constitutive expression of beta-galactosidase in doubly transgenic 2.5P-Cre/ROSA26 mice. In these mice, crescentic glomerulonephritis was induced with a previously described rabbit anti-glomerular basement membrane antiserum nephritis approach. Interestingly, beta-galactosidase-positive cells derived from podocytes adhered to the parietal basement membrane and populated glomerular crescents during the early phases of cellular crescent formation, accounting for at least one-fourth of the total cell mass. In cellular crescents, the proliferation marker Ki-67 was expressed in beta-galactosidase-positive and beta-galactosidase-negative cells, indicating that both cell types contributed to the formation of cellular crescents through proliferation in situ. Podocyte-specific antigens, including WT-1, synaptopodin, nephrin, and podocin, were not expressed by any cells in glomerular crescents, suggesting that podocytes underwent profound phenotypic changes in this nephritis model.

Mitotic position and morphology of committed precursor cells in the zebrafish retina adapt to architectural changes upon tissue maturation.

PubMed

Weber, Isabell P; Ramos, Ana P; Strzyz, Paulina J; Leung, Louis C; Young, Stephen; Norden, Caren

2014-04-24

The development of complex neuronal tissues like the vertebrate retina requires the tight orchestration of cell proliferation and differentiation. Although the complexity of transcription factors and signaling pathways involved in retinogenesis has been studied extensively, the influence of tissue maturation itself has not yet been systematically explored. Here, we present a quantitative analysis of mitotic events during zebrafish retinogenesis that reveals three types of committed neuronal precursors in addition to the previously known apical progenitors. The identified precursor types present at distinct developmental stages and exhibit different mitotic location (apical versus nonapical), cleavage plane orientation, and morphology. Interestingly, the emergence of nonapically dividing committed bipolar cell precursors can be linked to an increase in apical crowding caused by the developing photoreceptor cell layer. Furthermore, genetic interference with neuronal subset specification induces ectopic divisions of committed precursors, underlining the finding that progressing morphogenesis can effect precursor division position. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

FGF signalling controls the specification of hair placode-derived SOX9 positive progenitors to Merkel cells.

PubMed

Nguyen, Minh Binh; Cohen, Idan; Kumar, Vinod; Xu, Zijian; Bar, Carmit; Dauber-Decker, Katherine L; Tsai, Pai-Chi; Marangoni, Pauline; Klein, Ophir D; Hsu, Ya-Chieh; Chen, Ting; Mikkola, Marja L; Ezhkova, Elena

2018-06-13

Merkel cells are innervated mechanosensory cells responsible for light-touch sensations. In murine dorsal skin, Merkel cells are located in touch domes and found in the epidermis around primary hairs. While it has been shown that Merkel cells are skin epithelial cells, the progenitor cell population that gives rise to these cells is unknown. Here, we show that during embryogenesis, SOX9-positive (+) cells inside hair follicles, which were previously known to give rise to hair follicle stem cells (HFSCs) and cells of the hair follicle lineage, can also give rise to Merkel Cells. Interestingly, while SOX9 is critical for HFSC specification, it is dispensable for Merkel cell formation. Conversely, FGFR2 is required for Merkel cell formation but is dispensable for HFSCs. Together, our studies uncover SOX9(+) cells as precursors of Merkel cells and show the requirement for FGFR2-mediated epithelial signalling in Merkel cell specification.

Generation of Megakaryocytes and Platelets from Human Pluripotent Stem Cells.

PubMed

Pick, Marjorie

2016-01-01

Human pluripotent stem cells (hPSC) have the potential to produce any tissue type in the body and thus represent a source of cells for regenerative medicine. Here we have shown that human platelets can be produced from embryonic or induced pluripotent stem cells in a defined culture system. We describe a serum- and feeder-free culture system that enabled the generation of megakaryocyte (Mk) progenitors and functional platelets from hPSCs. After 13 days the differentiated population included precursor cells that formed colonies containing differentiated Mks, and after 20 days these Mks were able to fragment into platelet-like particles that were functional. This protocol represents an important step towards the generation of human platelets for therapeutic use.

Postnatal reduction of BDNF regulates the developmental remodeling of taste bud innervation.

PubMed

Huang, Tao; Ma, Liqun; Krimm, Robin F

2015-09-15

The refinement of innervation is a common developmental mechanism that serves to increase the specificity of connections following initial innervation. In the peripheral gustatory system, the extent to which innervation is refined and how refinement might be regulated is unclear. The initial innervation of taste buds is controlled by brain-derived neurotrophic factor (BDNF). Following initial innervation, taste receptor cells are added and become newly innervated. The connections between the taste receptor cells and nerve fibers are likely to be specific in order to retain peripheral coding mechanisms. Here, we explored the possibility that the down-regulation of BDNF regulates the refinement of taste bud innervation during postnatal development. An analysis of BDNF expression in Bdnf(lacZ/+) mice and real-time reverse transcription polymerase chain reaction (RT-PCR) revealed that BDNF was down-regulated between postnatal day (P) 5 and P10. This reduction in BDNF expression was due to a loss of precursor/progenitor cells that express BDNF, while the expression of BDNF in the subpopulations of taste receptor cells did not change. Gustatory innervation, which was identified by P2X3 immunohistochemistry, was lost around the perimeter where most progenitor/precursor cells are located. In addition, the density of innervation in the taste bud was reduced between P5 and P10, because taste buds increase in size without increasing innervation. This reduction of innervation density was blocked by the overexpression of BDNF in the precursor/progenitor population of taste bud cells. Together these findings indicate that the process of BDNF restriction to a subpopulation of taste receptor cells between P5 and P10, results in a refinement of gustatory innervation. We speculate that this refinement results in an increased specificity of connections between neurons and taste receptor cells during development. Copyright © 2015 Elsevier Inc. All rights reserved.

CD19 CAR T Cells for B Cell Malignancies After Allogeneic Transplant

ClinicalTrials.gov

2017-02-14

Philadelphia Chromosome Negative Adult Precursor Acute Lymphoblastic Leukemia; Philadelphia Chromosome Positive Adult Precursor Acute Lymphoblastic Leukemia; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Diffuse Large Cell Lymphoma; Recurrent Adult Immunoblastic Large Cell Lymphoma; Recurrent Mantle Cell Lymphoma; Refractory Chronic Lymphocytic Leukemia

Hedgehog-responsive candidate cell of origin for diffuse intrinsic pontine glioma

PubMed Central

Monje, Michelle; Mitra, Siddhartha S.; Freret, Morgan E.; Raveh, Tal B.; Kim, James; Masek, Marilyn; Attema, Joanne L.; Haddix, Terri; Edwards, Michael S. B.; Fisher, Paul G.; Weissman, Irving L.; Rowitch, David H.; Vogel, Hannes; Wong, Albert J.; Beachy, Philip A.

2011-01-01

Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive tumors of childhood that are almost universally fatal. Our understanding of this devastating cancer is limited by a dearth of available tissue for study and by the lack of a faithful animal model. Intriguingly, DIPGs are restricted to the ventral pons and occur during a narrow window of middle childhood, suggesting dysregulation of a postnatal neurodevelopmental process. Here, we report the identification of a previously undescribed population of immunophenotypic neural precursor cells in the human and murine brainstem whose temporal and spatial distributions correlate closely with the incidence of DIPG and highlight a candidate cell of origin. Using early postmortem DIPG tumor tissue, we have established in vitro and xenograft models and find that the Hedgehog (Hh) signaling pathway implicated in many developmental and oncogenic processes is active in DIPG tumor cells. Modulation of Hh pathway activity has functional consequences for DIPG self-renewal capacity in neurosphere culture. The Hh pathway also appears to be active in normal ventral pontine precursor-like cells of the mouse, and unregulated pathway activity results in hypertrophy of the ventral pons. Together, these findings provide a foundation for understanding the cellular and molecular origins of DIPG, and suggest that the Hh pathway represents a potential therapeutic target in this devastating pediatric tumor. PMID:21368213

High-LET radiation induces inflammation and persistent changes in markers of hippocampal neurogenesis.

PubMed

Rola, Radoslaw; Sarkissian, Vahe; Obenaus, Andre; Nelson, Gregory A; Otsuka, Shinji; Limoli, Charles L; Fike, John R

2005-10-01

Exposure to heavy-ion radiation is considered a potential health risk in long-term space travel. It may result in the loss of critical cellular components in complex systems like the central nervous system (CNS), which could lead to performance decrements that ultimately could compromise mission goals and long-term quality of life. Specific hippocampal-dependent cognitive impairment occurs after whole-body 56Fe-particle irradiation, and while the pathogenesis of this effect is not yet clear, it may involve damage to neural precursor cells in the hippocampal dentate gyrus. We irradiated mice with 1-3 Gy of 12C or 56Fe ions and 9 months later quantified proliferating cells and immature neurons in the dentate subgranular zone (SGZ). Our results showed that reductions in these cells were dependent on the dose and LET. When compared with data for mice that were studied 3 months after 56Fe-particle irradiation, our current data suggest that these changes are not only persistent but may worsen with time. Loss of precursor cells was also associated with altered neurogenesis and a robust inflammatory response. These results indicate that high-LET radiation has a significant and long-lasting effect on the neurogenic population in the hippocampus that involves cell loss and changes in the microenvironment.

Generation of amyloid-β is reduced by the interaction of calreticulin with amyloid precursor protein, presenilin and nicastrin.

PubMed

Stemmer, Nina; Strekalova, Elena; Djogo, Nevena; Plöger, Frank; Loers, Gabriele; Lutz, David; Buck, Friedrich; Michalak, Marek; Schachner, Melitta; Kleene, Ralf

2013-01-01

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer's disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca(2+)- and N-glycan-independent interaction is mediated by amino acids 330-344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β42 levels in culture supernatants, while transfection with the P-domain increases amyloid-β40 levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330-344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β40 and amyloid-β42 levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer's disease.

Inv(7)(q22q36) in refactory anemia with excess blasts

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

Rayburn, J.; Stegeman, D.; Berger, C.

1994-09-01

Morphological review of bone marrow from an 89 year-old male revealed an immature cell population with increased blasts (25% CD34 positive). However, the morphology was not sufficiently clear to discriminate lymphoid from myeloid precursors. Immunophenotypically, there was evidence for both lymphoid and myeloid derivation with dual expression of CD5 and CD20, aberrant expression of CD19 versus CD20, and an increased CD13 population. Twenty percent (20%) of the cells were TdT positive. Cytogenetically, an inversion of chromosome 7, inv(7)(q22q36), was observed in 9 of 20 cells. This abnormality has been reported only once previously, in association with refractory anemia with excessmore » blasts (RAEB). The patient, to date, has not developed an acute leukemic process, but remains in a myelodysplastic state, defined as RAEB.« less

Early T-cell precursor acute lymphoblastic leukaemia in children treated in AIEOP centres with AIEOP-BFM protocols: a retrospective analysis.

PubMed

Conter, Valentino; Valsecchi, Maria Grazia; Buldini, Barbara; Parasole, Rosanna; Locatelli, Franco; Colombini, Antonella; Rizzari, Carmelo; Putti, Maria Caterina; Barisone, Elena; Lo Nigro, Luca; Santoro, Nicola; Ziino, Ottavio; Pession, Andrea; Testi, Anna Maria; Micalizzi, Concetta; Casale, Fiorina; Pierani, Paolo; Cesaro, Simone; Cellini, Monica; Silvestri, Daniela; Cazzaniga, Giovanni; Biondi, Andrea; Basso, Giuseppe

2016-02-01

Early T-cell precursor acute lymphoblastic leukaemia was recently recognised as a distinct leukaemia and reported as associated with poor outcomes. We aimed to assess the outcome of early T-cell precursor acute lymphoblastic leukaemia in patients from the Italian Association of Pediatric Hematology Oncology (AIEOP) centres treated with AIEOP-Berlin-Frankfurt-Münster (AIEOP-BFM) protocols. In this retrospective analysis, we included all children aged from 1 to less than 18 years with early T-cell precursor acute lymphoblastic leukaemia immunophenotype diagnosed between Jan 1, 2008, and Oct 31, 2014, from AIEOP centres. Early T-cell precursors were defined as being CD1a and CD8 negative, CD5 weak positive or negative, and positive for at least one of the following antigens: CD34, CD117, HLADR, CD13, CD33, CD11b, or CD65. Treatment was based on AIEOP-BFM acute lymphoblastic leukaemia 2000 (NCT00613457) or AIEOP-BFM acute lymphoblastic leukaemia 2009 protocols (European Clinical Trials Database 2007-004270-43). The main differences in treatment and stratification of T-cell acute lymphoblastic leukaemia between the two protocols were that in the 2009 protocol only, pegylated L-asparaginase was substituted for Escherichia coli L-asparaginase, patients with prednisone poor response received an additional dose of cyclophosphamide at day 10 of phase IA, and high minimal residual disease at day 15 assessed by flow cytometry was used as a high-risk criterion. Outcomes were assessed in terms of event-free survival, disease-free survival, and overall survival. Early T-cell precursor acute lymphoblastic leukaemia was diagnosed in 49 patients. Compared with overall T-cell acute lymphoblastic leukaemia, it was associated with absence of molecular markers for PCR detection of minimal residual disease in 25 (56%) of 45 patients; prednisone poor response in 27 (55%) of 49 patients; high minimal residual disease at day 15 after starting therapy in 25 (64%) of 39 patients (bone marrow blasts ≥ 10%, by flow cytometry); no complete remission after phase IA in 7 (15%) of 46 patients (bone marrow blasts ≥ 5%, morphologically); and high PCR minimal residual disease (≥ 5 × 10(-4)) at day 33 after starting therapy in 17 (85%) of 20 patients with markers available. Overall, 38 (78%) of 49 patients are in continuous complete remission, including 13 of 18 after haemopoietic stem cell transplantation, with three deaths in induction, five deaths after haemopoietic stem cell transplantation, and three relapses. Severe adverse events in the 2009 study were reported in 10 (30%) of 33 patients with early T-cell precursor acute lymphoblastic leukaemia versus 24 (15%) of 164 patients without early T-cell precursor acute lymphoblastic leukaemia and life-threatening events in induction phase IA occurred in 4 (12%) of 33 patients with early T-cell precursor acute lymphoblastic leukaemia versus 7 (4%) of 164 patients without early T-cell precursor acute lymphoblastic leukaemia. No difference was seen in the subsequent consolidation phase IB of protocol I. Early T-cell precursor acute lymphoblastic leukaemia is characterised by poor early response to conventional induction treatment. Consolidation phase IB, based on cyclophosphamide, 6-mercaptopurine, and ara-C at conventional (non-high) doses is effective in reducing minimal residual disease. Although the number of patients and observational time are limited, patients with early T-cell precursor acute lymphoblastic leukaemia treated with current BFM stratification and treatment strategy have a favourable outcome compared with earlier reports. The role of innovative therapies and haemopoietic stem cell therapy in early T-cell precursor acute lymphoblastic leukaemia needs to be assessed. None. Copyright © 2016 Elsevier Ltd. All rights reserved.

Immune responses to epstein-barr virus in atomic bomb survivors: Study of precursor frequency of cytotoxic lymphocytes and titer levels of anti-Epstein-Barr virus-related antibodies

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

Kusunoki, Yoichiro; Kyoizumi, Seishi; Saito, Mayumi

Precursor frequencies of cytotoxic lymphocytes to autologous Epstein-Barr virus-transformed B cells and serum titers of anti-Epstein-Barr virus-related antibodies were measured in 68 atomic bomb survivors to clarify the immune mechanism controlling Epstein-Barr virus infection. The precursor frequency was negatively correlated with the titer of anti-early antigen lgG, which is probably produced at the stage of viral reactivation. A positive correlation between the precursor frequency and titer of anti-Epstein-Barr virus-associated nuclear antigen antibody was also observed, indicating that the precursor frequency reflects the degree of in vivo destruction by T cells of the virus-infected cells. These results suggest that T-cell memorymore » specific to Epstein-Barr virus keeps the virus under control and that the precursor frequency assay is useful for the evaluation of immune responses to Epstein-Barr virus. However, no significant effect of atomic bomb radiation on the precursor frequency was observed in the present study, probably due to the limited number of participants. 24 refs., 4 figs., 2 tabs.« less

Sparing of extraocular muscle in aging and muscular dystrophies: A myogenic precursor cell hypothesis

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

Kallestad, Kristen M.; Hebert, Sadie L.; McDonald, Abby A.

2011-04-01

The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry,more » EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin{sup -/-} (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a subpopulation of mpcs, the EOMCD34 cells, that are retained in significantly higher percentages in normal, mdx and DKO mice EOM, appear to be resistant to elevated levels of oxidative stress and toxins, and actively proliferate throughout life. Current studies are focused on further defining the EOMCD34 cell subtype molecularly, with the hopes that this may shed light on a cell type with potential therapeutic use in patients with sarcopenia, cachexia, or muscular dystrophy.« less

IGFBP-7 inhibits the differentiation of oligodendrocyte precursor cells via regulation of Wnt/β-Catenin signaling.

PubMed

Li, Nan; Han, Jinfeng; Tang, Jing; Ying, Yanqin

2018-06-01

Oligodendrocytes (OLs) are glial cells that form myelin sheaths in the central nervous system. Myelin sheath plays important role in nervous system and loss of it in neurodegenerative diseases can lead to impairment of movement. Understanding the signals and factors that regulate OL differentiation can help to address novel strategies for improving myelin repair in neurodegenerative diseases. The aim of this study was to investigate the role of insulin-like growth factor-binding proteins 7 (IGFBP-7) in differentiating OL precursor cells (OPCs). It was found that oligodendrocyte precursors undergoing differentiation were accompanied by selective expression of IGFBP-7. In addition, knockdown of IGFBP-7 promoted differentiation of oligodendrocytes and increased formation of myelin in cultured cells. In contrast, excessive expression of IGFBP-7 inhibited differentiation of oligodendrocytes. Furthermore, overexpression of IGFBP-7 in oligodendrocyte precursor cells increased transcription of Wnt target genes and promoted β-Catenin nuclear translocation. These findings suggest that IGFBP-7 negatively regulates differentiation of oligodendrocyte precursor cells via regulation of Wnt/β-Catenin signaling. © 2017 Wiley Periodicals, Inc.

Development of the Drosophila entero-endocrine lineage and its specification by the Notch signaling pathway

PubMed Central

Takashima, Shigeo; Adams, Katrina L.; Ortiz, Paola A.; Ying, Chong T.; Moridzadeh, Rameen; Younossi-Hartenstein, Amelia; Hartenstein, Volker

2013-01-01

In this paper we have investigated the developmental-genetic steps that shape the entero-endocrine system of Drosophila melanogaster from the embryo to the adult. The process starts in the endoderm of the early embryo where precursors of endocrine cells and enterocytes of the larval midgut, as well as progenitors of the adult midgut, are specified by a Notch signaling-dependent mechanism. In a second step that occurs during the late larval period, enterocytes and endocrine cells of a transient pupal midgut are selected from within the clusters of adult midgut progenitors. As in the embryo, activation of the Notch pathway triggers enterocyte differentiation, and inhibits cells from further proliferation or choosing the endocrine fate. The third step of entero-endocrine cell development takes place at a mid-pupal stage. Before this time point, the epithelial layer destined to become the adult midgut is devoid of endocrine cells. However, precursors of the intestinal midgut stem cells (pISCs) are already present. After an initial phase of symmetric divisions which causes an increase in their own population size, pISCs start to spin off cells that become postmitotic and express the endocrine fate marker, Prospero. Activation of Notch in pISCs forces these cells into an enterocyte fate. Loss of Notch function causes an increase in the proliferatory activity of pISCs, as well as a higher ratio of Prospero-positive cells. PMID:21382366

Purification of Immature Neuronal Cells from Neural Stem Cell Progeny

PubMed Central

Azari, Hassan; Osborne, Geoffrey W.; Yasuda, Takahiro; Golmohammadi, Mohammad G.; Rahman, Maryam; Deleyrolle, Loic P.; Esfandiari, Ebrahim; Adams, David J.; Scheffler, Bjorn; Steindler, Dennis A.; Reynolds, Brent A.

2011-01-01

Large-scale proliferation and multi-lineage differentiation capabilities make neural stem cells (NSCs) a promising renewable source of cells for therapeutic applications. However, the practical application for neuronal cell replacement is limited by heterogeneity of NSC progeny, relatively low yield of neurons, predominance of astrocytes, poor survival of donor cells following transplantation and the potential for uncontrolled proliferation of precursor cells. To address these impediments, we have developed a method for the generation of highly enriched immature neurons from murine NSC progeny. Adaptation of the standard differentiation procedure in concert with flow cytometry selection, using scattered light and positive fluorescent light selection based on cell surface antibody binding, provided a near pure (97%) immature neuron population. Using the purified neurons, we screened a panel of growth factors and found that bone morphogenetic protein-4 (BMP-4) demonstrated a strong survival effect on the cells in vitro, and enhanced their functional maturity. This effect was maintained following transplantation into the adult mouse striatum where we observed a 2-fold increase in the survival of the implanted cells and a 3-fold increase in NeuN expression. Additionally, based on the neural-colony forming cell assay (N-CFCA), we noted a 64 fold reduction of the bona fide NSC frequency in neuronal cell population and that implanted donor cells showed no signs of excessive or uncontrolled proliferation. The ability to provide defined neural cell populations from renewable sources such as NSC may find application for cell replacement therapies in the central nervous system. PMID:21687800

Derivation of Multipotent Mesenchymal Precursors from Human Embryonic Stem Cells

PubMed Central

Barberi, Tiziano; Willis, Lucy M; Socci, Nicholas D; Studer, Lorenz

2005-01-01

Background Human embryonic stem cells provide access to the earliest stages of human development and may serve as a source of specialized cells for regenerative medicine. Thus, it becomes crucial to develop protocols for the directed differentiation of embryonic stem cells into tissue-restricted precursors. Methods and Findings Here, we present culture conditions for the derivation of unlimited numbers of pure mesenchymal precursors from human embryonic stem cells and demonstrate multilineage differentiation into fat, cartilage, bone, and skeletal muscle cells. Conclusion Our findings will help to elucidate the mechanism of mesoderm specification during embryonic stem cell differentiation and provide a platform to efficiently generate specialized human mesenchymal cell types for future clinical applications. PMID:15971941

Cellular Functions of the Autism Risk Factor PTCHD1 in Mice.

PubMed

Tora, David; Gomez, Andrea M; Michaud, Jean-Francois; Yam, Patricia T; Charron, Frédéric; Scheiffele, Peter

2017-12-06

The gene patched domain containing 1 ( PTCHD1 ) is mutated in patients with autism spectrum disorders and intellectual disabilities and has been hypothesized to contribute to Sonic hedgehog (Shh) signaling and synapse formation. We identify a panel of Ptchd1-interacting proteins that include postsynaptic density proteins and the retromer complex, revealing a link to critical regulators of dendritic and postsynaptic trafficking. Ptchd1 knock-out (KO) male mice exhibit cognitive alterations, including defects in a novel object recognition task. To test whether Ptchd1 is required for Shh-dependent signaling, we examined two Shh-dependent cell populations that express high levels of Ptchd1 mRNA: cerebellar granule cell precursors and dentate granule cells in the hippocampus. We found that proliferation of these neuronal precursors was not altered significantly in Ptchd1 KO male mice. We used whole-cell electrophysiology and anatomical methods to assess synaptic function in Ptchd1-deficient dentate granule cells. In the absence of Ptchd1, we observed profound disruption in excitatory/inhibitory balance despite normal dendritic spine density on dentate granule cells. These findings support a critical role of the Ptchd1 protein in the dentate gyrus, but indicate that it is not required for structural synapse formation in dentate granule cells or for Shh-dependent neuronal precursor proliferation. SIGNIFICANCE STATEMENT The mechanisms underlying neuronal and cellular alterations resulting from patched domain containing 1 ( Ptchd1 ) gene mutations are unknown. The results from this study support an association with dendritic trafficking complexes of Ptchd1. Loss-of-function experiments do not support a role in sonic hedgehog-dependent signaling, but reveal a disruption of synaptic transmission in the mouse dentate gyrus. The findings will help to guide ongoing efforts to understand the etiology of neurodevelopmental disorders arising from Ptchd1 deficiency. Copyright © 2017 the authors 0270-6474/17/3711993-13$15.00/0.

Eph regulates dorsoventral asymmetry of the notochord plate and convergent extension-mediated notochord formation.

PubMed

Oda-Ishii, Izumi; Ishii, Yasuo; Mikawa, Takashi

2010-10-29

The notochord is a signaling center required for the patterning of the vertebrate embryonic midline, however, the molecular and cellular mechanisms involved in the formation of this essential embryonic tissue remain unclear. The urochordate Ciona intestinalis develops a simple notochord from 40 specific postmitotic mesodermal cells. The precursors intercalate mediolaterally and establish a single array of disk-shaped notochord cells along the midline. However, the role that notochord precursor polarization, particularly along the dorsoventral axis, plays in this morphogenetic process remains poorly understood. Here we show that the notochord preferentially accumulates an apical cell polarity marker, aPKC, ventrally and a basement membrane marker, laminin, dorsally. This asymmetric accumulation of apicobasal cell polarity markers along the embryonic dorsoventral axis was sustained in notochord precursors during convergence and extension. Further, of several members of the Eph gene family implicated in cellular and tissue morphogenesis, only Ci-Eph4 was predominantly expressed in the notochord throughout cell intercalation. Introduction of a dominant-negative Ci-Eph4 to notochord precursors diminished asymmetric accumulation of apicobasal cell polarity markers, leading to defective intercalation. In contrast, misexpression of a dominant-negative mutant of a planar cell polarity gene Dishevelled preserved asymmetric accumulation of aPKC and laminin in notochord precursors, although their intercalation was incomplete. Our data support a model in which in ascidian embryos Eph-dependent dorsoventral polarity of notochord precursors plays a crucial role in mediolateral cell intercalation and is required for proper notochord morphogenesis.

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

PubMed Central

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

2011-01-01

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

Taste Bud Homeostasis in Health, Disease, and Aging

PubMed Central

2014-01-01

The mammalian taste bud is an onion-shaped epithelial structure with 50–100 tightly packed cells, including taste receptor cells, supporting cells, and basal cells. Taste receptor cells detect nutrients and toxins in the oral cavity and transmit the sensory information to gustatory nerve endings in the buds. Supporting cells may play a role in the clearance of excess neurotransmitters after their release from taste receptor cells. Basal cells are precursor cells that differentiate into mature taste cells. Similar to other epithelial cells, taste cells turn over continuously, with an average life span of about 8–12 days. To maintain structural homeostasis in taste buds, new cells are generated to replace dying cells. Several recent studies using genetic lineage tracing methods have identified populations of progenitor/stem cells for taste buds, although contributions of these progenitor/stem cell populations to taste bud homeostasis have yet to be fully determined. Some regulatory factors of taste cell differentiation and degeneration have been identified, but our understanding of these aspects of taste bud homoeostasis remains limited. Many patients with various diseases develop taste disorders, including taste loss and taste distortion. Decline in taste function also occurs during aging. Recent studies suggest that disruption or alteration of taste bud homeostasis may contribute to taste dysfunction associated with disease and aging. PMID:24287552

Taste bud homeostasis in health, disease, and aging.

PubMed

Feng, Pu; Huang, Liquan; Wang, Hong

2014-01-01

The mammalian taste bud is an onion-shaped epithelial structure with 50-100 tightly packed cells, including taste receptor cells, supporting cells, and basal cells. Taste receptor cells detect nutrients and toxins in the oral cavity and transmit the sensory information to gustatory nerve endings in the buds. Supporting cells may play a role in the clearance of excess neurotransmitters after their release from taste receptor cells. Basal cells are precursor cells that differentiate into mature taste cells. Similar to other epithelial cells, taste cells turn over continuously, with an average life span of about 8-12 days. To maintain structural homeostasis in taste buds, new cells are generated to replace dying cells. Several recent studies using genetic lineage tracing methods have identified populations of progenitor/stem cells for taste buds, although contributions of these progenitor/stem cell populations to taste bud homeostasis have yet to be fully determined. Some regulatory factors of taste cell differentiation and degeneration have been identified, but our understanding of these aspects of taste bud homoeostasis remains limited. Many patients with various diseases develop taste disorders, including taste loss and taste distortion. Decline in taste function also occurs during aging. Recent studies suggest that disruption or alteration of taste bud homeostasis may contribute to taste dysfunction associated with disease and aging.

Differential requirements for activation and growth of unprimed cytotoxic and helper T lymphocytes.

PubMed

Gullberg, M; Pobor, G; Bandeira, A; Larsson, E L; Coutinho, A

1983-09-01

The requirements for activation and growth of T lymphocytes capable of mediating either cytolytic activity or help to B lymphocytes were studied in unprimed splenic T cell populations. The selectivity of expression of Lyt-2 antigens, the reactivity to soluble concanavalin A (Con A), to partially purified interleukin 2 (IL 2, T cell growth factor[s]) and to lectin-pulsed macrophages (M phi) were used in this analysis. Lectin-dependent cytotoxicity assays and a novel method that allows for the detection of all effector helper cells, regardless of their clonal specificities, were used for the functional identification of the responding T cells. The results show a marked contrast between cytolytic and helper T cells in their growth and activation requirements. Thus, while Lyt-2+ cytotoxic T lymphocyte precursors grow exponentially in IL 2 after a short pulse with soluble Con A in the absence of accessory cells, Lyt-2- helper cell precursors completely fail to proliferate under the same conditions and require the continuous presence of lectin-pulsed M phi for significant growth. Furthermore, addition of IL 2 to M phi-stimulated cultures of Lyt-2- cells has no effect. T cells which produce IL 2 have the same growth characteristics as helper cells. In both cases, effector helper functions could be expanded more than 10-fold on a per cell basis by a 5-day-culture period under those growth supporting conditions. The development of effector helper functions, however, was strongly inhibited by the presence of Lyt-2+ T cells.

Coordinated Regulation of Niche and Stem Cell Precursors by Hormonal Signaling

PubMed Central

Gancz, Dana; Lengil, Tamar; Gilboa, Lilach

2011-01-01

Stem cells and their niches constitute units that act cooperatively to achieve adult body homeostasis. How such units form and whether stem cell and niche precursors might be coordinated already during organogenesis are unknown. In fruit flies, primordial germ cells (PGCs), the precursors of germ line stem cells (GSCs), and somatic niche precursors develop within the larval ovary. Together they form the 16–20 GSC units of the adult ovary. We show that ecdysone receptors are required to coordinate the development of niche and GSC precursors. At early third instar, ecdysone receptors repress precocious differentiation of both niches and PGCs. Early repression is required for correct morphogenesis of the ovary and for protecting future GSCs from differentiation. At mid-third instar, ecdysone signaling is required for niche formation. Finally, and concurrent with the initiation of wandering behavior, ecdysone signaling initiates PGC differentiation by allowing the expression of the differentiation gene bag of marbles in PGCs that are not protected by the newly formed niches. All the ovarian functions of ecdysone receptors are mediated through early repression, and late activation, of the ecdysone target gene broad. These results show that, similar to mammals, a brain-gland-gonad axis controls the initiation of oogenesis in insects. They further exemplify how a physiological cue coordinates the formation of a stem cell unit within an organ: it is required for niche establishment and to ensure that precursor cells to adult stem cells remain undifferentiated until the niches can accommodate them. Similar principles might govern the formation of additional stem cell units during organogenesis. PMID:22131903

Live cell imaging reveals marked variability in myoblast proliferation and fate

PubMed Central

2013-01-01

Background During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear. Methods We have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line. Results Analyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls. Conclusion Our results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated. PMID:23638706

Activation-induced cytidine deaminase (AID) expression in human B-cell precursors is essential for central B-cell tolerance

PubMed Central

Cantaert, Tineke; Schickel, Jean-Nicolas; Bannock, Jason M.; Ng, Yen-Shing; Massad, Christopher; Oe, Tyler; Wu, Renee; Lavoie, Aubert; Walter, Jolan E.; Notarangelo, Luigi D.; Al-Herz, Waleed; Kilic, Sara Sebnem; Ochs, Hans D.; Nonoyama, Shigeaki; Durandy, Anne; Meffre, Eric

2015-01-01

SUMMARY Activation-induced cytidine deaminase (AID), the enzyme mediating class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B-cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B-cell intrinsic AID expression mediates central B-cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells. PMID:26546282

Alemtuzumab and Combination Chemotherapy in Treating Patients With Untreated Acute Lymphoblastic Leukemia

ClinicalTrials.gov

2014-03-20

Acute Undifferentiated Leukemia; B-cell Adult Acute Lymphoblastic Leukemia; B-cell Childhood Acute Lymphoblastic Leukemia; L1 Adult Acute Lymphoblastic Leukemia; L1 Childhood Acute Lymphoblastic Leukemia; L2 Adult Acute Lymphoblastic Leukemia; L2 Childhood Acute Lymphoblastic Leukemia; Philadelphia Chromosome Negative Adult Precursor Acute Lymphoblastic Leukemia; Philadelphia Chromosome Positive Adult Precursor Acute Lymphoblastic Leukemia; Philadelphia Chromosome Positive Childhood Precursor Acute Lymphoblastic Leukemia; T-cell Adult Acute Lymphoblastic Leukemia; T-cell Childhood Acute Lymphoblastic Leukemia; Untreated Adult Acute Lymphoblastic Leukemia; Untreated Childhood Acute Lymphoblastic Leukemia

Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

PubMed Central

Fricke, Stephan; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jörg

2014-01-01

Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies. PMID:25546418

Regulation of DM-20 mRNA expression and intracellular translocation of glutathione-S-transferase pi isoform during oligodendrocyte differentiation in the adult rat spinal cord.

PubMed

Kitada, Masaaki; Takeda, Kazuya; Dezawa, Mari

2016-07-01

We previously demonstrated that NG2-positive oligodendrocyte precursor cells (OPCs) do not express DM-20 mRNA and identified a distinct DM-20 mRNA-positive cell population expressing glutathione-S-transferase pi isoform (GST-pi) in the nucleus (GST-pi(Nuc)) of the adult rat spinal cord. As GST-pi intranuclear localization correlates with progenitor cell properties, we examined the differentiation status of this cell population under the intensive 5-bromo-2'-deoxyuridine (BrdU) administration method, consisting of intraperitoneal BrdU injections every 2 h for 48 h. We observed that a certain population of proliferating/proliferated cells expressed DM-20 mRNA, and sometimes two proliferating/proliferated cells were observed still attached to each other. We performed triple staining for BrdU, DM-20 mRNA, and NG2 and found pairs of neighboring BrdU-positive cells, which were considered to originate from the same progenitor cells and where both cells expressed DM-20 mRNA. Triple staining for BrdU, DM-20 mRNA, and GST-pi detected proliferating/proliferated cells exhibiting the GST-pi(Nuc)/DM-20 mRNA-positive expression pattern. These findings suggested the presence of a GST-pi(Nuc)/DM-20 mRNA-positive oligodendrocyte-lineage progenitor cell population in the adult rat spinal cord. However, we did not find any pair of neighboring BrdU-positive cells with this expression pattern. These observations collectively support the idea that GST-pi(Nuc)/DM-20 mRNA-expressing cells are the progeny of NG2-positive OPCs rather than a novel type of oligodendrocyte-lineage progenitor cells and that DM-20 mRNA expression is dynamically regulated during differentiation of OPCs into oligodendrocytes.

High-throughput combinatorial cell co-culture using microfluidics.

PubMed

Tumarkin, Ethan; Tzadu, Lsan; Csaszar, Elizabeth; Seo, Minseok; Zhang, Hong; Lee, Anna; Peerani, Raheem; Purpura, Kelly; Zandstra, Peter W; Kumacheva, Eugenia

2011-06-01

Co-culture strategies are foundational in cell biology. These systems, which serve as mimics of in vivo tissue niches, are typically poorly defined in terms of cell ratios, local cues and supportive cell-cell interactions. In the stem cell niche, the ability to screen cell-cell interactions and identify local supportive microenvironments has a broad range of applications in transplantation, tissue engineering and wound healing. We present a microfluidic platform for the high-throughput generation of hydrogel microbeads for cell co-culture. Encapsulation of different cell populations in microgels was achieved by introducing in a microfluidic device two streams of distinct cell suspensions, emulsifying the mixed suspension, and gelling the precursor droplets. The cellular composition in the microgels was controlled by varying the volumetric flow rates of the corresponding streams. We demonstrate one of the applications of the microfluidic method by co-encapsulating factor-dependent and responsive blood progenitor cell lines (MBA2 and M07e cells, respectively) at varying ratios, and show that in-bead paracrine secretion can modulate the viability of the factor dependent cells. Furthermore, we show the application of the method as a tool to screen the impact of specific growth factors on a primary human heterogeneous cell population. Co-encapsulation of IL-3 secreting MBA2 cells with umbilical cord blood cells revealed differential sub-population responsiveness to paracrine signals (CD14+ cells were particularly responsive to locally delivered IL-3). This microfluidic co-culture platform should enable high throughput screening of cell co-culture conditions, leading to new strategies to manipulate cell fate. This journal is © The Royal Society of Chemistry 2011

Excess fructose intake-induced hypertrophic visceral adipose tissue results from unbalanced precursor cell adipogenic signals.

PubMed

Zubiría, María G; Fariña, Juan P; Moreno, Griselda; Gagliardino, Juan J; Spinedi, Eduardo; Giovambattista, Andrés

2013-11-01

We studied the effect of feeding normal adult male rats with a commercial diet supplemented with fructose added to the drinking water (10% w/v; fructose-rich diet, FRD) on the adipogenic capacity of stromal-vascular fraction (SVF) cells isolated from visceral adipose tissue (VAT) pads. Animals received either the commercial diet or FRD ad libitum for 3 weeks; thereafter, we evaluated the in vitro proliferative and adipogenic capacities of their VAT SVF cells. FRD significantly increased plasma insulin, triglyceride and leptin levels, VAT mass/cell size, and the in vitro adipogenic capacity of SVF cells. Flow cytometry studies indicated that the VAT precursor cell population number did not differ between groups; however, the accelerated adipogenic process could result from an imbalance between endogenous pro- and anti-adipogenic SVF cell signals, which are clearly shifted towards the former. The increased insulin milieu and its intracellular mediator (insulin receptor substrate-1) in VAT pads, as well as the enhanced SVF cell expression of Zpf423 and peroxisome proliferator receptor-γ2 (all pro-adipogenic modulators), together with a decreased SVF cell concentration of anti-adipogenic factors (pre-adipocyte factor-1 and wingless-type MMTV-10b), strongly supports this assumption. We hypothesize that the VAT mass expansion recorded in FRD rats results from the combination of initial accelerated adipogenesis and final cell hypertrophy. It remains to be determined whether FRD administration over longer periods could perpetuate both processes, or whether cell hypertrophy itself remains responsible for a further VAT mass expansion, as observed in advanced/morbid obesity. © 2013 FEBS.

Distinctions Among Circulating Antibody Secreting Cell Populations, Including B-1 Cells, in Human Adult Peripheral Blood1

PubMed Central

Quách, Tâm D.; Rodríguez-Zhurbenko, Nely; Hopkins, Thomas J.; Guo, Xiaoti; Vázquez, Ana María Hernández; Li, Wentian; Rothstein, Thomas L.

2015-01-01

Human antibody secreting cell (ASC) populations in circulation are not well studied. In addition to B-1 (CD20+CD27+CD38lo/intCD43+) cell and the conventional plasmablast (CD20-CD27hiCD38hi) cell populations, here we identified a novel B cell population termed 20+38hi B cells (CD20+CD27hiCD38hi) that spontaneously secretes antibody. At steady state, 20+38hi B cells are distinct from plasmablasts on the basis of CD20 expression, amount of antibody production, frequency of mutation, and diversity of B cell receptor repertoire. However, cytokine treatment of 20+38hi B cells induces loss of CD20 and acquisition of CD138, suggesting that 20+38hi B cells are precursors to plasmablasts, or pre-plasmablasts. We then evaluated similarities and differences between CD20+CD27+CD38lo/intCD43+ B-1 cells, CD20+CD27hiCD38hi 20+38hi B cells, CD20-CD27hiCD38hi plasmablasts, and CD20+CD27+CD38lo/intCD43- memory B cells. We found that B-1 cells differ from 20+38hi B cells and plasmablasts in numbers of ways, including antigen expression, morphological appearance, transcriptional profiling, antibody skewing, antibody repertoire, and secretory response to stimulation. In terms of gene expression, B-1 cells align more closely with memory B cells than with 20+38hi B cells or plasmablasts, but differ in that memory B cells do not express antibody secretion related genes. We found that, B-1 cell antibodies utilize Vh4-34, which is often associated with autoreactivity, 3 to 6-fold more often than other B cell populations. Along with selective production of IgM anti-PC, this data suggests that human B-1 cells might be preferentially selected for autoreactivity/natural-specificity. In sum, our results indicate that human healthy adult peripheral blood at steady state consists of 3 distinct ASC populations. PMID:26740107

Quantifying the Labeling and the Levels of Plant Cell Wall Precursors Using Ion Chromatography Tandem Mass Spectrometry1[W][OA

PubMed Central

Alonso, Ana P.; Piasecki, Rebecca J.; Wang, Yan; LaClair, Russell W.; Shachar-Hill, Yair

2010-01-01

The biosynthesis of cell wall polymers involves enormous fluxes through central metabolism that are not fully delineated and whose regulation is poorly understood. We have established and validated a liquid chromatography tandem mass spectrometry method using multiple reaction monitoring mode to separate and quantify the levels of plant cell wall precursors. Target analytes were identified by their parent/daughter ions and retention times. The method allows the quantification of precursors at low picomole quantities with linear responses up to the nanomole quantity range. When applying the technique to Arabidopsis (Arabidopsis thaliana) T87 cell cultures, 16 hexose-phosphates (hexose-Ps) and nucleotide-sugars (NDP-sugars) involved in cell wall biosynthesis were separately quantified. Using hexose-P and NDP-sugar standards, we have shown that hot water extraction allows good recovery of the target metabolites (over 86%). This method is applicable to quantifying the levels of hexose-Ps and NDP-sugars in different plant tissues, such as Arabidopsis T87 cells in culture and fenugreek (Trigonella foenum-graecum) endosperm tissue, showing higher levels of galacto-mannan precursors in fenugreek endosperm. In Arabidopsis cells incubated with [U-13CFru]sucrose, the method was used to track the labeling pattern in cell wall precursors. As the fragmentation of hexose-Ps and NDP-sugars results in high yields of [PO3]−/or [H2PO4]− ions, mass isotopomers can be quantified directly from the intensity of selected tandem mass spectrometry transitions. The ability to directly measure 13C labeling in cell wall precursors makes possible metabolic flux analysis of cell wall biosynthesis based on dynamic labeling experiments. PMID:20442274

Effects of Low Level Radiation exposure on Neurogenesis and Cognitive Function: Mechanisms and Prevention

DTIC Science & Technology

2005-09-01

precursor cells in culture with uX-lipoic acid reverses the density dependent changes observed in culture; this compound may provide an effective means...inhibited growth of precursor cells in vitro; - Antioxidant treatment of neural precursor cells in culture with a-lipoic acid (ALA) reverses the...with a single lO-Gy dose, and tissues avidin-biotinylated pemxidase complex; GFAP, glial fibrillary acidic protein; DAB, 3,3’- were collected from 6 to

A Novel View of the Adult Stem Cell Compartment From the Perspective of a Quiescent Population of Very Small Embryonic-Like Stem Cells.

PubMed

Ratajczak, Mariusz Z; Ratajczak, Janina; Suszynska, Malwina; Miller, Donald M; Kucia, Magda; Shin, Dong-Myung

2017-01-06

Evidence has accumulated that adult hematopoietic tissues and other organs contain a population of dormant stem cells (SCs) that are more primitive than other, already restricted, monopotent tissue-committed SCs (TCSCs). These observations raise several questions, such as the developmental origin of these cells, their true pluripotent or multipotent nature, which surface markers they express, how they can be efficiently isolated from adult tissues, and what role they play in the adult organism. The phenotype of these cells and expression of some genes characteristic of embryonic SCs, epiblast SCs, and primordial germ cells suggests their early-embryonic deposition in developing tissues as precursors of adult SCs. In this review, we will critically discuss all these questions and the concept that small dormant SCs related to migratory primordial germ cells, described as very small embryonic-like SCs, are deposited during embryogenesis in bone marrow and other organs as a backup population for adult tissue-committed SCs and are involved in several processes related to tissue or organ rejuvenation, aging, and cancerogenesis. The most recent results on successful ex vivo expansion of human very small embryonic-like SC in chemically defined media free from feeder-layer cells open up new and exciting possibilities for their application in regenerative medicine. © 2017 American Heart Association, Inc.

The chemokine CXCL16 induces migration and invasion of glial precursor cells via its receptor CXCR6.

PubMed

Hattermann, Kirsten; Ludwig, Andreas; Gieselmann, Volkmar; Held-Feindt, Janka; Mentlein, Rolf

2008-09-01

Chemokines are implicated in developmental and inflammatory processes in the brain. The transmembrane chemokine CXCL16 is produced in brain endothelial and reactive astroglial cells and released by shedding. Its receptor CXCR6 is detected during brain development highest at postnatal day 6, found in glial precursor cells differentiated from neural stem cells and in an A2B5-positive glial precursor cell line. Their stimulation by soluble CXCL16 induces the PI3-kinase/Akt and Erk pathways resulting in the activation of the transcription factor AP-1. As biological responses, soluble CXCL16 upregulates its own receptor, increases cell proliferation, stimulates cell migration in wound-healing and in spheroid confrontation assays. Invasion of CXCR6-positive glial cells into CXCL16-expressing spheroids can be blocked by sheddase inhibitors and CXCL16-antibody. Since CXCL16 is induced by cytokines at sites of inflammation, neurodegeneration, ischemia and malignant transformation, it should attract CXCR6-positive glial precursor cells, enhance their invasion and proliferation and thus favor astrogliosis.

Fabrication of solution processed 3D nanostructured CuInGaS₂ thin film solar cells.

PubMed

Chu, Van Ben; Cho, Jin Woo; Park, Se Jin; Hwang, Yun Jeong; Park, Hoo Keun; Do, Young Rag; Min, Byoung Koun

2014-03-28

In this study we demonstrate the fabrication of CuInGaS₂ (CIGS) thin film solar cells with a three-dimensional (3D) nanostructure based on indium tin oxide (ITO) nanorod films and precursor solutions (Cu, In and Ga nitrates in alcohol). To obtain solution processed 3D nanostructured CIGS thin film solar cells, two different precursor solutions were applied to complete gap filling in ITO nanorods and achieve the desirable absorber film thickness. Specifically, a coating of precursor solution without polymer binder material was first applied to fill the gap between ITO nanorods followed by deposition of the second precursor solution in the presence of a binder to generate an absorber film thickness of ∼1.3 μm. A solar cell device with a (Al, Ni)/AZO/i-ZnO/CdS/CIGS/ITO nanorod/glass structure was constructed using the CIGS film, and the highest power conversion efficiency was measured to be ∼6.3% at standard irradiation conditions, which was 22.5% higher than the planar type of CIGS solar cell on ITO substrate fabricated using the same precursor solutions.

Plasmacytoid Dendritic Cell Dynamics Tune Interferon-Alfa Production in SIV-Infected Cynomolgus Macaques

PubMed Central

Bruel, Timothée; Dupuy, Stéphanie; Démoulins, Thomas; Rogez-Kreuz, Christine; Dutrieux, Jacques; Corneau, Aurélien; Cosma, Antonio; Cheynier, Rémi; Dereuddre-Bosquet, Nathalie; Le Grand, Roger; Vaslin, Bruno

2014-01-01

IFN-I production is a characteristic of HIV/SIV primary infections. However, acute IFN-I plasma concentrations rapidly decline thereafter. Plasmacytoid dendritic cells (pDC) are key players in this production but primary infection is associated with decreased responsiveness of pDC to TLR 7 and 9 triggering. IFNα production during primary SIV infection contrasts with increased pDC death, renewal and dysfunction. We investigated the contribution of pDC dynamics to both acute IFNα production and the rapid return of IFNα concentrations to pre-infection levels during acute-to-chronic transition. Nine cynomolgus macaques were infected with SIVmac251 and IFNα-producing cells were quantified and characterized. The plasma IFN-I peak was temporally associated with the presence of IFNα+ pDC in tissues but IFN-I production was not detectable during the acute-to-chronic transition despite persistent immune activation. No IFNα+ cells other than pDC were detected by intracellular staining. Blood-pDC and peripheral lymph node-pDC both lost IFNα− production ability in parallel. In blood, this phenomenon correlated with an increase in the counts of Ki67+-pDC precursors with no IFNα production ability. In tissues, it was associated with increase of both activated pDC and KI67+-pDC precursors, none of these being IFNα+ in vivo. Our findings also indicate that activation/death-driven pDC renewal rapidly blunts acute IFNα production in vivo: pDC sub-populations with no IFNα-production ability rapidly increase and shrinkage of IFNα production thus involves both early pDC exhaustion, and increase of pDC precursors. PMID:24497833

An essential role of variant histone H3.3 for ectomesenchyme potential of the cranial neural crest.

PubMed

Cox, Samuel G; Kim, Hyunjung; Garnett, Aaron Timothy; Medeiros, Daniel Meulemans; An, Woojin; Crump, J Gage

2012-09-01

The neural crest (NC) is a vertebrate-specific cell population that exhibits remarkable multipotency. Although derived from the neural plate border (NPB) ectoderm, cranial NC (CNC) cells contribute not only to the peripheral nervous system but also to the ectomesenchymal precursors of the head skeleton. To date, the developmental basis for such broad potential has remained elusive. Here, we show that the replacement histone H3.3 is essential during early CNC development for these cells to generate ectomesenchyme and head pigment precursors. In a forward genetic screen in zebrafish, we identified a dominant D123N mutation in h3f3a, one of five zebrafish variant histone H3.3 genes, that eliminates the CNC-derived head skeleton and a subset of pigment cells yet leaves other CNC derivatives and trunk NC intact. Analyses of nucleosome assembly indicate that mutant D123N H3.3 interferes with H3.3 nucleosomal incorporation by forming aberrant H3 homodimers. Consistent with CNC defects arising from insufficient H3.3 incorporation into chromatin, supplying exogenous wild-type H3.3 rescues head skeletal development in mutants. Surprisingly, embryo-wide expression of dominant mutant H3.3 had little effect on embryonic development outside CNC, indicating an unexpectedly specific sensitivity of CNC to defects in H3.3 incorporation. Whereas previous studies had implicated H3.3 in large-scale histone replacement events that generate totipotency during germ line development, our work has revealed an additional role of H3.3 in the broad potential of the ectoderm-derived CNC, including the ability to make the mesoderm-like ectomesenchymal precursors of the head skeleton.

An Essential Role of Variant Histone H3.3 for Ectomesenchyme Potential of the Cranial Neural Crest

PubMed Central

Cox, Samuel G.; Kim, Hyunjung; Garnett, Aaron Timothy; Medeiros, Daniel Meulemans; An, Woojin; Crump, J. Gage

2012-01-01

The neural crest (NC) is a vertebrate-specific cell population that exhibits remarkable multipotency. Although derived from the neural plate border (NPB) ectoderm, cranial NC (CNC) cells contribute not only to the peripheral nervous system but also to the ectomesenchymal precursors of the head skeleton. To date, the developmental basis for such broad potential has remained elusive. Here, we show that the replacement histone H3.3 is essential during early CNC development for these cells to generate ectomesenchyme and head pigment precursors. In a forward genetic screen in zebrafish, we identified a dominant D123N mutation in h3f3a, one of five zebrafish variant histone H3.3 genes, that eliminates the CNC–derived head skeleton and a subset of pigment cells yet leaves other CNC derivatives and trunk NC intact. Analyses of nucleosome assembly indicate that mutant D123N H3.3 interferes with H3.3 nucleosomal incorporation by forming aberrant H3 homodimers. Consistent with CNC defects arising from insufficient H3.3 incorporation into chromatin, supplying exogenous wild-type H3.3 rescues head skeletal development in mutants. Surprisingly, embryo-wide expression of dominant mutant H3.3 had little effect on embryonic development outside CNC, indicating an unexpectedly specific sensitivity of CNC to defects in H3.3 incorporation. Whereas previous studies had implicated H3.3 in large-scale histone replacement events that generate totipotency during germ line development, our work has revealed an additional role of H3.3 in the broad potential of the ectoderm-derived CNC, including the ability to make the mesoderm-like ectomesenchymal precursors of the head skeleton. PMID:23028350

Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

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

Manceur, Aziza P.; Donnelly Centre, University of Toronto, Toronto, Ontario; Tseng, Michael

2011-09-10

The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B)more » inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.« less

Neurotoxicity of a Fragment of the Amyloid Precursor Associated with Alzheimer's Disease

NASA Astrophysics Data System (ADS)

Yankner, Bruce A.; Dawes, Linda R.; Fisher, Shannon; Villa-Komaroff, Lydia; Oster-Granite, Mary Lou; Neve, Rachael L.

1989-07-01

Amyloid deposition in senile plaques and the cerebral vasculature is a marker of Alzheimer's disease. Whether amyloid itself contributes to the neurodegenerative process or is simply a by-product of that process is unknown. Pheochromocytoma (PC12) and fibroblast (NIH 3T3) cell lines were transfected with portions of the gene for the human amyloid precursor protein. Stable PC12 cell transfectants expressing a specific amyloid-containing fragment of the precursor protein gradually degenerated when induced to differentiate into neuronal cells with nerve growth factor. Conditioned medium from these cells was toxic to neurons in primary hippocampal cultures, and the toxic agent could be removed by immunoabsorption with an antibody directed against the amyloid polypeptide. Thus, a peptide derived from the amyloid precursor may be neurotoxic.

HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen

DOE PAGES

Jardine, Joseph G.; Kulp, Daniel W.; Havenar-Daughton, Colin; ...

2016-03-25

Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. We employed deep mutational scanning and multi-target optimization to develop a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen asmore » a candidate human vaccine prime. Lastly, these methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.« less

HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen

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

Jardine, Joseph G.; Kulp, Daniel W.; Havenar-Daughton, Colin

Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. We employed deep mutational scanning and multi-target optimization to develop a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen asmore » a candidate human vaccine prime. Lastly, these methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.« less

HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen.

PubMed

Jardine, Joseph G; Kulp, Daniel W; Havenar-Daughton, Colin; Sarkar, Anita; Briney, Bryan; Sok, Devin; Sesterhenn, Fabian; Ereño-Orbea, June; Kalyuzhniy, Oleksandr; Deresa, Isaiah; Hu, Xiaozhen; Spencer, Skye; Jones, Meaghan; Georgeson, Erik; Adachi, Yumiko; Kubitz, Michael; deCamp, Allan C; Julien, Jean-Philippe; Wilson, Ian A; Burton, Dennis R; Crotty, Shane; Schief, William R

2016-03-25

Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. Using deep mutational scanning and multitarget optimization, we developed a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens. Copyright © 2016, American Association for the Advancement of Science.

Lymphoid tissue and plasmacytoid dendritic cells and macrophages do not share a common macrophage-dendritic cell-restricted progenitor.

PubMed

Sathe, Priyanka; Metcalf, Donald; Vremec, David; Naik, Shalin H; Langdon, Wallace Y; Huntington, Nicholas D; Wu, Li; Shortman, Ken

2014-07-17

The relationship between dendritic cells (DCs) and macrophages is often debated. Here we ask whether steady-state, lymphoid-tissue-resident conventional DCs (cDCs), plasmacytoid DCs (pDCs), and macrophages share a common macrophage-DC-restricted precursor (MDP). Using new clonal culture assays combined with adoptive transfer, we found that MDP fractions isolated by previous strategies are dominated by precursors of macrophages and monocytes, include some multipotent precursors of other hematopoietic lineages, but contain few precursors of resident cDCs and pDCs and no detectable common precursors restricted to these DC types and macrophages. Overall we find no evidence for a common restricted MDP leading to both macrophages and FL-dependent, resident cDCs and pDCs. Copyright © 2014 Elsevier Inc. All rights reserved.

Retained Myogenic Potency of Human Satellite Cells from Torn Rotator Cuff Muscles Despite Fatty Infiltration.

PubMed

Koide, Masashi; Hagiwara, Yoshihiro; Tsuchiya, Masahiro; Kanzaki, Makoto; Hatakeyama, Hiroyasu; Tanaka, Yukinori; Minowa, Takashi; Takemura, Taro; Ando, Akira; Sekiguchi, Takuya; Yabe, Yutaka; Itoi, Eiji

2018-01-01

Rotator cuff tears (RCTs) are a common shoulder problem in the elderly that can lead to both muscle atrophy and fatty infiltration due to less physical load. Satellite cells, quiescent cells under the basal lamina of skeletal muscle fibers, play a major role in muscle regeneration. However, the myogenic potency of human satellite cells in muscles with fatty infiltration is unclear due to the difficulty in isolating from small samples, and the mechanism of the progression of fatty infiltration has not been elucidated. The purpose of this study was to analyze the population of myogenic and adipogenic cells in disused supraspinatus (SSP) and intact subscapularis (SSC) muscles of the RCTs from the same patients using fluorescence-activated cell sorting. The microstructure of the muscle with fatty infiltration was observed as a whole mount condition under multi-photon microscopy. Myogenic differentiation potential and gene expression were evaluated in satellite cells. The results showed that the SSP muscle with greater fatty infiltration surrounded by collagen fibers compared with the SSC muscle under multi-photon microscopy. A positive correlation was observed between the ratio of muscle volume to fat volume and the ratio of myogenic precursor to adipogenic precursor. Although no difference was observed in the myogenic potential between the two groups in cell culture, satellite cells in the disused SSP muscle showed higher intrinsic myogenic gene expression than those in the intact SSC muscle. Our results indicate that satellite cells from the disused SSP retain sufficient potential of muscle growth despite the fatty infiltration.

Dual origin, development, and fate of bovine pancreatic islets

PubMed Central

Merkwitz, Claudia; Lochhead, Paul; Böttger, Jan; Matz-Soja, Madlen; Sakurai, Michiharu; Gebhardt, Rolf; Ricken, Albert M

2013-01-01

Endocrine cells are evident at an early stage in bovine pancreatic development when the pancreas still consists of primitive epithelial cords. At this stage, the endocrine cells are interspersed between the precursor cells destined to form the ductulo-acinar trees of later exocrine lobules. We here demonstrate that, in bovine fetuses of crown rump length ≥ 11 cm, the endocrine cells become increasingly segregated from the developing exocrine pancreas by assembly into two units that differ in histogenesis, architecture, and fate. Small numbers of ‘perilobular giant islets’ are distinguishable from larger numbers of ‘intralobular small islets’. The two types of islets arise in parallel from the ends of the ductal tree. Aside from differences in number, location, and size, the giant and small islets differ in cellular composition (predominantly insulin-synthesising cells vs. mixtures of endocrine cells), morphology (epithelial trabeculae with gyriform and rosette-like appearance vs. compact circular arrangements of endocrine cells), and in their relationships to intrapancreatic ganglia and nerves. A further difference becomes apparent during the antenatal period; while the ‘interlobular small islets’ persist in the pancreata of calves and adult cattle, the perilobular giant islets are subject to regression, characterised by involution of the parenchyma, extensive haemorrhage, leukocyte infiltration (myeloid and T-cells) and progressive fibrotic replacement. In conclusion, epithelial precursor cells of the ductolo-acinar tree may give rise to populations of pancreatic islets with different histomorphology, cellular composition and fates. This should be taken into account when using these cells for the generation of pancreatic islets for transplantation therapy. PMID:23171225

Definition of Drosophila hemocyte subsets by cell-type specific antigens.

PubMed

Kurucz, Eva; Váczi, B; Márkus, R; Laurinyecz, Barbara; Vilmos, P; Zsámboki, J; Csorba, Kinga; Gateff, Elisabeth; Hultmark, D; Andó, I

2007-01-01

We analyzed the heterogeneity of Drosophila hemocytes on the basis of the expression of cell-type specific antigens. The antigens characterize distinct subsets which partially overlap with those defined by morphological criteria. On the basis of the expression or the lack of expression of blood cell antigens the following hemocyte populations have been defined: crystal cells, plasmatocytes, lamellocytes and precursor cells. The expression of the antigens and thus the different cell types are developmentally regulated. The hemocytes are arranged in four main compartments: the circulating blood cells, the sessile tissue, the lymph glands and the posterior hematopoietic tissue. Each hemocyte compartment has a specific and characteristic composition of the various cell types. The described markers represent the first successful attempt to define hemocyte lineages by immunological markers in Drosophila and help to define morphologically, functionally, spatially and developmentally distinct subsets of hemocytes.

Wnt/β-catenin and FGF signalling direct the specification and maintenance of a neuromesodermal axial progenitor in ensembles of mouse embryonic stem cells

PubMed Central

Turner, David A.; Hayward, Penelope C.; Baillie-Johnson, Peter; Rué, Pau; Broome, Rebecca; Faunes, Fernando; Martinez Arias, Alfonso

2014-01-01

The development of the central nervous system is known to result from two sequential events. First, an inductive event of the mesoderm on the overlying ectoderm that generates a neural plate that, after rolling into a neural tube, acts as the main source of neural progenitors. Second, the axial regionalization of the neural plate that will result in the specification of neurons with different anteroposterior identities. Although this description of the process applies with ease to amphibians and fish, it is more difficult to confirm in amniote embryos. Here, a specialized population of cells emerges at the end of gastrulation that, under the influence of Wnt and FGF signalling, expands and generates the spinal cord and the paraxial mesoderm. This population is known as the long-term neuromesodermal precursor (NMp). Here, we show that controlled increases of Wnt/β-catenin and FGF signalling during adherent culture differentiation of mouse embryonic stem cells (mESCs) generates a population with many of the properties of the NMp. A single-cell analysis of gene expression within this population reveals signatures that are characteristic of stem cell populations. Furthermore, when this activation is triggered in three-dimensional aggregates of mESCs, the population self-organizes macroscopically and undergoes growth and axial elongation that mimics some of the features of the embryonic spinal cord and paraxial mesoderm. We use both adherent and three-dimensional cultures of mESCs to probe the establishment and maintenance of NMps and their differentiation. PMID:25371361

CLONING AND CHARACTERIZATION OF OSTEOCLAST PRECURSORS FROM THE RAW264.7 CELL LINE

PubMed Central

Cuetara, Bethany L. V.; Crotti, Tania N.; O'Donoghue, Anthony J.

2006-01-01

SUMMARY Osteoclasts are bone-resorbing cells that differentiate from macrophage precursors in response to receptor activator of NF-κB (RANKL). In vitro models of osteoclast differentiation are principally based on primary cell culture, which are poorly suited to molecular and transgene studies due to the limitations associated with the use of primary macrophage. RAW264.7 is a transfectable macrophage cell line with the capacity to form osteoclast-like cells. In the present study we have identified osteoclast precursors among clones of RAW264.7 cells. RAW264.7 cell were cloned by limiting dilution and induced to osteoclast differentiation by treatment with recombinant RANKL. Individual RAW264.7 cell clones formed tartrate resistant acid phosphatase (TRAP) positive multinuclear cells to various degrees with RANKL treatment. All clones tested expressed the RANKL receptor RANK. Each of the clones expressed the osteoclast marker genes TRAP and cathepsin-K mRNA with RANKL treatment. However, we noted that only select clones were able to form large, well-spread, TRAP positive multinuclear cells. Clones capable of forming large TRAP positive multinuclear cells also expressed β3 integrin and calcitonin receptor mRNAs and were capable of resorbing a mineralized matrix. All clones tested activated NF-κB with RANKL treatment. cDNA expression profiling of osteoclast precursor RAW264.7 cell clones demonstrates appropriate expression of a large number of genes before and after osteoclastic differentiation. These osteoclast precursor RAW264.7 cell clones provide a valuable model for dissecting the cellular and molecular regulation of osteoclast differentiation and activation. PMID:16948499

GAS6/Mer axis regulates the homing and survival of the E2A/PBX1 positive B-cell precursor acute lymphoblastic leukemia in the bone marrow niche

PubMed Central

Shiozawa, Yusuke; Pedersen, Elisabeth A.; Taichman, Russell S.

2009-01-01

Despite improvements in current combinational chemotherapy regimens, the prognosis of the (1;19)(q23;p13) translocation (E2A/PBX1) positive B-cell precursor acute lymphoblastic leukemia (ALL) is poor in pediatric leukemia patients. In this study, we examined the roles of GAS6/Mer axis in the interactions between E2A/PBX1 positive B-cell precursor ALL cells and the osteoblastic niche in the bone marrow. The data show that primary human osteoblasts secrete GAS6 in response to the Mer-over-expressed E2A/PBX1 positive ALL cells through MAPK signaling pathway and that leukemia cells migrate toward GAS6 using pathways activated by Mer. Importantly, GAS6 supports the survival and prevents apoptosis from chemotherapy of E2A/PBX1 positive ALL cells by inducing dormancy. Together, these data suggest that GAS6/Mer axis regulates the homing and survival of the E2A/PBX1 positive B-cell precursor ALL in the bone marrow niche. PMID:19922767

Somatic and germinal cells' interrelationship in the course of seminiferous tubule maturation in man.

PubMed

Kula, K; Romer, T E; Wlodarczyk, W P

1980-02-01

Certain successive phases of seminiferous tubule maturation were observed in a transsection of a Leydig cell adenoma-bearing testis of a boy with precocious puberty. Massively accumulated Leydig cells may stimulate the maturation of Sertoli cells, as indicated by progressive replacement of Sertoli cell precursors by mature Sertoli cells at a distance closer to the adenoma. On the other hand, tubules less advanced in maturation contained a higher number of somatic cells than those more advanced in maturation. Leydig-cell-dependent maturation of Sertoli cells may be in competition with Certoli cell multiplication, or numerous undifferentiated somatic cells may undergo a natural elimination in the course of tubular maturation. An inverse relation between the number of Sertoli cell precursors and the number of meiotic spermatocytes suggests that quantitative reduction of Sertoli cell precursors may be important for the intratubular milieu necessary for the onset of the first meiosis in man.

Platelets, lymphocytes and erythrocytes from Alzheimer's disease patients: the quest for blood cell-based biomarkers.

PubMed

Pluta, Ryszard; Ułamek-Kozioł, Marzena; Januszewski, Sławomir; Czuczwar, Stanisław J

2018-01-01

In elderly population, Alzheimer's disease is a common neurodegenerative disorder and accounts for about 70% of all cases of dementia. The neurodegenerative processes of this disease start presumably 20 years ahead of the clinical beginning of the disorder. The postmortem histopathological examination, brains from Alzheimer's disease patients with characteristic features like amyloid plaques and neurofibrillary tangles, neuronal and synaptic disintegration confirm the final diagnosis of Alzheimer's disease. Senile plaques are composed of -amyloid peptide, deriving from the amyloid protein precursor, which is present not only in the brain tissue, but also in other non-neuronal tissues. Some investigations reported that platelets possess amyloid protein precursor and all the enzymatic activities required for the metabolism of this protein throughout the same pathways present in the brain. Thus, platelets may be a good peripheral blood cell-based biomarker to study the onset of Alzheimer's disease. Another line of research indicated molecular and cellular aberrations in blood lymphocytes and erythrocytes from Alzheimer's disease patients and emphasizes the systemic nature of the disease. In this review, we will summarize the recent knowledge on the involvement and/or response of platelets, lymphocytes and red blood cells in the circulation during Alzheimer's disease development. The facts will be reviewed with the special possibility for applying the above blood cells as Alzheimer's disease preclinical and antemortem blood cell-based biomarkers.

Differences in irradiation susceptibility and turnover between mucosal and connective tissue-type mast cells of mice

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

Fukuzumi, T.; Waki, N.; Kanakura, Y.

Although precursors of mast cells are derived from the bone marrow, phenotypes of mast cells are influenced by the tissues in which final differentiation occurs. Connective tissue-type mast cells (CTMC) and mucosal mast cells (MMC) are different in morphological, biochemical, immunological, and functional criteria. The purpose of the present study was to obtain information about the differentiation process of MMC. First, we compared changes in irradiation susceptibility in mice during the differentiation process of CTMC and MMC. The decrease in irradiation susceptibility was remarkable in the CTMC differentiation process, but it was moderate in that of MMC. Some morphologically identifiablemore » CTMC in the peritoneal cavity had proliferative potential and were highly radioresistant, whereas such a radioresistant population of MMC was not detectable in the gastric mucosa. Second, we estimated the turnover of CTMC and MMC by determining the proportion of mast cells that were labeled with continuously administered bromodeoxyuridine. The turnover of MMC was significantly faster than that of CTMC. The absence of the radioresistant mast cell population in the gastric mucosa appeared to be related to the short life span of MMC.« less

An estimation of the frequency of precursor cells which generate cytotoxic lymphocytes

PubMed Central

1976-01-01

The cell-mediated immune response has been generated in vitro with a polyacrylamide culture system which allows the segregation of foci (clones?) of cytotoxic lymphocytes. Using the method of limiting dilutions, the frequency of precursor cells in CBA spleen cells able to generate a cytotoxic response against DBA mastocytoma is estimated at 1 per 1,700 cells. PMID:1083894

VpreB gene expression in hematopoietic malignancies: a lineage- and stage-restricted marker for B-cell precursor leukemias.

PubMed

Bauer, S R; Kubagawa, H; Maclennan, I; Melchers, F

1991-09-15

We show here that analysis of VpreB gene transcription can be a specific way to identify acute leukemias of cells at very early stages of B-cell development. Northern blot analysis of RNAs from 63 leukemia samples showed that VpreB RNA was present in malignancies of precursor B cells, the expression being a feature of both common acute lymphoblastic leukemia (ALL) (CD10+) and null ALL (CD10-). It was absent from malignancies of mature B cells (surface Ig positive), from acute leukemias of the T-cell lineage and granulocyte-macrophage lineages, and from normal tonsil B and T lymphocytes. Chronic myeloid leukemia blast crises of the B-precursor-cell type expressed the VpreB gene while myeloid blast crises did not. VpreB RNA was also expressed in the neoplastic cells of one of three patients with acute undifferentiated leukemias. These data show that VpreB RNA expression is a marker of the malignant forms of precursor B cells, and that it appears at least as early as cytoplasmic CD22 and CD19 in tumors of the B-cell lineage.

Epidermal keratinocytes initiate wound healing and pro-inflammatory immune responses following percutaneous schistosome infection

PubMed Central

Bourke, Claire D.; Prendergast, Catriona T.; Sanin, David E.; Oulton, Tate E.; Hall, Rebecca J.; Mountford, Adrian P.

2015-01-01

Keratinocytes constitute the majority of cells in the skin’s epidermis, the first line of defence against percutaneous pathogens. Schistosome larvae (cercariae) actively penetrate the epidermis to establish infection, however the response of keratinocytes to invading cercariae has not been investigated. Here we address the hypothesis that cercariae activate epidermal keratinocytes to promote the development of a pro-inflammatory immune response in the skin. C57BL/6 mice were exposed to Schistosoma mansoni cercariae via each pinna and non-haematopoietic cells isolated from epidermal tissue were characterised for the presence of different keratinocyte sub-sets at 6, 24 and 96 h p.i. We identified an expansion of epidermal keratinocyte precursors (CD45−, CD326−, CD34+) within 24 h of infection relative to naïve animals. Following infection, cells within the precursor population displayed a more differentiated phenotype (α6integrin−) than in uninfected skin. Parallel immunohistochemical analysis of pinnae cryosections showed that this expansion corresponded to an increase in the intensity of CD34 staining, specifically in the basal bulge region of hair follicles of infected mice, and a higher frequency of keratinocyte Ki67+ nuclei in both the hair follicle and interfollicular epidermis. Expression of pro-inflammatory cytokine and stress-associated keratin 6b genes was also transiently upregulated in the epidermal tissue of infected mice. In vitro exposure of keratinocyte precursors isolated from neonatal mouse skin to excretory/secretory antigens released by penetrating cercariae elicited IL-1α and IL-1β production, supporting a role for keratinocyte precursors in initiating cutaneous inflammatory immune responses. Together, these observations indicate that S.mansoni cercariae and their excretory/secretory products act directly upon epidermal keratinocytes, which respond by initiating barrier repair and pro-inflammatory mechanisms similar to those observed in epidermal wound healing. PMID:25575749

The cell adhesion molecules Echinoid and Friend of Echinoid coordinate cell adhesion and cell signaling to regulate the fidelity of ommatidial rotation in the Drosophila eye.

PubMed

Fetting, Jennifer L; Spencer, Susan A; Wolff, Tanya

2009-10-01

Directed cellular movements are a universal feature of morphogenesis in multicellular organisms. Differential adhesion between the stationary and motile cells promotes these cellular movements to effect spatial patterning of cells. A prominent feature of Drosophila eye development is the 90 degrees rotational movement of the multicellular ommatidial precursors within a matrix of stationary cells. We demonstrate that the cell adhesion molecules Echinoid (Ed) and Friend of Echinoid (Fred) act throughout ommatidial rotation to modulate the degree of ommatidial precursor movement. We propose that differential levels of Ed and Fred between stationary and rotating cells at the initiation of rotation create a permissive environment for cell movement, and that uniform levels in these two populations later contribute to stopping the movement. Based on genetic data, we propose that ed and fred impart a second, independent, ;brake-like' contribution to this process via Egfr signaling. Ed and Fred are localized in largely distinct and dynamic patterns throughout rotation. However, ed and fred are required in only a subset of cells - photoreceptors R1, R7 and R6 - for normal rotation, cells that have only recently been linked to a role in planar cell polarity (PCP). This work also provides the first demonstration of a requirement for cone cells in the ommatidial rotation aspect of PCP. ed and fred also genetically interact with the PCP genes, but affect only the degree-of-rotation aspect of the PCP phenotype. Significantly, we demonstrate that at least one PCP protein, Stbm, is required in R7 to control the degree of ommatidial rotation.

Differential gene expression in notochord and nerve cord fate segregation in the Ciona intestinalis embryo.

PubMed

Kobayashi, Kenji; Yamada, Lixy; Satou, Yutaka; Satoh, Nori

2013-09-01

During early embryogenesis, embryonic cells gradually restrict their developmental potential and are eventually destined to give rise to one type of cells. Molecular mechanisms underlying developmental fate restriction are one of the major research subjects within developmental biology. In this article, this subject was addressed by combining blastomere isolation with microarray analysis. During the 6th cleavage of the Ciona intestinalis embryo, from the 32-cell to the 64-cell stage, four mother cells divide into daughter cells with two distinct fates, one giving rise to notochord precursor cells and the other to nerve cord precursors. Approximately 2,200 each of notochord and nerve cord precursor cells were isolated, and their mRNA expression profiles were compared by microarray. This analysis identified 106 and 68 genes, respectively, that are differentially expressed in notochord and nerve cord precursor cells. These included not only genes for transcription factors and signaling molecules but also those with generalized functions observed in many types of cells. In addition, whole-mount in situ hybridization showed dynamic spatial expression profiles of these genes during segregation of the two fates: partitioning of transcripts present in the mother cells into either type of daughter cells, and initiation of preferential gene expression in either type of cells. Copyright © 2013 Wiley Periodicals, Inc.

CHARACTERISTICS OF IMMUNOLOGICAL MEMORY IN MICE

PubMed Central

Black, S. J.; Inchley, C. J.

1974-01-01

The kinetics of the generation of primed IgM and IgG antibody-forming cell precursors, and of helper T-cell populations, were analyzed in mice whose primary responses to high and low doses of SRBC were arrested at intervals by the immunosuppressive agents cyclophosphamide monohydrate and specific antibody. The extent to which immunological memory was established in these animals before blockade of the primary response was assessed by the hemolytic plaque assay following challenge 12 wk after priming. The presence of IgG B-memory cells and T-memory cells in suppressed mice was further investigated by the transfer into these animals of syngeneic SRBC-stimulated thymocytes or anti-θ-treated spleen cells. It was found that the progenitors of secondary IgM-synthesizing cells were primed almost immediately after injection of antigen, and that early blockade of the primary response resulted in a raised IgM response after challenge. On the other hand, priming for a secondary IgG response took at least 4 days, and was dose-dependent, although helper T populations for a secondary IgG response appeared 3 days after antigen injection. It appeared that both IgM and IgG memory cells may be considered as Y cells in terms of the X-Y-Z scheme of lymphocyte activation, but that the two populations are generated at different times after exposure to antigen. The size of either Y-cell population at any given time is dependent upon the amount of antigen available to provoke differentiation to antibody-forming Z cells, and the IgM Y-cell population in particular is likely to be depleted during the course of a normal 1° response. When IgM Y cells were maintained for long periods as a result of immunosuppression, their secondary antibody response was independent of the primed T cells necessary for a secondary IgG response. PMID:4602981

Improved Single-Source Precursors for Solar-Cell Absorbers

NASA Technical Reports Server (NTRS)

Banger, Kulbinder K.; Harris, Jerry; Hepp, Aloysius

2007-01-01

Improved single-source precursor compounds have been invented for use in spray chemical vapor deposition (spray CVD) of chalcopyrite semiconductor absorber layers of thin-film cells. A "single-source precursor compound" is a single molecular compound that contains all the required elements, which when used under the spray CVD conditions, thermally decomposes to form CuIn(x)Ga(1-x)S(y)Se(2-y).

Zinc Deficiency Induces Apoptosis via Mitochondrial p53- and Caspase-Dependent Pathways in Human Neuronal Precursor Cells

ERIC Educational Resources Information Center

Seth, Rohit; Corniola, Rikki S.; Gower-Winter, Shannon D.; Morgan, Thomas J., Jr.; Bishop, Brian; Levenson, Cathy W.

2015-01-01

Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent…

A dynamic dual role of IL-2 signaling in the two-step differentiation process of adaptive regulatory T cells.

PubMed

Guo, Zhiyong; Khattar, Mithun; Schroder, Paul M; Miyahara, Yoshihiro; Wang, Guohua; He, Xiaoshung; Chen, Wenhao; Stepkowski, Stanislaw M

2013-04-01

The molecular mechanism of the extrathymic generation of adaptive, or inducible, CD4(+)Foxp3(+) regulatory T cells (iTregs) remains incompletely defined. We show that exposure of splenic CD4(+)CD25(+)Foxp3(-) cells to IL-2, but not other common γ-chain cytokines, resulted in Stat5 phosphorylation and induced Foxp3 expression in ∼10% of the cells. Thus, IL-2/Stat5 signaling may be critical for Foxp3 induction in peripheral CD4(+)CD25(+)Foxp3(-) iTreg precursors. In this study, to further define the role of IL-2 in the formation of iTreg precursors as well as their subsequent Foxp3 expression, we designed a two-step iTreg differentiation model. During the initial "conditioning" step, CD4(+)CD25(-)Foxp3(-) naive T cells were activated by TCR stimulation. Inhibition of IL-2 signaling via Jak3-Stat5 was required during this step to generate CD4(+)CD25(+)Foxp3(-) cells containing iTreg precursors. During the subsequent Foxp3-induction step driven by cytokines, IL-2 was the most potent cytokine to induce Foxp3 expression in these iTreg precursors. This two-step method generated a large number of iTregs with relatively stable expression of Foxp3, which were able to prevent CD4(+)CD45RB(high) cell-mediated colitis in Rag1(-/-) mice. In consideration of this information, whereas initial inhibition of IL-2 signaling upon T cell priming generates iTreg precursors, subsequent activation of IL-2 signaling in these precursors induces the expression of Foxp3. These findings advance the understanding of iTreg differentiation and may facilitate the therapeutic use of iTregs in immune disorders.

Sindbis virus glycoproteins are abnormally glycosylated in Chinese hamster ovary cells deprived of glucose.

PubMed

Davidson, S K; Hunt, L A

1985-07-01

We have previously demonstrated that Sindbis virus infection of Chinese hamster ovary (CHO) cells altered the protein glycosylation machinery of the cell, so that both normal, full-size (nine mannose-containing) oligosaccharides and abnormal, "truncated' (five mannose-containing) oligosaccharides are transferred from lipid-linked precursors to newly synthesized viral membrane glycoproteins. In the present studies, we have examined the precursor oligosaccharides on viral glycoproteins that were pulse-labelled with [3H]mannose in the presence or absence of glucose, since glucose starvation of uninfected CHO cells has been reported to induce synthesis of truncated precursor oligosaccharides. Pulse-labelling in the absence of glucose led to a greater than 10-fold increase in the relative amount of the truncated precursor oligosaccharides being transferred to the newly synthesized viral glycoproteins and to an apparent underglycosylation of some precursor viral polypeptides, with some asparaginyl sites not acquiring covalently linked oligosaccharides. The mature virion glycoproteins from CHO cells which were pulse-labelled in the absence of glucose and then 'chased' in the presence of glucose contained proportionately more unusual Man3GlcNAc2-size oligosaccharides. These small neutral-type oligosaccharides were apparently not as good a substrate for further processing into complex acidic-type oligosaccharides as the normal Man5GlcNAc2 intermediate that results from the full-size precursor oligosaccharides.

Differential and directional estrogenic signaling pathways induced by enterolignans and their precursors

PubMed Central

Zhu, Yun; Kawaguchi, Kayoko; Kiyama, Ryoiti

2017-01-01

Mammalian lignans or enterolignans are metabolites of plant lignans, an important category of phytochemicals. Although they are known to be associated with estrogenic activity, cell signaling pathways leading to specific cell functions, and especially the differences among lignans, have not been explored. We examined the estrogenic activity of enterolignans and their precursor plant lignans and cell signaling pathways for some cell functions, cell cycle and chemokine secretion. We used DNA microarray-based gene expression profiling in human breast cancer MCF-7 cells to examine the similarities, as well as the differences, among enterolignans, enterolactone and enterodiol, and their precursors, matairesinol, pinoresinol and sesamin. The profiles showed moderate to high levels of correlation (R values: 0.44 to 0.81) with that of estrogen (17β-estradiol or E2). Significant correlations were observed among lignans (R values: 0.77 to 0.97), and the correlations were higher for cell functions related to enzymes, signaling, proliferation and transport. All the enterolignans/precursors examined showed activation of the Erk1/2 and PI3K/Akt pathways, indicating the involvement of rapid signaling through the non-genomic estrogen signaling pathway. However, when their effects on specific cell functions, cell cycle progression and chemokine (MCP-1) secretion were examined, positive effects were observed only for enterolactone, suggesting that signals are given in certain directions at a position closer to cell functions. We hypothesized that, while estrogen signaling is initiated by the enterolignans/precursors examined, their signals are differentially and directionally modulated later in the pathways, resulting in the differences at the cell function level. PMID:28152041

Progressive CD4+ central–memory T cell decline results in CD4+ effector–memory insufficiency and overt disease in chronic SIV infection

PubMed Central

Okoye, Afam; Meier-Schellersheim, Martin; Brenchley, Jason M.; Hagen, Shoko I.; Walker, Joshua M.; Rohankhedkar, Mukta; Lum, Richard; Edgar, John B.; Planer, Shannon L.; Legasse, Alfred; Sylwester, Andrew W.; Piatak, Michael; Lifson, Jeffrey D.; Maino, Vernon C.; Sodora, Donald L.; Douek, Daniel C.; Axthelm, Michael K.; Grossman, Zvi; Picker, Louis J.

2007-01-01

Primary simian immunodeficiency virus (SIV) infections of rhesus macaques result in the dramatic depletion of CD4+ CCR5+ effector–memory T (TEM) cells from extra-lymphoid effector sites, but in most infections, an increased rate of CD4+ memory T cell proliferation appears to prevent collapse of effector site CD4+ TEM cell populations and acute-phase AIDS. Eventually, persistent SIV replication results in chronic-phase AIDS, but the responsible mechanisms remain controversial. Here, we demonstrate that in the chronic phase of progressive SIV infection, effector site CD4+ TEM cell populations manifest a slow, continuous decline, and that the degree of this depletion remains a highly significant correlate of late-onset AIDS. We further show that due to persistent immune activation, effector site CD4+ TEM cells are predominantly short-lived, and that their homeostasis is strikingly dependent on the production of new CD4+ TEM cells from central–memory T (TCM) cell precursors. The instability of effector site CD4+ TEM cell populations over time was not explained by increasing destruction of these cells, but rather was attributable to progressive reduction in their production, secondary to decreasing numbers of CCR5− CD4+ TCM cells. These data suggest that although CD4+ TEM cell depletion is a proximate mechanism of immunodeficiency, the tempo of this depletion and the timing of disease onset are largely determined by destruction, failing production, and gradual decline of CD4+ TCM cells. PMID:17724130

Progressive CD4+ central memory T cell decline results in CD4+ effector memory insufficiency and overt disease in chronic SIV infection.

PubMed

Okoye, Afam; Meier-Schellersheim, Martin; Brenchley, Jason M; Hagen, Shoko I; Walker, Joshua M; Rohankhedkar, Mukta; Lum, Richard; Edgar, John B; Planer, Shannon L; Legasse, Alfred; Sylwester, Andrew W; Piatak, Michael; Lifson, Jeffrey D; Maino, Vernon C; Sodora, Donald L; Douek, Daniel C; Axthelm, Michael K; Grossman, Zvi; Picker, Louis J

2007-09-03

Primary simian immunodeficiency virus (SIV) infections of rhesus macaques result in the dramatic depletion of CD4(+) CCR5(+) effector-memory T (T(EM)) cells from extra-lymphoid effector sites, but in most infections, an increased rate of CD4(+) memory T cell proliferation appears to prevent collapse of effector site CD4(+) T(EM) cell populations and acute-phase AIDS. Eventually, persistent SIV replication results in chronic-phase AIDS, but the responsible mechanisms remain controversial. Here, we demonstrate that in the chronic phase of progressive SIV infection, effector site CD4(+) T(EM) cell populations manifest a slow, continuous decline, and that the degree of this depletion remains a highly significant correlate of late-onset AIDS. We further show that due to persistent immune activation, effector site CD4(+) T(EM) cells are predominantly short-lived, and that their homeostasis is strikingly dependent on the production of new CD4(+) T(EM) cells from central-memory T (T(CM)) cell precursors. The instability of effector site CD4(+) T(EM) cell populations over time was not explained by increasing destruction of these cells, but rather was attributable to progressive reduction in their production, secondary to decreasing numbers of CCR5(-) CD4(+) T(CM) cells. These data suggest that although CD4(+) T(EM) cell depletion is a proximate mechanism of immunodeficiency, the tempo of this depletion and the timing of disease onset are largely determined by destruction, failing production, and gradual decline of CD4(+) T(CM) cells.

Imaging cardiac extracellular matrices: a blueprint for regeneration

PubMed Central

Jung, Jangwook P.; Squirrell, Jayne M.; Lyons, Gary E.; Eliceiri, Kevin W.; Ogle, Brenda M.

2013-01-01

Once damaged, cardiac tissue does not readily repair and is therefore a primary target of regenerative therapies. One regenerative approach is the development of scaffolds that functionally mimic the cardiac extracellular matrix (ECM) to deliver stem cells or cardiac precursor populations to the heart. Technological advances in micro/nanotechnology, stem cell biology, biomaterials and tissue decellularization have propelled this promising approach forward. Surprisingly, technological advances in optical imaging methods have not been fully utilized in the field of cardiac regeneration. Here, we describe and provide examples to demonstrate how advanced imaging techniques could revolutionize how ECM-mimicking cardiac tissues are informed and evaluated. PMID:22209562

Carbon-based composite electrocatalysts for low temperature fuel cells

DOEpatents

Popov, Branko N [Columbia, SC; Lee, Jog-Won [Columbia, SC; Subramanian, Nalini P [Kennesaw, GA; Kumaraguru, Swaminatha P [Honeoye Falls, NY; Colon-Mercado, Hector R [Columbia, SC; Nallathambi, Vijayadurga [T-Nagar, IN; Li, Xuguang [Columbia, SC; Wu, Gang [West Columbia, SC

2009-12-08

A process for synthesis of a catalyst is provided. The process includes providing a carbon precursor material, oxidizing the carbon precursor material whereby an oxygen functional group is introduced into the carbon precursor material, and adding a nitrogen functional group into the oxidized carbon precursor material.

Cells of origin in the embryonic nerve roots for NF1-associated plexiform neurofibroma

PubMed Central

Chen, Zhiguo; Liu, Chiachi; Patel, Amish J.; Liao, Chung-Ping; Wang, Yong; Le, Lu Q.

2014-01-01

Summary Neurofibromatosis type 1 is a tumor-predisposing genetic disorder. Plexiform neurofibromas are common NF1 tumors carrying a risk of malignant transformation, which is typically fatal. Little is known about mechanisms mediating initiation and identity of specific cell-type that gives rise to neurofibromas. Using cell-lineage tracing, we identify a population of GAP43+ PLP+ precursors in embryonic nerve roots as the cells of origin for these tumors and report a non-germline model of neurofibroma for preclinical drug screening to identify effective therapies. The identity of tumor cell-of-origin and facility for isolation and expansion provides fertile ground for continued analysis to define intrinsic and extrinsic factors critical for neurofibromagenesis. It also provides unique approaches to develop therapies to prevent neurofibroma formation in NF1 patients. PMID:25446898

Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy.

PubMed

Igarashi, Yuka; Uchiyama, Toru; Minegishi, Tomoko; Takahashi, Sirirat; Watanabe, Nobuyuki; Kawai, Toshinao; Yamada, Masafumi; Ariga, Tadashi; Onodera, Masafumi

2017-09-15

Clinical improvement in stem cell gene therapy (SCGT) for primary immunodeficiencies depends on the engraftment levels of genetically corrected cells, and tracing the transgene in each hematopoietic lineage is therefore extremely important in evaluating the efficacy of SCGT. We established a single cell-based droplet digital PCR (sc-ddPCR) method consisting of the encapsulation of a single cell into each droplet, followed by emulsion PCR with primers and probes specific for the transgene. A fluorescent signal in a droplet indicates the presence of a single cell carrying the target gene in its genome, and this system can clearly determine the ratio of transgene-positive cells in the entire population at the genomic level. Using sc-ddPCR, we analyzed the engraftment of vector-transduced cells in two patients with severe combined immunodeficiency (SCID) who were treated with SCGT. Sufficient engraftment of the transduced cells was limited to the T cell lineage in peripheral blood (PB), and a small percentage of CD34 + cells exhibited vector integration in bone marrow, indicating that the transgene-positive cells in PB might have differentiated from a small population of stem cells or lineage-restricted precursor cells. sc-ddPCR is a simplified and powerful tool for the detailed assessment of transgene-positive cell distribution in patients treated with SCGT.

Subnuclear localization, rates and effectiveness of UVC-induced unscheduled DNA synthesis visualized by fluorescence widefield, confocal and super-resolution microscopy.

PubMed

Pierzyńska-Mach, Agnieszka; Szczurek, Aleksander; Cella Zanacchi, Francesca; Pennacchietti, Francesca; Drukała, Justyna; Diaspro, Alberto; Cremer, Christoph; Darzynkiewicz, Zbigniew; Dobrucki, Jurek W

2016-01-01

Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2'-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from healthy subjects, we demonstrate that the sub-nuclear pattern of UDS detected via incorporation of EdU is different from that when BrdU is used as DNA precursor. EdU incorporation occurs evenly throughout chromatin, as opposed to just a few small and large repair foci detected by BrdU. We attribute this difference to the fact that BrdU antibody is of much larger size than EdU, and its accessibility to the incorporated precursor requires the presence of denatured sections of DNA. It appears that under the standard conditions of immunocytochemical detection of BrdU only fragments of DNA of various length are being denatured. We argue that, compared with BrdU, the UDS pattern visualized by EdU constitutes a more faithful representation of sub-nuclear distribution of the final stage of nucleotide excision repair induced by UVC. Using the optimized integrated EdU detection procedure we also measured the relative amount of the DNA precursor incorporated by cells during UDS following exposure to various doses of UVC. Also described is the high degree of heterogeneity in terms of the UVC-induced EdU incorporation per cell, presumably reflecting various DNA repair efficiencies or differences in the level of endogenous dT competing with EdU within a population of normal human fibroblasts.

Subnuclear localization, rates and effectiveness of UVC-induced unscheduled DNA synthesis visualized by fluorescence widefield, confocal and super-resolution microscopy

PubMed Central

Pierzyńska-Mach, Agnieszka; Szczurek, Aleksander; Cella Zanacchi, Francesca; Pennacchietti, Francesca; Drukała, Justyna; Diaspro, Alberto; Cremer, Christoph; Darzynkiewicz, Zbigniew; Dobrucki, Jurek W.

2016-01-01

ABSTRACT Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2′-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from healthy subjects, we demonstrate that the sub-nuclear pattern of UDS detected via incorporation of EdU is different from that when BrdU is used as DNA precursor. EdU incorporation occurs evenly throughout chromatin, as opposed to just a few small and large repair foci detected by BrdU. We attribute this difference to the fact that BrdU antibody is of much larger size than EdU, and its accessibility to the incorporated precursor requires the presence of denatured sections of DNA. It appears that under the standard conditions of immunocytochemical detection of BrdU only fragments of DNA of various length are being denatured. We argue that, compared with BrdU, the UDS pattern visualized by EdU constitutes a more faithful representation of sub-nuclear distribution of the final stage of nucleotide excision repair induced by UVC. Using the optimized integrated EdU detection procedure we also measured the relative amount of the DNA precursor incorporated by cells during UDS following exposure to various doses of UVC. Also described is the high degree of heterogeneity in terms of the UVC-induced EdU incorporation per cell, presumably reflecting various DNA repair efficiencies or differences in the level of endogenous dT competing with EdU within a population of normal human fibroblasts. PMID:27097376

Isolating RNA from precursor and mature melanocytes from human vitiligo and normal skin using laser capture microdissection.

PubMed

Goldstein, Nathaniel B; Koster, Maranke I; Hoaglin, Laura G; Wright, Michael J; Robinson, Steven E; Robinson, William A; Roop, Dennis R; Norris, David A; Birlea, Stanca A

2016-10-01

To characterize the gene expression profile of regenerated melanocytes in the narrow band UVB (NBUVB)-treated vitiligo epidermis and their precursors in the hair follicle, we present here a strategy of RNA isolation from in situ melanocytes using human frozen skin. We developed a rapid immunostaining protocol using the NKI-beteb antibody, which labels differentiated and precursor melanocytes, followed by fluorescent laser capture microdissection. This technique enabled the direct isolation, from melanocyte and adjacent keratinocyte populations, of satisfactory quality RNA that was successfully amplified and analysed by qRT-PCR. The melanocyte-specific gene transcripts TYR, DCT, TYRP1 and PMEL were significantly upregulated in our NBUVB-treated melanocyte samples as compared with the keratinocyte samples, while keratinocyte-specific genes (KRT5 and KRT14) were expressed significantly higher in the keratinocyte samples as compared with the melanocyte samples. Furthermore, in both NBUVB-treated vitiligo skin and normal skin, when bulge melanocytes were compared with epidermal melanocytes, we found significantly lower expression of melanocyte-specific genes and significantly higher expression of three melanocytic stem cell genes (SOX9, WIF1 and SFRP1), while ALCAM and ALDH1A1 transcripts did not show significant variation. We found significantly higher expression of melanocyte-specific genes in the epidermis of NBUVB-treated vitiligo, as compared to the normal skin. When comparing bulge melanocyte samples from untreated vitiligo, NBUVB-treated vitiligo and normal skin, we did not find significant differences in the expression of melanocyte-specific genes or melanocytic stem cell genes. These techniques offer valuable opportunities to study melanocytes and their precursors in vitiligo and other pigmentation disorders. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

High CD45 surface expression determines relapse risk in children with precursor B-cell and T-cell acute lymphoblastic leukemia treated according to the ALL-BFM 2000 protocol

PubMed Central

Cario, Gunnar; Rhein, Peter; Mitlöhner, Rita; Zimmermann, Martin; Bandapalli, Obul R.; Romey, Renja; Moericke, Anja; Ludwig, Wolf-Dieter; Ratei, Richard; Muckenthaler, Martina U.; Kulozik, Andreas E.; Schrappe, Martin; Stanulla, Martin; Karawajew, Leonid

2014-01-01

Further improvement of outcome in childhood acute lymphoblastic leukemia could be achieved by identifying additional high-risk patients who may benefit from intensified treatment. We earlier identified PTPRC (CD45) gene expression as a potential new stratification marker and now analyzed the prognostic relevance of CD45 protein expression. CD45 was measured by flow cytometry in 1065 patients treated according to the ALL-BFM-2000 protocol. The 75th percentile was used as cut-off to distinguish a CD45-high from a CD45-low group. As mean CD45 expression was significantly higher in T-cell acute lymphoblastic leukemia than in B-cell-precursor acute lymphoblastic leukemia (P<0.0001), the analysis was performed separately in both groups. In B-cell-precursor acute lymphoblastic leukemia we observed a significant association of a high CD45 expression with older age, high initial white blood cell count, ETV6/RUNX1 negativity, absence of high hyperdiploidy (P<0.0001), MLL/AF4 positivity (P=0.002), BCR/ABL1 positivity (P=0.007), prednisone poor response (P=0.002) and minimal residual disease (P<0.0001). In T-cell acute lymphoblastic leukemia we observed a significant association with initial white blood cell count (P=0.0003), prednisone poor response (P=0.01), and minimal residual disease (P=0.02). Compared to CD45-low patients, CD45-high patients had a lower event-free survival rate (B-cell-precursor acute lymphoblastic leukemia: 72±3% versus 86±1%, P<0.0001; T-cell acute lymphoblastic leukemia: 60±8% versus 78±4%, P=0.02), which was mainly attributable to a higher cumulative relapse incidence (B-cell-precursor acute lymphoblastic leukemia: 22±3% versus 11±1%, P<0.0001; T-cell acute lymphoblastic leukemia: 31±8% versus 11±3%, P=0.003) and kept its significance in multivariate analysis considering sex, age, initial white blood cell count, and minimal residual disease in B-cell-precursor- and T-cell acute lymphoblastic leukemia, and additionally presence of ETV6/RUNX1, MLL/AF4 and BCR/ABL1 rearrangements in B-cell-precursor acute lymphoblastic leukemia (P=0.002 and P=0.025, respectively). Consideration of CD45 expression may serve as an additional stratification tool in BFM-based protocols. (ClinicalTrials.gov identifier: NCT00430118) PMID:23911702

High CD45 surface expression determines relapse risk in children with precursor B-cell and T-cell acute lymphoblastic leukemia treated according to the ALL-BFM 2000 protocol.

PubMed

Cario, Gunnar; Rhein, Peter; Mitlöhner, Rita; Zimmermann, Martin; Bandapalli, Obul R; Romey, Renja; Moericke, Anja; Ludwig, Wolf-Dieter; Ratei, Richard; Muckenthaler, Martina U; Kulozik, Andreas E; Schrappe, Martin; Stanulla, Martin; Karawajew, Leonid

2014-01-01

Further improvement of outcome in childhood acute lymphoblastic leukemia could be achieved by identifying additional high-risk patients who may benefit from intensified treatment. We earlier identified PTPRC (CD45) gene expression as a potential new stratification marker and now analyzed the prognostic relevance of CD45 protein expression. CD45 was measured by flow cytometry in 1065 patients treated according to the ALL-BFM-2000 protocol. The 75(th) percentile was used as cut-off to distinguish a CD45-high from a CD45-low group. As mean CD45 expression was significantly higher in T-cell acute lymphoblastic leukemia than in B-cell-precursor acute lymphoblastic leukemia (P<0.0001), the analysis was performed separately in both groups. In B-cell-precursor acute lymphoblastic leukemia we observed a significant association of a high CD45 expression with older age, high initial white blood cell count, ETV6/RUNX1 negativity, absence of high hyperdiploidy (P<0.0001), MLL/AF4 positivity (P=0.002), BCR/ABL1 positivity (P=0.007), prednisone poor response (P=0.002) and minimal residual disease (P<0.0001). In T-cell acute lymphoblastic leukemia we observed a significant association with initial white blood cell count (P=0.0003), prednisone poor response (P=0.01), and minimal residual disease (P=0.02). Compared to CD45-low patients, CD45-high patients had a lower event-free survival rate (B-cell-precursor acute lymphoblastic leukemia: 72 ± 3% versus 86 ± 1%, P<0.0001; T-cell acute lymphoblastic leukemia: 60 ± 8% versus 78 ± 4%, P=0.02), which was mainly attributable to a higher cumulative relapse incidence (B-cell-precursor acute lymphoblastic leukemia: 22 ± 3% versus 11 ± 1%, P<0.0001; T-cell acute lymphoblastic leukemia: 31 ± 8% versus 11 ± 3%, P=0.003) and kept its significance in multivariate analysis considering sex, age, initial white blood cell count, and minimal residual disease in B-cell-precursor- and T-cell acute lymphoblastic leukemia, and additionally presence of ETV6/RUNX1, MLL/AF4 and BCR/ABL1 rearrangements in B-cell-precursor acute lymphoblastic leukemia (P=0.002 and P=0.025, respectively). Consideration of CD45 expression may serve as an additional stratification tool in BFM-based protocols. (ClinicalTrials.gov identifier: NCT00430118).

Induction of tolerance towards TNP entails down-regulation of an autoimmune attack.

PubMed Central

Zöller, M; Andrighetto, G

1988-01-01

In order to follow the process of induction and maintainance of tolerance, BALB/c mice were tolerized by free hapten, and effector and regulatory cell interactions were analysed by limiting-dilution (LD) cultures. Injection of trinitrobenzenesulphonic acid (TNBS) resulted, predominantly, in the activation and expansion of self-reactive cytotoxic T cells (CTL), which were observed transiently at frequencies comparable to allo-specific CTL. In addition, self-reactive helper T cells (Th) were activated and expanded in tolerized mice. TNP-specific reactivity was difficult to evaluate, since cytotoxic activity against haptenized self followed the pattern of self-reactivity throughout the test period. But in LD cultures determining proliferation, two populations of Th responding to TNP-self were observed, while only one Th population could be detected in response to self. Expansion/activation of Th and CTL precursors (CTLp) was followed by activation of suppressor T cells (Ts). The suppressor population could be divided into two subpopulations, one interfering with Th, the second interacting directly with CTL (veto cells). The results indicate that during the induction of tolerance, animals pass through an autoimmune attack, with expansion and activation of self-reactive clones (CTL, Th). The final status of non-responsiveness towards TNP is not due to the deletion of effector or regulatory cells, but results from the establishment of a steady state of dominance of self-reactive and TNP-self-reactive suppression. PMID:2965095

Single cell cultures of Drosophila neuroectodermal and mesectodermal central nervous system progenitors reveal different degrees of developmental autonomy.

PubMed

Lüer, Karin; Technau, Gerhard M

2009-08-03

The Drosophila embryonic central nervous system (CNS) develops from two sets of progenitor cells, neuroblasts and ventral midline progenitors, which behave differently in many respects. Neuroblasts derive from the neurogenic region of the ectoderm and form the lateral parts of the CNS. Ventral midline precursors are formed by two rows of mesectodermal cells and build the CNS midline. There is plenty of evidence that individual identities are conferred to precursor cells by positional information in the ectoderm. It is unclear, however, how far the precursors can maintain their identities and developmental properties in the absence of normal external signals. To separate the respective contributions of autonomous properties versus extrinsic signals during their further development, we isolated individual midline precursors and neuroectodermal precursors at the pre-mitotic gastrula stage, traced their development in vitro, and analyzed the characteristics of their lineages in comparison with those described for the embryo. Although individually cultured mesectodermal cells exhibit basic characteristics of CNS midline progenitors, the clones produced by these progenitors differ from their in situ counterparts with regard to cell numbers, expression of molecular markers, and the separation of neuronal and glial fate. In contrast, clones derived from individually cultured precursors taken from specific dorsoventral zones of the neuroectoderm develop striking similarities to the lineages of neuroblasts that normally delaminate from these zones and develop in situ. This in vitro analysis allows for the first time a comparison of the developmental capacities in situ and in vitro of individual neural precursors of defined spatial and temporal origin. The data reveal that cells isolated at the pre-mitotic and pre-delamination stage express characteristics of the progenitor type appropriate to their site of origin in the embryo. However, presumptive neuroblasts, once specified in the neuroectoderm, exhibit a higher degree of autonomy regarding generation of their lineages compared to mesectodermal midline progenitors.

Pluripotent stem cell-derived radial glia-like cells as stable intermediate for efficient generation of human oligodendrocytes.

PubMed

Gorris, Raphaela; Fischer, Julia; Erwes, Kim Lina; Kesavan, Jaideep; Peterson, Daniel A; Alexander, Michael; Nöthen, Markus M; Peitz, Michael; Quandel, Tamara; Karus, Michael; Brüstle, Oliver

2015-12-01

Neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) represent an attractive tool for the in vitro generation of various neural cell types. However, the developmentally early NPCs emerging during hPSC differentiation typically show a strong propensity for neuronal differentiation, with more limited potential for generating astrocytes and, in particular, for generating oligodendrocytes. This phenomenon corresponds well to the consecutive and protracted generation of neurons and GLIA during normal human development. To obtain a more gliogenic NPC type, we combined growth factor-mediated expansion with pre-exposure to the differentiation-inducing agent retinoic acid and subsequent immunoisolation of CD133-positive cells. This protocol yields an adherent and self-renewing population of hindbrain/spinal cord radial glia (RG)-like neural precursor cells (RGL-NPCs) expressing typical neural stem cell markers such as nestin, ASCL1, SOX2, and PAX6 as well as RG markers BLBP, GLAST, vimentin, and GFAP. While RGL-NPCs maintain the ability for tripotential differentiation into neurons, astrocytes, and oligodendrocytes, they exhibit greatly enhanced propensity for oligodendrocyte generation. Under defined differentiation conditions promoting the expression of the major oligodendrocyte fate-determinants OLIG1/2, NKX6.2, NKX2.2, and SOX10, RGL-NPCs efficiently convert into NG2-positive oligodendroglial progenitor cells (OPCs) and are subsequently capable of in vivo myelination. Representing a stable intermediate between PSCs and OPCs, RGL-NPCs expedite the generation of PSC-derived oligodendrocytes with O4-, 4860-, and myelin basic protein (MBP)-positive cells that already appear within 7 weeks following growth factor withdrawal-induced differentiation. Thus, RGL-NPCs may serve as robust tool for time-efficient generation of human oligodendrocytes from embryonic and induced pluripotent stem cells. © 2015 Wiley Periodicals, Inc.

Comparative Analysis of the Subventricular Zone in Rat, Ferret and Macaque: Evidence for an Outer Subventricular Zone in Rodents

PubMed Central

Camacho, Jasmin; Antczak, Jared L.; Prakash, Anish N.; Cziep, Matthew E.; Walker, Anita I.; Noctor, Stephen C.

2012-01-01

The mammalian cerebral cortex arises from precursor cells that reside in a proliferative region surrounding the lateral ventricles of the developing brain. Recent work has shown that precursor cells in the subventricular zone (SVZ) provide a major contribution to prenatal cortical neurogenesis, and that the SVZ is significantly thicker in gyrencephalic mammals such as primates than it is in lissencephalic mammals including rodents. Identifying characteristics that are shared by or that distinguish cortical precursor cells across mammalian species will shed light on factors that regulate cortical neurogenesis and may point toward mechanisms that underlie the evolutionary expansion of the neocortex in gyrencephalic mammals. We immunostained sections of the developing cerebral cortex from lissencephalic rats, and from gyrencephalic ferrets and macaques to compare the distribution of precursor cell types in each species. We also performed time-lapse imaging of precursor cells in the developing rat neocortex. We show that the distribution of Pax6+ and Tbr2+ precursor cells is similar in lissencephalic rat and gyrencephalic ferret, and different in the gyrencephalic cortex of macaque. We show that mitotic Pax6+ translocating radial glial cells (tRG) are present in the cerebral cortex of each species during and after neurogenesis, demonstrating that the function of Pax6+ tRG cells is not restricted to neurogenesis. Furthermore, we show that Olig2 expression distinguishes two distinct subtypes of Pax6+ tRG cells. Finally we present a novel method for discriminating the inner and outer SVZ across mammalian species and show that the key cytoarchitectural features and cell types that define the outer SVZ in developing primates are present in the developing rat neocortex. Our data demonstrate that the developing rat cerebral cortex possesses an outer subventricular zone during late stages of cortical neurogenesis and that the developing rodent cortex shares important features with that of primates. PMID:22272298

Comparative analysis of the subventricular zone in rat, ferret and macaque: evidence for an outer subventricular zone in rodents.

PubMed

Martínez-Cerdeño, Verónica; Cunningham, Christopher L; Camacho, Jasmin; Antczak, Jared L; Prakash, Anish N; Cziep, Matthew E; Walker, Anita I; Noctor, Stephen C

2012-01-01

The mammalian cerebral cortex arises from precursor cells that reside in a proliferative region surrounding the lateral ventricles of the developing brain. Recent work has shown that precursor cells in the subventricular zone (SVZ) provide a major contribution to prenatal cortical neurogenesis, and that the SVZ is significantly thicker in gyrencephalic mammals such as primates than it is in lissencephalic mammals including rodents. Identifying characteristics that are shared by or that distinguish cortical precursor cells across mammalian species will shed light on factors that regulate cortical neurogenesis and may point toward mechanisms that underlie the evolutionary expansion of the neocortex in gyrencephalic mammals. We immunostained sections of the developing cerebral cortex from lissencephalic rats, and from gyrencephalic ferrets and macaques to compare the distribution of precursor cell types in each species. We also performed time-lapse imaging of precursor cells in the developing rat neocortex. We show that the distribution of Pax6+ and Tbr2+ precursor cells is similar in lissencephalic rat and gyrencephalic ferret, and different in the gyrencephalic cortex of macaque. We show that mitotic Pax6+ translocating radial glial cells (tRG) are present in the cerebral cortex of each species during and after neurogenesis, demonstrating that the function of Pax6+ tRG cells is not restricted to neurogenesis. Furthermore, we show that Olig2 expression distinguishes two distinct subtypes of Pax6+ tRG cells. Finally we present a novel method for discriminating the inner and outer SVZ across mammalian species and show that the key cytoarchitectural features and cell types that define the outer SVZ in developing primates are present in the developing rat neocortex. Our data demonstrate that the developing rat cerebral cortex possesses an outer subventricular zone during late stages of cortical neurogenesis and that the developing rodent cortex shares important features with that of primates.

Alcohol-Induced Molecular Dysregulation in Human Embryonic Stem Cell-Derived Neural Precursor Cells

PubMed Central

Kim, Yi Young; Roubal, Ivan; Lee, Youn Soo; Kim, Jin Seok; Hoang, Michael; Mathiyakom, Nathan; Kim, Yong

2016-01-01

Adverse effect of alcohol on neural function has been well documented. Especially, the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models, which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described, the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol’s effect on JAK-STAT signaling pathway, neuroactive ligand-receptor interaction, Toll-like receptor (TLR) signaling pathway, cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of P2RX3, which is associated with nociception, a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs, but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event associated with alcohol-related peripheral neuropathy of an enhanced nociceptive response. PMID:27682028

Subcellular Distribution of Glutathione Precursors in Arabidopsis thaliana

PubMed Central

Koffler, Barbara Eva; Maier, Romana; Zechmann, Bernd

2011-01-01

Abstract Glutathione is an important antioxidant and has many important functions in plant development, growth and defense. Glutathione synthesis and degradation is highly compartment-specific and relies on the subcellular availability of its precursors, cysteine, glutamate, glycine and γ-glutamylcysteine especially in plastids and the cytosol which are considered as the main centers for glutathione synthesis. The availability of glutathione precursors within these cell compartments is therefore of great importance for successful plant development and defense. The aim of this study was to investigate the compartment-specific importance of glutathione precursors in Arabidopsis thaliana. The subcellular distribution was compared between wild type plants (Col-0), plants with impaired glutathione synthesis (glutathione deficient pad2-1 mutant, wild type plants treated with buthionine sulfoximine), and one complemented line (OE3) with restored glutathione synthesis. Immunocytohistochemistry revealed that the inhibition of glutathione synthesis induced the accumulation of the glutathione precursors cysteine, glutamate and glycine in most cell compartments including plastids and the cytosol. A strong decrease could be observed in γ-glutamylcysteine (γ-EC) contents in these cell compartments. These experiments demonstrated that the inhibition of γ-glutamylcysteine synthetase (GSH1) – the first enzyme of glutathione synthesis – causes a reduction of γ-EC levels and an accumulation of all other glutathione precursors within the cells. PMID:22050910

Vascular Mural Cells Promote Noradrenergic Differentiation of Embryonic Sympathetic Neurons.

PubMed

Fortuna, Vitor; Pardanaud, Luc; Brunet, Isabelle; Ola, Roxana; Ristori, Emma; Santoro, Massimo M; Nicoli, Stefania; Eichmann, Anne

2015-06-23

The sympathetic nervous system controls smooth muscle tone and heart rate in the cardiovascular system. Postganglionic sympathetic neurons (SNs) develop in close proximity to the dorsal aorta (DA) and innervate visceral smooth muscle targets. Here, we use the zebrafish embryo to ask whether the DA is required for SN development. We show that noradrenergic (NA) differentiation of SN precursors temporally coincides with vascular mural cell (VMC) recruitment to the DA and vascular maturation. Blocking vascular maturation inhibits VMC recruitment and blocks NA differentiation of SN precursors. Inhibition of platelet-derived growth factor receptor (PDGFR) signaling prevents VMC differentiation and also blocks NA differentiation of SN precursors. NA differentiation is normal in cloche mutants that are devoid of endothelial cells but have VMCs. Thus, PDGFR-mediated mural cell recruitment mediates neurovascular interactions between the aorta and sympathetic precursors and promotes their noradrenergic differentiation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Ezh2 phosphorylation state determines its capacity to maintain CD8+ T memory precursors for antitumor immunity.

PubMed

He, Shan; Liu, Yongnian; Meng, Lijun; Sun, Hongxing; Wang, Ying; Ji, Yun; Purushe, Janaki; Chen, Pan; Li, Changhong; Madzo, Jozef; Issa, Jean-Pierre; Soboloff, Jonathan; Reshef, Ran; Moore, Bethany; Gattinoni, Luca; Zhang, Yi

2017-12-14

Memory T cells sustain effector T-cell production while self-renewing in reaction to persistent antigen; yet, excessive expansion reduces memory potential and impairs antitumor immunity. Epigenetic mechanisms are thought to be important for balancing effector and memory differentiation; however, the epigenetic regulator(s) underpinning this process remains unknown. Herein, we show that the histone methyltransferase Ezh2 controls CD8 + T memory precursor formation and antitumor activity. Ezh2 activates Id3 while silencing Id2, Prdm1 and Eomes, promoting the expansion of memory precursor cells and their differentiation into functional memory cells. Akt activation phosphorylates Ezh2 and decreases its control of these transcriptional programs, causing enhanced effector differentiation at the expense of T memory precursors. Engineering T cells with an Akt-insensitive Ezh2 mutant markedly improves their memory potential and capability of controlling tumor growth compared to transiently inhibiting Akt. These findings establish Akt-mediated phosphorylation of Ezh2 as a critical target to potentiate antitumor immunotherapeutic strategies.

Two separate defects affecting true naive or virtual memory T cell precursors combine to reduce naive T cell responses with aging.

PubMed

Renkema, Kristin R; Li, Gang; Wu, Angela; Smithey, Megan J; Nikolich-Žugich, Janko

2014-01-01

Naive T cell responses are eroded with aging. We and others have recently shown that unimmunized old mice lose ≥ 70% of Ag-specific CD8 T cell precursors and that many of the remaining precursors acquire a virtual (central) memory (VM; CD44(hi)CD62L(hi)) phenotype. In this study, we demonstrate that unimmunized TCR transgenic (TCRTg) mice also undergo massive VM conversion with age, exhibiting rapid effector function upon both TCR and cytokine triggering. Age-related VM conversion in TCRTg mice directly depended on replacement of the original TCRTg specificity by endogenous TCRα rearrangements, indicating that TCR signals must be critical in VM conversion. Importantly, we found that VM conversion had adverse functional effects in both old wild-type and old TCRTg mice; that is, old VM, but not old true naive, T cells exhibited blunted TCR-mediated, but not IL-15-mediated, proliferation. This selective proliferative senescence correlated with increased apoptosis in old VM cells in response to peptide, but decreased apoptosis in response to homeostatic cytokines IL-7 and IL-15. Our results identify TCR as the key factor in differential maintenance and function of Ag-specific precursors in unimmunized mice with aging, and they demonstrate that two separate age-related defects--drastic reduction in true naive T cell precursors and impaired proliferative capacity of their VM cousins--combine to reduce naive T cell responses with aging.

Quantifying the importance of pMHC valency, total pMHC dose and frequency on nanoparticle therapeutic efficacy.

PubMed

Sugarman, Jordan; Tsai, Sue; Santamaria, Pere; Khadra, Anmar

2013-05-01

Nanoparticles (NPs) coated with β-cell-specific peptide major histocompatibility complex (pMHC) class I molecules can effectively restore normoglycemia in spontaneously diabetic nonobese diabetic mice. They do so by expanding pools of cognate memory autoreactive regulatory CD8+ T cells that arise from naive low-avidity T-cell precursors to therapeutic levels. Here we develop our previously constructed mathematical model to explore the effects of compound design parameters (NP dose and pMHC valency) on therapeutic efficacy with the underlying hypothesis that the functional correlates of the therapeutic response (expansion of autoregulatory T cells and deletion of autoantigen-loaded antigen-presenting cells by these T cells) are biphasic. We show, using bifurcation analysis, that the model exhibits a 'resonance'-like behavior for a given range of NP dose in which bistability between the healthy state (possessing zero level of effector T-cell population) and autoimmune state (possessing elevated level of the same population) disappears. A heterogeneous population of model mice subjected to several treatment protocols under these new conditions is conducted to quantify both the average percentage of autoregulatory T cells in responsive and nonresponsive model mice, and the average valency-dependent minimal optimal dose needed for effective therapy. Our results reveal that a moderate increase (≥1.6-fold) in the NP-dependent expansion rate of autoregulatory T-cell population leads to a significant increase in the efficacy and the area corresponding to the effective treatment regimen, provided that NP dose ≥8 μg. We expect the model developed here to generalize to other autoimmune diseases and serve as a computational tool to understand and optimize pMHC-NP-based therapies.

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

Maternal Forced Swimming Reduces Cell Proliferation in the Postnatal Dentate Gyrus of Mouse Offspring

PubMed Central

Wasinski, Frederick; Estrela, Gabriel R.; Arakaki, Aline M.; Bader, Michael; Alenina, Natalia; Klempin, Friederike; Araújo, Ronaldo C.

2016-01-01

Physical exercise positively affects the metabolism and induces proliferation of precursor cells in the adult brain. Maternal exercise likewise provokes adaptations early in the offspring. Using a high-intensity swimming protocol that comprises forced swim training before and during pregnancy, we determined the effect of maternal swimming on the mouse offspring's neurogenesis. Our data demonstrate decreased proliferation in sublayers of the postnatal dentate gyrus in offspring of swimming mother at postnatal day (P) 8 accompanied with decreased survival of newly generated cells 4 weeks later. The reduction in cell numbers was predominantly seen in the hilus and molecular layer. At P35, the reduced amount of cells was also reflected by a decrease in the population of newly generated immature and mature neurons of the granule cell layer. Our data suggest that forced maternal swimming at high-intensity has a negative effect on the neurogenic niche development in postnatal offspring. PMID:27621701

Eomeshi NK Cells in Human Liver Are Long-Lived and Do Not Recirculate but Can Be Replenished from the Circulation

PubMed Central

Cuff, Antonia O.; Robertson, Francis P.; Stegmann, Kerstin A.; Pallett, Laura J.; Maini, Mala K.; Davidson, Brian R.

2016-01-01

Human liver contains an Eomeshi population of NK cells that is not present in the blood. In this study, we show that these cells are characterized by a molecular signature that mediates their retention in the liver. By examining liver transplants where donors and recipients are HLA mismatched, we distinguish between donor liver–derived and recipient-derived leukocytes to show that Eomeslo NK cells circulate freely whereas Eomeshi NK cells are unable to leave the liver. Furthermore, Eomeshi NK cells are retained in the liver for up to 13 y. Therefore, Eomeshi NK cells are long-lived liver-resident cells. We go on to show that Eomeshi NK cells can be recruited from the circulation during adult life and that circulating Eomeslo NK cells are able to upregulate Eomes and molecules mediating liver retention under cytokine conditions similar to those in the liver. This suggests that circulating NK cells are a precursor of their liver-resident counterparts. PMID:27798170

Mouse model of CADASIL reveals novel insights into Notch3 function in adult hippocampal neurogenesis.

PubMed

Ehret, Fanny; Vogler, Steffen; Pojar, Sherin; Elliott, David A; Bradke, Frank; Steiner, Barbara; Kempermann, Gerd

2015-03-01

Could impaired adult hippocampal neurogenesis be a relevant mechanism underlying CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy)? Memory symptoms in CADASIL, the most common hereditary form of vascular dementia, are usually thought to be primarily due to vascular degeneration and white matter lacunes. Since adult hippocampal neurogenesis, a process essential for the integration of new spatial memory occurs in a highly vascularized niche, we considered dysregulation of adult neurogenesis as a potential mechanism for the manifestation of dementia in CADASIL. Analysis in aged mice overexpressing Notch3 with a CADASIL mutation, revealed vascular deficits in arteries of the hippocampal fissure but not in the niche of the dentate gyrus. At 12 months of age, cell proliferation and survival of newborn neurons were reduced not only in CADASIL mice but also in transgenic controls overexpressing wild type Notch3. At 6 months, hippocampal neurogenesis was altered in CADASIL mice independent of overt vascular abnormalities in the fissure. Further, we identified Notch3 expression in hippocampal precursor cells and maturing neurons in vivo as well as in cultured hippocampal precursor cells. Overexpression and knockdown experiments showed that Notch3 signaling negatively regulated precursor cell proliferation. Notch3 overexpression also led to deficits in KCl-induced precursor cell activation. This suggests a cell-autonomous effect of Notch3 signaling in the regulation of precursor proliferation and activation and a loss-of-function effect in CADASIL. Consequently, besides vascular damage, aberrant precursor cell proliferation and differentiation due to Notch3 dysfunction might be an additional independent mechanism for the development of hippocampal dysfunction in CADASIL. Copyright © 2014. Published by Elsevier Inc.

Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac-derived macrophages.

PubMed

Hoeffel, Guillaume; Wang, Yilin; Greter, Melanie; See, Peter; Teo, Pearline; Malleret, Benoit; Leboeuf, Marylène; Low, Donovan; Oller, Guillaume; Almeida, Francisca; Choy, Sharon H Y; Grisotto, Marcos; Renia, Laurent; Conway, Simon J; Stanley, E Richard; Chan, Jerry K Y; Ng, Lai Guan; Samokhvalov, Igor M; Merad, Miriam; Ginhoux, Florent

2012-06-04

Langerhans cells (LCs) are the dendritic cells (DCs) of the epidermis, forming one of the first hematopoietic lines of defense against skin pathogens. In contrast to other DCs, LCs arise from hematopoietic precursors that seed the skin before birth. However, the origin of these embryonic precursors remains unclear. Using in vivo lineage tracing, we identify a first wave of yolk sac (YS)-derived primitive myeloid progenitors that seed the skin before the onset of fetal liver hematopoiesis. YS progenitors migrate to the embryo proper, including the prospective skin, where they give rise to LC precursors, and the brain rudiment, where they give rise to microglial cells. However, in contrast to microglia, which remain of YS origin throughout life, YS-derived LC precursors are largely replaced by fetal liver monocytes during late embryogenesis. Consequently, adult LCs derive predominantly from fetal liver monocyte-derived cells with a minor contribution of YS-derived cells. Altogether, we establish that adult LCs have a dual origin, bridging early embryonic and late fetal myeloid development.

Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac–derived macrophages

PubMed Central

Hoeffel, Guillaume; Wang, Yilin; Greter, Melanie; See, Peter; Teo, Pearline; Malleret, Benoit; Leboeuf, Marylène; Low, Donovan; Oller, Guillaume; Almeida, Francisca; Choy, Sharon H.Y.; Grisotto, Marcos; Renia, Laurent; Conway, Simon J.; Stanley, E. Richard; Chan, Jerry K.Y.; Ng, Lai Guan; Samokhvalov, Igor M.

2012-01-01

Langerhans cells (LCs) are the dendritic cells (DCs) of the epidermis, forming one of the first hematopoietic lines of defense against skin pathogens. In contrast to other DCs, LCs arise from hematopoietic precursors that seed the skin before birth. However, the origin of these embryonic precursors remains unclear. Using in vivo lineage tracing, we identify a first wave of yolk sac (YS)–derived primitive myeloid progenitors that seed the skin before the onset of fetal liver hematopoiesis. YS progenitors migrate to the embryo proper, including the prospective skin, where they give rise to LC precursors, and the brain rudiment, where they give rise to microglial cells. However, in contrast to microglia, which remain of YS origin throughout life, YS-derived LC precursors are largely replaced by fetal liver monocytes during late embryogenesis. Consequently, adult LCs derive predominantly from fetal liver monocyte-derived cells with a minor contribution of YS-derived cells. Altogether, we establish that adult LCs have a dual origin, bridging early embryonic and late fetal myeloid development. PMID:22565823

cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness

PubMed Central

Wiley, Luke A.; Burnight, Erin R.; DeLuca, Adam P.; Anfinson, Kristin R.; Cranston, Cathryn M.; Kaalberg, Emily E.; Penticoff, Jessica A.; Affatigato, Louisa M.; Mullins, Robert F.; Stone, Edwin M.; Tucker, Budd A.

2016-01-01

Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans. PMID:27471043

Evaluation of Accessory Lacrimal Gland in Muller's Muscle Conjunctival Resection Specimens for Precursor Cell Markers and Biological Markers of Dry Eye Disease.

PubMed

Ali, Marwan; Shah, Dhara; Pasha, Zeeshan; Jassim, Sarmad H; Jassim Jaboori, Assraa; Setabutr, Pete; Aakalu, Vinay K

2017-04-01

The accessory lacrimal glands (ALGs) are an understudied component of the tear functional unit, even though they are important in the development of dry eye syndrome (DES). To advance our understanding of aging changes, regenerative potential, and histologic correlates to human characteristics, we investigated human ALG tissue from surgical samples to determine the presence or absence of progenitor cell markers and lacrimal epithelial markers and to correlate marker expression to relevant patient characteristics. ALG tissues obtained from Muller's muscle conjunctival resection (MMCR) specimens were created using tissue microarrays (TMAs). Immunofluorescence staining of MMCR sections was performed using primary antibodies specific to cell protein markers. Cell marker localization in TMAs was then assessed by two blinded observers using a standardized scoring system. Patient characteristics including age, race, and status of ocular surface health were then compared against expression of stem cell markers. Human ALG expressed a number of epithelial markers, and in particular, histatin-1 was well correlated with the expression of epithelial markers and was present in most acini. In addition, we noted the presence of precursor cell markers nestin, ABCG2, and CD90 in ALG tissue. There was a decrease in precursor cell marker expression with increasing age. Finally, we noted that a negative association was present between histatin-1 expression and DES. Thus, we report for the first time that human ALG tissues contain precursor marker-positive cells and that this marker expression may decrease with increasing age. Moreover, histatin-1 expression may be decreased in DES. Future studies will be performed to use these cell markers to isolate and culture lacrimal epithelial cells from heterogeneous tissues, determine the relevance of histatin-1 expression to DES, and isolate candidate precursor cells from ALG tissue.

Differential effects of Notch ligands Delta-1 and Jagged-1 in human lymphoid differentiation.

PubMed

Jaleco, A C; Neves, H; Hooijberg, E; Gameiro, P; Clode, N; Haury, M; Henrique, D; Parreira, L

2001-10-01

Notch signaling is known to differentially affect the development of lymphoid B and T cell lineages, but it remains unclear whether such effects are specifically dependent on distinct Notch ligands. Using a cell coculture assay we observed that the Notch ligand Delta-1 completely inhibits the differentiation of human hematopoietic progenitors into the B cell lineage while promoting the emergence of cells with a phenotype of T cell/natural killer (NK) precursors. In contrast, Jagged-1 did not disturb either B or T cell/NK development. Furthermore, cells cultured in the presence of either Delta-1 or Jagged-1 can acquire a phenotype of NK cells, and Delta-1, but not Jagged-1, permits the emergence of a de novo cell population coexpressing CD4 and CD8. Our results thus indicate that distinct Notch ligands can mediate differential effects of Notch signaling and provide a useful system to further address cell-fate decision processes in lymphopoiesis.

Differential Effects of Notch Ligands Delta-1 and Jagged-1 in Human Lymphoid Differentiation

PubMed Central

Jaleco, Ana C.; Neves, Hélia; Hooijberg, Erik; Gameiro, Paula; Clode, Nuno; Haury, Matthias; Henrique, Domingos; Parreira, Leonor

2001-01-01

Notch signaling is known to differentially affect the development of lymphoid B and T cell lineages, but it remains unclear whether such effects are specifically dependent on distinct Notch ligands. Using a cell coculture assay we observed that the Notch ligand Delta-1 completely inhibits the differentiation of human hematopoietic progenitors into the B cell lineage while promoting the emergence of cells with a phenotype of T cell/natural killer (NK) precursors. In contrast, Jagged-1 did not disturb either B or T cell/NK development. Furthermore, cells cultured in the presence of either Delta-1 or Jagged-1 can acquire a phenotype of NK cells, and Delta-1, but not Jagged-1, permits the emergence of a de novo cell population coexpressing CD4 and CD8. Our results thus indicate that distinct Notch ligands can mediate differential effects of Notch signaling and provide a useful system to further address cell-fate decision processes in lymphopoiesis. PMID:11581320

Replacement of Lost Lgr5-Positive Stem Cells through Plasticity of Their Enterocyte-Lineage Daughters.

PubMed

Tetteh, Paul W; Basak, Onur; Farin, Henner F; Wiebrands, Kay; Kretzschmar, Kai; Begthel, Harry; van den Born, Maaike; Korving, Jeroen; de Sauvage, Frederic; van Es, Johan H; van Oudenaarden, Alexander; Clevers, Hans

2016-02-04

Intestinal crypts display robust regeneration upon injury. The relatively rare secretory precursors can replace lost stem cells, but it is unknown if the abundant enterocyte progenitors that express the Alkaline phosphate intestinal (Alpi) gene also have this capacity. We created an Alpi-IRES-CreERT2 (Alpi(CreER)) knockin allele for lineage tracing. Marked clones consist entirely of enterocytes and are all lost from villus tips within days. Genetic fate-mapping of Alpi(+) cells before or during targeted ablation of Lgr5-expressing stem cells generated numerous long-lived crypt-villus "ribbons," indicative of dedifferentiation of enterocyte precursors into Lgr5(+) stems. By single-cell analysis of dedifferentiating enterocytes, we observed the generation of Paneth-like cells and proliferative stem cells. We conclude that the highly proliferative, short-lived enterocyte precursors serve as a large reservoir of potential stem cells during crypt regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

In vivo modulation of thymus-derived lymphocytes with monoclonal antibodies in mice. III. Spontaneous and natural cytotoxic effector cells.

PubMed Central

Herbert, A G; Le Gros, G S; Bidawid, S; Watson, J D

1984-01-01

Cytotoxic effector cell populations in murine spleen can be characterized by the phenotype of the cytotoxic cells or the nature of target cells. Lytic events can be antigen-specific, MHC-restricted and clonal, or target cell-specific but apparently non-MHC-restricted. Two cytotoxic effectors of this latter category are spontaneous and natural killers. Normal spleen cells from (BALB/c X DBA/2J)F1 mice (CDF1) cultured without added antigen develop a population of Thy-1+ spontaneous cytotoxic lymphocytes (SCTL) that lyse the DBA/2J mastocytoma P815, as well as the BALB/c-derived plasmacytomas MOPC-11 and SP2/0. Cold target competition experiments reveal the BALB/c-derived plasmacytomas MOPC-11, SP2/0, J558 and the A strain-derived T cell lymphoma YAC-1, but not normal lymphoblasts, block the lysis of P815 target cells. Thus, while these tumour cells appear to express common antigens which are recognized by SCTL cells, plasmacytomas such as J558 are not susceptible to lysis by SCTL. The relationship of SCTL to natural killer (NK) cells was examined. In-vivo treatment of mice with monoclonal anti-Thy-1 antibody leads to a rapid loss of SCTL and precursors from the spleen, but there is a concomitant increase in NK cell activity. PMID:6607213

MicroRNAs Promote Granule Cell Expansion in the Cerebellum Through Gli2.

PubMed

Constantin, Lena; Wainwright, Brandon J

2015-12-01

MicroRNAs (miRNAs) are important regulators of cerebellar function and homeostasis. Their deregulation results in cerebellar neuronal degeneration and spinocerebellar ataxia type 1 and contributes to medulloblastoma. Canonical miRNA processing involves Dicer, which cleaves precursor miRNAs into mature double-stranded RNA duplexes. In order to address the role of miRNAs in cerebellar granule cell precursor development, loxP-flanked exons of Dicer1 were conditionally inactivated using the granule cell precursor-specific Atoh1-Cre recombinase. A reduction of 87% in Dicer1 transcript was achieved in this conditional Dicer knockdown model. Although knockdown resulted in normal survival, mice had disruptions to the cortical layering of the anterior cerebellum, which resulted from the premature differentiation of granule cell precursors in this region during neonatal development. This defect manifested as a thinner external granular layer with ectopic mature granule cells, and a depleted internal granular layer. We found that expression of the activator components of the Hedgehog-Patched pathway, the Gli family of transcription factors, was perturbed in conditional Dicer knockdown mice. We propose that loss of Gli2 mRNA mediated the anterior-restricted defect in conditional Dicer knockdown mice and, as proof of principle, were able to show that miR-106b positively regulated Gli2 mRNA expression. These findings confirm the importance of miRNAs as positive mediators of Hedgehog-Patched signalling during granule cell precursor development.

Hedgehog signalling stimulates precursor cell accumulation and impairs epithelial maturation in the murine oesophagus.

PubMed

van Dop, Willemijn A; Rosekrans, Sanne L; Uhmann, Anja; Jaks, Viljar; Offerhaus, G Johan A; van den Bergh Weerman, Marius A; Kasper, Maria; Heijmans, Jarom; Hardwick, James C H; Verspaget, Hein W; Hommes, Daan W; Toftgård, Rune; Hahn, Heidi; van den Brink, Gijs R

2013-03-01

In the intestine Hedgehog (Hh) signalling is directed from epithelium to mesenchyme and negatively regulates epithelial precursor cell fate. The role of Hh signalling in the oesophagus has not been studied in vivo. Here the authors examined the role of Hh signalling in epithelial homeostasis of oesophagus. The authors used transgenic mice in which the Hh receptor Patched1 (Ptch1) could be conditionally inactivated in a body-wide manner and mice in which Gli1 could be induced specifically in the epithelium of the skin and oesophagus. Effects on epithelial homeostasis of the oesophagus were examined using immunohistochemistry, in situ hybridisation, transmission electron microscopy and real-time PCR. Hh signalling was examined in patients with oesophageal squamous cell carcinoma (SCC) by quantitative real-time PCR. Sonic Hh is signalled in an autocrine manner in the basal layer of the oesophagus. Activation of Hh signalling resulted in an expansion of the epithelial precursor cell compartment and failure of epithelial maturation and migration. Levels of Hh targets GLI1, HHIP and PTCH1 were increased in SCC compared with normal tissue from the same patients. Here the authors find that Hh signalling positively regulates the precursor cell compartment in the oesophageal epithelium in an autocrine manner. Since Hh signalling targets precursor cells in the oesophageal epithelium and signalling is increased in SCCs, Hh signalling may be involved in oesophageal SCC formation.

The PD-1: PD-L1 pathway promotes development of brain-resident memory T cells following acute viral encephalitis.

PubMed

Prasad, Sujata; Hu, Shuxian; Sheng, Wen S; Chauhan, Priyanka; Singh, Amar; Lokensgard, James R

2017-04-13

Previous work from our laboratory has demonstrated that during acute viral brain infection, glial cells modulate antiviral T cell effector responses through the PD-1: PD-L1 pathway, thereby limiting the deleterious consequences of unrestrained neuroinflammation. Here, we evaluated the PD-1: PD-L1 pathway in development of brain-resident memory T cells (bT RM ) following murine cytomegalovirus (MCMV) infection. Flow cytometric analysis of immune cells was performed at 7, 14, and 30 days post-infection (dpi) to assess the shift of brain-infiltrating CD8 + T cell populations from short-lived effector cells (SLEC) to memory precursor effector cells (MPEC), as well as generation of bT RMs . In wild-type (WT) animals, we observed a switch in the phenotype of brain-infiltrating CD8 + T cell populations from KLRG1 + CD127 - (SLEC) to KLRG1 - CD127 + (MPEC) during transition from acute through chronic phases of infection. At 14 and 30 dpi, the majority of CD8 + T cells expressed CD127, a marker of memory cells. In contrast, fewer CD8 + T cells expressed CD127 within brains of infected, PD-L1 knockout (KO) animals. Notably, in WT mice, a large population of CD8 + T cells was phenotyped as CD103 + CD69 + , markers of bT RM , and differences were observed in the numbers of these cells when compared to PD-L1 KOs. Immunohistochemical studies revealed that brain-resident CD103 + bT RM cells were localized to the parenchyma. Higher frequencies of CXCR3 were also observed among WT animals in contrast to PD-L1 KOs. Taken together, our results indicate that bT RMs are present within the CNS following viral infection and the PD-1: PD-L1 pathway plays a role in the generation of this brain-resident population.

GDNF facilitates differentiation of the adult dentate gyrus-derived neural precursor cells into astrocytes via STAT3

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

Boku, Shuken, E-mail: shuboku@med.hokudai.ac.jp; Nakagawa, Shin; Takamura, Naoki

2013-05-17

Highlights: •GDNF has no effect on ADP proliferation and apoptosis. •GDNF increases ADP differentiation into astrocyte. •A specific inhibitor of STAT3 decreases the astrogliogenic effect of GDNF. •STAT3 knockdown by lentiviral shRNA vector also decreases the astrogliogenic effect of GDNF. •GDNF increases the phosphorylation of STAT3. -- Abstract: While the pro-neurogenic actions of antidepressants in the adult hippocampal dentate gyrus (DG) are thought to be one of the mechanisms through which antidepressants exert their therapeutic actions, antidepressants do not increase proliferation of neural precursor cells derived from the adult DG. Because previous studies showed that antidepressants increase the expression andmore » secretion of glial cell line-derived neurotrophic factor (GDNF) in C6 glioma cells derived from rat astrocytes and GDNF increases neurogenesis in adult DG in vivo, we investigated the effects of GDNF on the proliferation, differentiation and apoptosis of cultured neural precursor cells derived from the adult DG. Data showed that GDNF facilitated the differentiation of neural precursor cells into astrocytes but had no effect on their proliferation or apoptosis. Moreover, GDNF increased the phosphorylation of STAT3, and both a specific inhibitor of STAT3 and lentiviral shRNA for STAT3 decreased their differentiation into astrocytes. Taken together, our findings suggest that GDNF facilitates astrogliogenesis from neural precursor cells in adult DG through activating STAT3 and that this action might indirectly affect neurogenesis.« less

Non-PGM cathode catalysts for fuel cell application derived from heat treated heteroatomic amines precursors

DOEpatents

Serov, Alexey; Halevi, Barr; Artyushkova, Kateryna; Atanassov, Plamen B; Martinez, Ulises A

2017-04-25

A method of preparing M-N--C catalysts utilizing a sacrificial support approach and inexpensive and readily available polymer precursors as the source of nitrogen and carbon is disclosed. Exemplary polymer precursors include non-porphyrin precursors with no initial catalytic activity. Examples of suitable non-catalytic non-porphyrin precursors include, but are not necessarily limited to low molecular weight precursors that form complexes with iron such as 4-aminoantipirine, phenylenediamine, hydroxysuccinimide, ethanolamine, and the like.

Live imaging of heart tube development in mouse reveals alternating phases of cardiac differentiation and morphogenesis

PubMed Central

Ivanovitch, Kenzo; Temiño, Susana

2017-01-01

During vertebrate heart development, two progenitor populations, first and second heart fields (FHF, SHF), sequentially contribute to longitudinal subdivisions of the heart tube (HT), with the FHF contributing the left ventricle and part of the atria, and the SHF the rest of the heart. Here, we study the dynamics of cardiac differentiation and morphogenesis by tracking individual cells in live analysis of mouse embryos. We report that during an initial phase, FHF precursors differentiate rapidly to form a cardiac crescent, while limited morphogenesis takes place. In a second phase, no differentiation occurs while extensive morphogenesis, including splanchnic mesoderm sliding over the endoderm, results in HT formation. In a third phase, cardiac precursor differentiation resumes and contributes to SHF-derived regions and the dorsal closure of the HT. These results reveal tissue-level coordination between morphogenesis and differentiation during HT formation and provide a new framework to understand heart development. PMID:29202929

Fluctuations in Blood Marginal Zone B-Cell Frequencies May Reflect Migratory Patterns Associated with HIV-1 Disease Progression Status

PubMed Central

Poudrier, Johanne; Roger, Michel

2016-01-01

We have previously shown that overexpression of BLyS/BAFF was associated with increased relative frequencies of innate “precursor” marginal zone (MZ)-like B-cells in the blood of HIV-1-infected rapid and classic progressors. However, along with relatively normal BLyS/BAFF expression levels, these cells remain unaltered in elite-controllers (EC), rather, percentages of more mature MZ-like B-cells are decreased in the blood of these individuals. Fluctuations in frequencies of blood MZ-like B-cell populations may reflect migratory patterns associated with disease progression status, suggesting an important role for these cells in HIV-1 pathogenesis. We have therefore longitudinally measured plasma levels of B-tropic chemokines by ELISA-based technology as well as their ligands by flow-cytometry on blood B-cell populations of HIV-1-infected individuals with different rates of disease progression and uninfected controls. Migration potential of B-cell populations from these individuals were determined by chemotaxis assays. We found important modulations of CXCL13-CXCR5, CXCL12-CXCR4/CXCR7, CCL20-CCR6 and CCL25-CCR9 chemokine-axes and increased cell migration patterns in HIV progressors. Interestingly, frequencies of CCR6 expressing cells were significantly elevated within the precursor MZ-like population, consistent with increased migration in response to CCL20. Although we found little modulation of chemokine-axes in EC, cell migration was greater than that observed for uninfected controls, especially for MZ-like B-cells. Overall the immune response against HIV-1 may involve recruitment of MZ-like B-cells to peripheral sites. Moreover, our findings suggest that “regulated” attraction of these cells in a preserved BLyS/BAFF non-inflammatory environment, such as encountered in EC could be beneficial to the battle and even control of HIV. PMID:27203285

Wnt signaling induces proliferation of sensory precursors in the postnatal mouse cochlea.

PubMed

Chai, Renjie; Kuo, Bryan; Wang, Tian; Liaw, Eric J; Xia, Anping; Jan, Taha A; Liu, Zhiyong; Taketo, Makoto M; Oghalai, John S; Nusse, Roeland; Zuo, Jian; Cheng, Alan G

2012-05-22

Inner ear hair cells are specialized sensory cells essential for auditory function. Previous studies have shown that the sensory epithelium is postmitotic, but it harbors cells that can behave as progenitor cells in vitro, including the ability to form new hair cells. Lgr5, a Wnt target gene, marks distinct supporting cell types in the neonatal cochlea. Here, we tested the hypothesis that Lgr5(+) cells are Wnt-responsive sensory precursor cells. In contrast to their quiescent in vivo behavior, Lgr5(+) cells isolated by flow cytometry from neonatal Lgr5(EGFP-CreERT2/+) mice proliferated and formed clonal colonies. After 10 d in culture, new sensory cells formed and displayed specific hair cell markers (myo7a, calretinin, parvalbumin, myo6) and stereocilia-like structures expressing F-actin and espin. In comparison with other supporting cells, Lgr5(+) cells were enriched precursors to myo7a(+) cells, most of which formed without mitotic division. Treatment with Wnt agonists increased proliferation and colony-formation capacity. Conversely, small-molecule inhibitors of Wnt signaling suppressed proliferation without compromising the myo7a(+) cells formed by direct differentiation. In vivo lineage tracing supported the idea that Lgr5(+) cells give rise to myo7a(+) hair cells in the neonatal Lgr5(EGFP-CreERT2/+) cochlea. In addition, overexpression of β-catenin initiated proliferation and led to transient expansion of Lgr5(+) cells within the cochlear sensory epithelium. These results suggest that Lgr5 marks sensory precursors and that Wnt signaling can promote their proliferation and provide mechanistic insights into Wnt-responsive progenitor cells during sensory organ development.

Intratumoral conversion of adrenal androgen precursors drives androgen receptor-activated cell growth in prostate cancer more potently than de novo steroidogenesis.

PubMed

Kumagai, Jinpei; Hofland, Johannes; Erkens-Schulze, Sigrun; Dits, Natasja F J; Steenbergen, Jacobie; Jenster, Guido; Homma, Yukio; de Jong, Frank H; van Weerden, Wytske M

2013-11-01

Despite an initial response to hormonal therapy, patients with advanced prostate cancer (PC) almost always progress to castration-resistant disease (CRPC). Although serum testosterone (T) is reduced by androgen deprivation therapy, intratumoral T levels in CRPC are comparable to those in prostate tissue of eugonadal men. These levels could originate from intratumoral conversion of adrenal androgens and/or from de novo steroid synthesis. However, the relative contribution of de novo steroidogenesis to AR-driven cell growth is unknown. The relative contribution of androgen biosynthetic pathways to activate androgen receptor (AR)-regulated cell growth and expression of PSA, FKBP5, and TMPRSS2 was studied at physiologically relevant levels of adrenal androgen precursors and intermediates of de novo androgen biosynthesis in human prostate cancer cell lines, PC346C, VCaP, and LNCaP. In PC346C and VCaP, responses to pregnenolone and progesterone were absent or minimal, while large effects of adrenal androgen precursors were found. VCaP CRPC clones overexpressing CYP17A1 did not acquire an increased ability to use pregnenolone or progesterone to activate AR. In contrast, all precursors stimulated growth and gene expression in LNCaP cells, presumably resulting from the mutated AR in these cells. Our data indicate that at physiological levels of T precursors PC cells can generally convert adrenal androgens, while de novo steroidogenesis is not generally possible in PC cells and is not able to support AR transactivation and PC growth. © 2013 Wiley Periodicals, Inc.

Enhanced production of L-DOPA in cell cultures of Mucuna pruriens L. and Mucuna prurita H.

PubMed

Raghavendra, S; Kumar, V; Ramesh, C K; Khan, M H Moinuddin

2012-01-01

A comparative study on the production of 3,4-dihydroxyphenylalanine (L-DOPA) was carried out in cell cultures of two Mucuna species by elicitor treatment and precursor feeding. The influence of elicitors and the precursor molecule on L-DOPA production, polyphenol oxidase (PPO) and tyrosinase activities was also studied. Callus cultures were initiated in Mucuna pruriens L. and Mucuna prurita H. on MS medium supplemented with BAP and IAA at different concentrations. Suspension cultures were established in MS liquid medium supplemented with BAP, IAA, the elicitors methyl jasmonate, chitin and pectin or the precursor L-tyrosine at different concentrations for L-DOPA production. Compared to the controls, several-fold increases in L-DOPA concentration were observed in elicitor-treated and precursor-fed suspension cultures of both plant species. L-DOPA concentrations were comparatively higher in precursor-fed cultures than those receiving elicitor treatments. A parallel increase in tyrosinase and PPO levels was also observed. Loss of cell viability was observed at high concentrations of elicitor-treated cultures, whereas L-tyrosine did not cause any cell death. Compared to elicitor treatments, precursor feeding resulted in higher concentrations of L-DOPA production and tyrosinase activity. The efficacy of L-DOPA production was found to be higher for suspension cultures of M. pruriens compared to M. prurita in all treatments.

CuInSe₂ thin-film solar cells with 7.72 % efficiency prepared via direct coating of a metal salts/alcohol-based precursor solution.

PubMed

Ahn, Sejin; Son, Tae Hwa; Cho, Ara; Gwak, Jihye; Yun, Jae Ho; Shin, Keeshik; Ahn, Seoung Kyu; Park, Sang Hyun; Yoon, Kyunghoon

2012-09-01

A simple direct solution coating process for forming CuInSe₂ (CIS) thin films was described, employing a low-cost and environmentally friendly precursor solution. The precursor solution was prepared by mixing metal acetates, ethanol, and ethanolamine. The facile formation of a precursor solution without the need to prefabricate nanoparticles enables a rapid and easy processing, and the high stability of the solution in air further ensures the precursor preparation and the film deposition in ambient conditions without a glove box. The thin film solar cell fabricated with the absorber film prepared by this route showed an initial conversion efficiency of as high as 7.72 %. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A CCR2+ myeloid cell niche required for pancreatic β cell growth

PubMed Central

Mussar, Kristin; Pardike, Stephanie; Hohl, Tobias M.; Hardiman, Gary; Cirulli, Vincenzo

2017-01-01

Organ-specific patterns of myeloid cells may contribute tissue-specific growth and/or regenerative potentials. The perinatal stage of pancreas development marks a time characterized by maximal proliferation of pancreatic islets, ensuring the maintenance of glucose homeostasis throughout life. Ontogenically distinct CX3CR1+ and CCR2+ macrophage populations have been reported in the adult pancreas, but their functional contribution to islet cell growth at birth remains unknown. Here, we uncovered a temporally restricted requirement for CCR2+ myeloid cells in the perinatal proliferation of the endocrine pancreatic epithelium. CCR2+ macrophages are transiently enriched over CX3CR1+ subsets in the neonatal pancreas through both local expansion and recruitment of immature precursors. Using CCR2-specific depletion models, we show that loss of this myeloid population leads to a striking reduction in β cell proliferation, dysfunctional islet phenotypes, and glucose intolerance in newborns. Replenishment of pancreatic CCR2+ myeloid compartments by adoptive transfer rescues these defects. Gene profiling identifies pancreatic CCR2+ myeloid cells as a prominent source of IGF2, which contributes to IGF1R-mediated islet proliferation. These findings uncover proproliferative functions of CCR2+ myeloid subsets and identify myeloid-dependent regulation of IGF signaling as a local cue supporting pancreatic proliferation. PMID:28768911

Functional expression of calcium-permeable canonical transient receptor potential 4-containing channels promotes migration of medulloblastoma cells.

PubMed

Wei, Wei-Chun; Huang, Wan-Chen; Lin, Yu-Ping; Becker, Esther B E; Ansorge, Olaf; Flockerzi, Veit; Conti, Daniele; Cenacchi, Giovanna; Glitsch, Maike D

2017-08-15

The proton sensing ovarian cancer G protein coupled receptor 1 (OGR1, aka GPR68) promotes expression of the canonical transient receptor potential channel subunit TRPC4 in normal and transformed cerebellar granule precursor (DAOY) cells. OGR1 and TRPC4 are prominently expressed in healthy cerebellar tissue throughout postnatal development and in primary cerebellar medulloblastoma tissues. Activation of TRPC4-containing channels in DAOY cells, but not non-transformed granule precursor cells, results in prominent increases in [Ca 2+ ] i and promotes cell motility in wound healing and transwell migration assays. Medulloblastoma cells not arising from granule precursor cells show neither prominent rises in [Ca 2+ ] i nor enhanced motility in response to TRPC4 activation unless they overexpressTRPC4. Our results suggest that OGR1 enhances expression of TRPC4-containing channels that contribute to enhanced invasion and metastasis of granule precursor-derived human medulloblastoma. Aberrant intracellular Ca 2+ signalling contributes to the formation and progression of a range of distinct pathologies including cancers. Rises in intracellular Ca 2+ concentration occur in response to Ca 2+ influx through plasma membrane channels and Ca 2+ release from intracellular Ca 2+ stores, which can be mobilized in response to activation of cell surface receptors. Ovarian cancer G protein coupled receptor 1 (OGR1, aka GPR68) is a proton-sensing G q -coupled receptor that is most highly expressed in cerebellum. Medulloblastoma (MB) is the most common paediatric brain tumour that arises from cerebellar precursor cells. We found that nine distinct human MB samples all expressed OGR1. In both normal granule cells and the transformed human cerebellar granule cell line DAOY, OGR1 promoted expression of the proton-potentiated member of the canonical transient receptor potential (TRPC) channel family, TRPC4. Consistent with a role for TRPC4 in MB, we found that all MB samples also expressed TRPC4. In DAOY cells, activation of TRPC4-containing channels resulted in large Ca 2+ influx and enhanced migration, while in normal cerebellar granule (precursor) cells and MB cells not derived from granule precursors, only small levels of Ca 2+ influx and no enhanced migration were observed. Our results suggest that OGR1-dependent increases in TRPC4 expression may favour formation of highly Ca 2+ -permeable TRPC4-containing channels that promote transformed granule cell migration. Increased motility of cancer cells is a prerequisite for cancer invasion and metastasis, and our findings may point towards a key role for TRPC4 in progression of certain types of MB. © 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Microglia modulate hippocampal neural precursor activity in response to exercise and aging.

PubMed

Vukovic, Jana; Colditz, Michael J; Blackmore, Daniel G; Ruitenberg, Marc J; Bartlett, Perry F

2012-05-09

Exercise has been shown to positively augment adult hippocampal neurogenesis; however, the cellular and molecular pathways mediating this effect remain largely unknown. Previous studies have suggested that microglia may have the ability to differentially instruct neurogenesis in the adult brain. Here, we used transgenic Csf1r-GFP mice to investigate whether hippocampal microglia directly influence the activation of neural precursor cells. Our results revealed that an exercise-induced increase in neural precursor cell activity was mediated via endogenous microglia and abolished when these cells were selectively removed from hippocampal cultures. Conversely, microglia from the hippocampi of animals that had exercised were able to activate latent neural precursor cells when added to neurosphere preparations from sedentary mice. We also investigated the role of CX(3)CL1, a chemokine that is known to provide a more neuroprotective microglial phenotype. Intraparenchymal infusion of a blocking antibody against the CX(3)CL1 receptor, CX(3)CR1, but not control IgG, dramatically reduced the neurosphere formation frequency in mice that had exercised. While an increase in soluble CX(3)CL1 was observed following running, reduced levels of this chemokine were found in the aged brain. Lower levels of CX(3)CL1 with advancing age correlated with the natural decline in neural precursor cell activity, a state that could be partially alleviated through removal of microglia. These findings provide the first direct evidence that endogenous microglia can exert a dual and opposing influence on neural precursor cell activity within the hippocampus, and that signaling through the CX(3)CL1-CX(3)CR1 axis critically contributes toward this process.

TOO MANY MOUTHS promotes cell fate progression in stomatal development of Arabidopsis stems.

PubMed

Bhave, Neela S; Veley, Kira M; Nadeau, Jeanette A; Lucas, Jessica R; Bhave, Sanjay L; Sack, Fred D

2009-01-01

Mutations in TOO MANY MOUTHS (TMM), which encodes a receptor-like protein, cause stomatal patterning defects in Arabidopsis leaves but eliminate stomatal formation in stems. Stomatal development in wild-type and tmm stems was analyzed to define TMM function. Epidermal cells in young tmm stems underwent many asymmetric divisions characteristic of entry into the stomatal pathway. The resulting precursor cells, meristemoids, appropriately expressed cell fate markers such as pTMM:GFP. However, instead of progressing developmentally by forming a guard mother cell, the meristemoids arrested, dedifferentiated, and enlarged. Thus asymmetric divisions are necessary but not sufficient for stomatal formation in stems, and TMM promotes the fate and developmental progression of early precursor cells. Comparable developmental and mature stomatal phenotypes were also found in tmm hypocotyls and in the proximal flower stalk. TMM is also a positive regulator of meristemoid division in leaves suggesting that TMM generally promotes meristemoid activity. Our results are consistent with a model in which TMM interacts with other proteins to modulate precursor cell fate and progression in an organ and domain-specific manner. Finally, the consistent presence of a small number of dedifferentiated meristemoids in mature wild-type stems suggests that precursor cell arrest is a normal feature of Arabidopsis stem development.

Thymic emigration revisited

PubMed Central

McCaughtry, Tom M.; Wilken, Matthew S.; Hogquist, Kristin A.

2007-01-01

Conventional αβ T cell precursors undergo positive selection in the thymic cortex. When this is successful, they migrate to the medulla and are exposed to tissue-specific antigens (TSA) for purposes of central tolerance, and they undergo maturation to become functionally responsive T cells. It is commonly understood that thymocytes spend up to 2 wk in the medulla undergoing these final maturation steps before emigrating to peripheral lymphoid tissues. In addition, emigration is thought to occur via a stochastic mechanism whereby some progenitors leave early and others leave late—a so-called “lucky dip” process. However, recent research has revealed that medullary thymocytes are a heterogeneous mix of naive αβ T cell precursors, memory T cells, natural killer T cells, and regulatory T cells. Given this, we revisited the question of how long it takes naive αβ T cell precursors to emigrate. We combined the following three approaches to study this question: BrdU labeling, intrathymic injection of a cellular tag, and RAG2p-GFP reporter mice. We established that, on average, naive αβ T cell precursors emigrate only 4–5 d after becoming single-positive (SP) thymocytes. Furthermore, emigration occurs via a strict “conveyor belt” mechanism, where the oldest thymocytes leave first. PMID:17908937

Effects of detergents on ribosomal precursor subunits of Bacillus megaterium.

PubMed

Body, A; Brownstein, B H

1978-01-01

Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction.

Effects of Detergents on Ribosomal Precursor Subunits of Bacillus megaterium

PubMed Central

Body, Barbara A.; Brownstein, Bernard H.

1978-01-01

Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction. PMID:412833

Case report of precursor B-cell lymphoblastic lymphoma presenting as syncope and cardiac mass in a nonimmunocompromised child.

PubMed

Hahn, Barry; Rao, Sudha; Shah, Binita

2007-08-01

We report the case of a previously healthy, 10-year-old boy who presented to the emergency department with a syncopal episode. In the emergency department, the patient was diagnosed with a right atrial mass, later identified as a precursor B-cell lymphoblastic lymphoma (LL). Most causes of syncope in children are not life threatening. In most cases, it indicates a predisposition to vasovagal episodes. Lymphomas account for approximately 7% of malignancies among children younger than 20 years, are more common in white males and immunocompromised patients, and are predominantly tumors of T-cell origin. Children with non-Hodgkin lymphoma usually present with extranodal disease, most frequently involving the abdomen (31%), mediastinum (26%), or head and neck (29%). Our patient was unique in that he was a nonimmunocompromised, black boy, presenting with syncope in the setting of a large atrial mass identified as a precursor B-cell LL. To our knowledge, there are no reported cases of precursor B-cell LL presenting as syncope and a cardiac mass.

Heterogeneity of Clonal Expansion and Maturation-Linked Mutation Acquisition in Hematopoietic Progenitors in Human Acute Myeloid Leukemia

PubMed Central

Walter, Roland B.; Laszlo, George S.; Lionberger, Jack M.; Pollard, Jessica A.; Harrington, Kimberly H.; Gudgeon, Chelsea J.; Othus, Megan; Rafii, Shahin; Meshinchi, Soheil; Appelbaum, Frederick R.; Bernstein, Irwin D.

2014-01-01

Recent technological advances led to an appreciation of the genetic complexity of human acute myeloid leukemia (AML) but underlying progenitor cells remain poorly understood because their rarity precludes direct study. We developed a co-culture method integrating hypoxia, aryl hydrocarbon receptor inhibition, and micro-environmental support via human endothelial cells to isolate these cells. X-chromosome inactivation studies of the least mature precursors derived following prolonged culture of CD34+/CD33− cells revealed polyclonal growth in highly curable AMLs, suggesting mutations necessary for clonal expansion were acquired in more mature progenitors. Consistently, in core-binding factor (CBF) leukemias with known complementing mutations, immature precursors derived following prolonged culture of CD34+/CD33− cells harbored neither mutation or the CBF mutation alone, whereas more mature precursors often carried both mutations. These results were in contrast to those with leukemias with poor prognosis that showed clonal dominance in the least mature precursors. These data indicate heterogeneity among progenitors in human AML that may have prognostic and therapeutic implications. PMID:24721792

Distal Regeneration Involves the Age Dependent Activity of Branchial Sac Stem Cells in the Ascidian Ciona intestinalis.

PubMed

Jeffery, William R

2015-02-01

Tunicates have high capacities for regeneration but the underlying mechanisms and their relationship to life cycle progression are not well understood. Here we investigate the regeneration of distal structures in the ascidian tunicate Ciona intestinalis . Analysis of regenerative potential along the proximal-distal body axis indicated that distal organs, such as the siphons, their pigmented sensory organs, and the neural complex, could only be replaced from body fragments containing the branchial sac. Distal regeneration involves the formation of a blastema composed of cells that undergo cell proliferation prior to differentiation and cells that differentiate without cell proliferation. Both cell types originate in the branchial sac and appear in the blastema at different times after distal injury. Whereas the branchial sac stem cells are present in young animals, they are depleted in old animals that have lost their regeneration capacity. Thus Ciona adults contain a population of age-related stem cells located in the branchial sac that are a source of precursors for distal body regeneration.

Evolution of New miRNAs and Cerebro-Cortical Development.

PubMed

Kosik, Kenneth S; Nowakowski, Tomasz

2018-04-04

The noncoding portion of the genome, including microRNAs, has been fertile evolutionary soil for cortical development in primates. A major contribution to cortical expansion in primates is the generation of novel precursor cell populations. Because miRNA expression profiles track closely with cell identity, it is likely that numerous novel microRNAs have contributed to cellular diversity in the brain. The tools to determine the genomic context within which novel microRNAs emerge and how they become integrated into molecular circuitry are now in hand. Expected final online publication date for the Annual Review of Neuroscience Volume 41 is July 8, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Defining disease with laser precision: laser capture microdissection in gastroenterology.

PubMed

Blatt, Richard; Srinivasan, Shanthi

2008-08-01

Laser capture microdissection (LCM) is an efficient and precise method for obtaining pure cell populations or specific cells of interest from a given tissue sample. LCM has been applied to animal and human gastroenterology research in analyzing the protein, DNA, and RNA from all organs of the gastrointestinal system. There are numerous potential applications for this technology in gastroenterology research, including malignancies of the esophagus, stomach, colon, biliary tract, and liver. This technology can also be used to study gastrointestinal infections, inflammatory bowel disease, pancreatitis, motility, malabsorption, and radiation enteropathy. LCM has multiple advantages when compared with conventional methods of microdissection, and this technology can be exploited to identify precursors to disease, diagnostic biomarkers, and therapeutic interventions.

A Novel Source of Cultured Podocytes

PubMed Central

Da Sacco, Stefano; Lemley, Kevin V.; Sedrakyan, Sargis; Zanusso, Ilenia; Petrosyan, Astgik; Peti-Peterdi, Janos; Burford, James; De Filippo, Roger E.; Perin, Laura

2013-01-01

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies. PMID:24349133

A new pro-migratory activity on human myogenic precursor cells for a synthetic peptide within the E domain of the mechano growth factor

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

Mills, Philippe; Lafreniere, Jean-Francois; Benabdallah, Basma Fattouma

2007-02-01

Duchenne muscular dystrophy (DMD) is an inherited disease that leads to progressive muscle wasting. Myogenic precursor cell transplantation is an approach that can introduce the normal dystrophin gene in the muscle fibers of the patients. Unfortunately, these myogenic precursor cells do not migrate well in the muscle and thus many injections have to be done to enable a good graft success. Recent reports have shown that there is extensive splicing of the IGF-1 gene in muscles. The MGF isoform contains a C-terminal 24 amino acids peptide in the E domain (MGF-Ct24E) that has intrinsic properties. It can promote the proliferationmore » while delaying the differentiation of C{sub 2}C{sub 12} cells. Here, we demonstrated that this synthetic peptide is a motogenic factor for human precursor myogenic cells in vitro and in vivo. Indeed, MGF-Ct24E peptide can modulate members of the fibrinolytic and metalloproteinase systems, which are implicated in the migration of myogenic cells. MGF-Ct24E peptide enhances the expression of u-PA, u-PAR and MMP-7 while reducing PAI-1 activity. Moreover, it has no effect on the gelatinases MMP-2 and -9. Those combined effects can favour cell migration. Finally, we present some results suggesting that the MGF-Ct24E peptide induces these cell responses through a mechanism that does not involve the IGF-1 receptor. Thus, this MGF-Ct24E peptide has a new pro-migratory activity on human myogenic precursor cells that may be helpful in the treatment of DMD. Those results reinforce the possibility that the IGF-1Ec isoform may produce an E domain peptide that can act as a cytokine.« less

Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells.

PubMed

Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E; Ziegler, Mathias; Nikiforov, Andrey

2015-11-06

NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells*

PubMed Central

Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E.; Ziegler, Mathias; Nikiforov, Andrey

2015-01-01

NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5′-nucleotidases (5′-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5′-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5′-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors. PMID:26385918

Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248.

PubMed

Bagley, Rebecca G; Rouleau, Cecile; St Martin, Thia; Boutin, Paula; Weber, William; Ruzek, Melanie; Honma, Nakayuki; Nacht, Mariana; Shankara, Srinivas; Kataoka, Shiro; Ishida, Isao; Roberts, Bruce L; Teicher, Beverly A

2008-08-01

Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.

KIT D816V-mutated bone marrow mesenchymal stem cells in indolent systemic mastocytosis are associated with disease progression.

PubMed

Garcia-Montero, Andres C; Jara-Acevedo, Maria; Alvarez-Twose, Ivan; Teodosio, Cristina; Sanchez-Muñoz, Laura; Muñiz, Carmen; Muñoz-Gonzalez, Javier I; Mayado, Andrea; Matito, Almudena; Caldas, Carolina; Morgado, Jose M; Escribano, Luis; Orfao, Alberto

2016-02-11

Multilineage involvement of bone marrow (BM) hematopoiesis by the somatic KIT D816V mutation is present in a subset of adult indolent systemic mastocytosis (ISM) patients in association with a poorer prognosis. Here, we investigated the potential involvement of BM mesenchymal stem cells (MSCs) from ISM patients by the KIT D816V mutation and its potential impact on disease progression and outcome. This mutation was investigated in highly purified BM MSCs and other BM cell populations from 83 ISM patients followed for a median of 116 months. KIT D816V-mutated MSCs were detected in 22 of 83 cases. All MSC-mutated patients had multilineage KIT mutation (100% vs 30%, P = .0001) and they more frequently showed involvement of lymphoid plus myeloid BM cells (59% vs 22%; P = .03) and a polyclonal pattern of inactivation of the X-chromosome of KIT-mutated BM mast cells (64% vs 0%; P = .01) vs other multilineage ISM cases. Moreover, presence of KIT-mutated MSCs was associated with more advanced disease features, a greater rate of disease progression (50% vs 17%; P = .04), and a shorter progression-free survival (P ≤ .003). Overall, these results support the notion that ISM patients with mutated MSCs may have acquired the KIT mutation in a common pluripotent progenitor cell, prior to differentiation into MSCs and hematopoietic precursor cells, before the X-chromosome inactivation process occurs. From a clinical point of view, acquisition of the KIT mutation in an earlier BM precursor cell confers a significantly greater risk for disease progression and a poorer outcome. © 2016 by The American Society of Hematology.

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

PubMed Central

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

2013-01-01

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

An early thymic precursor phenotype predicts outcome exclusively in HOXA-overexpressing adult T-cell acute lymphoblastic leukemia: a Group for Research in Adult Acute Lymphoblastic Leukemia study.

PubMed

Bond, Jonathan; Marchand, Tony; Touzart, Aurore; Cieslak, Agata; Trinquand, Amélie; Sutton, Laurent; Radford-Weiss, Isabelle; Lhermitte, Ludovic; Spicuglia, Salvatore; Dombret, Hervé; Macintyre, Elizabeth; Ifrah, Norbert; Hamel, Jean-François; Asnafi, Vahid

2016-06-01

Gene expression studies have consistently identified a HOXA-overexpressing cluster of T-cell acute lymphoblastic leukemias, but it is unclear whether these constitute a homogeneous clinical entity, and the biological consequences of HOXA overexpression have not been systematically examined. We characterized the biology and outcome of 55 HOXA-positive cases among 209 patients with adult T-cell acute lymphoblastic leukemia uniformly treated during the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003 and -2005 studies. HOXA-positive patients had markedly higher rates of an early thymic precursor-like immunophenotype (40.8% versus 14.5%, P=0.0004), chemoresistance (59.3% versus 40.8%, P=0.026) and positivity for minimal residual disease (48.5% versus 23.5%, P=0.01) than the HOXA-negative group. These differences were due to particularly high frequencies of chemoresistant early thymic precursor-like acute lymphoblastic leukemia in HOXA-positive cases harboring fusion oncoproteins that transactivate HOXA Strikingly, the presence of an early thymic precursor-like immunophenotype was associated with marked outcome differences within the HOXA-positive group (5-year overall survival 31.2% in HOXA-positive early thymic precursor versus 66.7% in HOXA-positive non-early thymic precursor, P=0.03), but not in HOXA-negative cases (5-year overall survival 74.2% in HOXA-negative early thymic precursor versus 57.2% in HOXA-negative non-early thymic precursor, P=0.44). Multivariate analysis further revealed that HOXA positivity independently affected event-free survival (P=0.053) and relapse risk (P=0.039) of chemoresistant T-cell acute lymphoblastic leukemia. These results show that the underlying mechanism of HOXA deregulation dictates the clinico-biological phenotype, and that the negative prognosis of early thymic precursor acute lymphoblastic leukemia is exclusive to HOXA-positive patients, suggesting that early treatment intensification is currently suboptimal for therapeutic rescue of HOXA-positive chemoresistant adult early thymic precursor acute lymphoblastic leukemia. The GRAALL-2003 and -2005 studies were registered at http://www.clinicaltrials.gov as #NCT00222027 and #NCT00327678, respectively. Copyright© Ferrata Storti Foundation.

Heat shock protein-90 beta is expressed at the surface of multipotential mesenchymal precursor cells: generation of a novel monoclonal antibody, STRO-4, with specificity for mesenchymal precursor cells from human and ovine tissues.

PubMed

Gronthos, Stan; McCarty, Rosa; Mrozik, Krzysztof; Fitter, Stephen; Paton, Sharon; Menicanin, Danijela; Itescu, Silviu; Bartold, P Mark; Xian, Cory; Zannettino, Andrew C W

2009-11-01

Mesenchymal stromal cells (MSCs) and their precursor cells (MPCs) can proliferate and differentiate into multiple mesodermal and some ectodermal and endodermal tissues. Culture-expanded MSCs are currently being evaluated as a possible cell therapy to replace/repair injured or diseased tissues. While a number of mAb reagents with specificity to human MSCs, including STRO-1, STRO-3 (BLK ALP), CD71 (SH2, SH3), CD106 (VCAM-1), CD166, and CD271, have facilitated the isolation of purified populations of human MSCs from primary tissues, few if any mAb reagents have been described that can be used to isolate equivalent cells from other species. This is of particular relevance when assessing the tissue regenerative efficacy of MSCs in large immunocompetent, preclinical animal models of disease. In light of this, we sought to generate novel monoclonal antibodies (mAb) with specific reactivity against a cell surface molecule that is expressed at high levels by MSCs from different species. Using CD106 (VCAM-1)-selected ovine MSCs as an immunogen, mAb-producing hybridomas were selected for their reactivity to both human and ovine MSCs. One such hybridoma, termed STRO-4, produced an IgG mAb that reacted with <5% of human and ovine bone marrow (BM) mononuclear cells. As a single selection reagent, STRO-4 mAb was able to enrich colony-forming fibroblasts (CFU-F) in both human and ovine BM by 16- and 8-folds, respectively. Cells isolated with STRO-4 exhibited reactivity with markers commonly associated with MSCs isolated by plastic adherence including CD29, CD44, and CD166. Moreover, when placed in inductive culture conditions in vitro, STRO-4(+) MSCs exhibited multilineage differentiation potential and were capable of forming a mineralized matrix, lipid-filled adipocytes, and chondrocytes capable of forming a glycosaminoglycan-rich matrix. Biochemical analysis revealed that STRO-4 identified the beta isoform of heat shock protein-90 (Hsp90beta). In addition to identifying an antibody reagent that identifies a highly conserved epitope expressed by MSCs from different species, our study also points to a potential role for Hsp90beta in MSC biology.

Dissecting the molecular pathways of (testicular) germ cell tumour pathogenesis; from initiation to treatment-resistance.

PubMed

Looijenga, L H J; Gillis, A J M; Stoop, H; Biermann, K; Oosterhuis, J W

2011-08-01

Human type II germ cell tumours (GCTs) originate from an embryonic germ cell, either as a primordial germ cell or gonocyte. This start determines the biological as well as clinical characteristics of this type of cancer, amongst others their totipotency as well as their overall (exceptional) sensitivity to DNA damaging agents. The histology of the precursor lesion, either carcinoma in situ or gonadoblastoma, depends on the level of testicularization (i.e. testis formation) of the gonad. The impact of either intrinsic (genetic) - and environmental factors involved in the pathogenesis is demonstrated by disorders of sex development as well as testicular dysgenesis syndrome as risk factors, including cryptorchidism, hypospadias and disturbed fertility as parameters. This knowledge allows identification of individuals at risk for development of this type of cancer, being a population of interest for screening. Factors known to regulate pluripotency during embryogenesis are proven to be of diagnostic value for type II GCTs, including OCT3/4, even applicable for non-invasive screening. In addition, presence of stem cell factor, also known as KITLG, allows distinction between delayed matured germ cells and the earliest stages of malignant transformation. This is of special interest because of the identified association between development of type II GCTs of the testis and a limited number of single nucleotide polymorphisms, including some likely related to KITL. Transition from the precursor lesion to an invasive cancer is associated with gain of the short arm of chromosome 12, in which multiple genes might be involved, including KRAS2 and possibly NANOG (pseudogenes). While most precursor lesions will progress to an invasive cancer, only a limited number of cancers will develop treatment resistance. Putative explanatory mechanisms are identified, including presence of microsatellite instability, BRAF mutations, apoptosis suppression and p21 sub-cellular localization. It remains to be investigated how these different pathways integrate to each other and how informative they are at the patient-individual level. Further understanding will allow development of more targeted treatment, which will benefit quality of life of these young cancer patients. © 2011 The Authors. International Journal of Andrology © 2011 European Academy of Andrology.

Measles Virus Nucleocapsid (MVNP) Gene Expression and RANK Receptor Signaling in Osteoclast Precursors, Osteoclast Inhibitors Peptide Therapy for Pagets Disease

DTIC Science & Technology

2007-10-01

OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC a. REPORT U b. ABSTRACT U c . THIS PAGE U UU 27 19b. TELEPHONE NUMBER...and c -Jun kinase activity in osteoclast precursor cells (4). Our hypothesis is that MVNP expression in osteoclast precursors modulates the status...transcription factors such as c - Fos, NFATc1 critical for OCL differentiation were significantly decreased in OIP-1 transgenic mice derived preosteoclast cells

Tissue-specific differentiation of a circulating CCR9- pDC-like common dendritic cell precursor.

PubMed

Schlitzer, Andreas; Heiseke, Alexander F; Einwächter, Henrik; Reindl, Wolfgang; Schiemann, Matthias; Manta, Calin-Petru; See, Peter; Niess, Jan-Hendrik; Suter, Tobias; Ginhoux, Florent; Krug, Anne B

2012-06-21

The ontogenic relationship between the common dendritic cell (DC) progenitor (CDP), the committed conventional DC precursor (pre-cDC), and cDC subpopulations in lymphoid and nonlymphoid tissues has been largely unraveled. In contrast, the sequential steps of plasmacytoid DC (pDC) development are less defined, and it is unknown at which developmental stage and location final commitment to the pDC lineage occurs. Here we show that CCR9(-) pDCs from murine BM which enter the circulation and peripheral tissues have a common DC precursor function in vivo in the steady state, in contrast to CCR9(+) pDCs which are terminally differentiated. On adoptive transfer, the fate of CCR9(-) pDC-like precursors is governed by the tissues they enter. In the BM and liver, most transferred CCR9(-) pDC-like precursors differentiate into CCR9(+) pDCs, whereas in peripheral lymphoid organs, lung, and intestine, they additionally give rise to cDCs. CCR9(-) pDC-like precursors which are distinct from pre-cDCs can be generated from the CDP. Thus, CCR9(-) pDC-like cells are novel CDP-derived circulating DC precursors with pDC and cDC potential. Their final differentiation into functionally distinct pDCs and cDCs depends on tissue-specific factors allowing adaptation to local requirements under homeostatic conditions.

Ghrelin precursor gene polymorphism and methamphetamine dependence in the Korean population.

PubMed

Yoon, Su-Jung; Pae, Chi-Un; Lee, Heejin; Choi, Bomoon; Kim, Tae-Suk; Lyoo, In Kyoon; Kwon, Do-Hoon; Kim, Dai-Jin

2005-12-01

Ghrelin is a recently isolated brain-gut peptide that has growth hormone-releasing and appetite-inducing activities. Several recent studies have suggested that ghrelin plays a major role in the pathophysiology of drug-seeking behavior and anxiety. Therefore, we assessed the effect of the ghrelin precursor polymorphism on methamphetamine dependence in the Korean population. One hundred and eighteen patients with methamphetamine dependence, according to the Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) criteria, and the 144 healthy controls were enrolled in this study. Genotyping for the ghrelin precursor polymorphism was performed by the polymerase chain reaction-restriction fragment length polymorphism-based technique. The genotypic and allelic distributions of the ghrelin precursor polymorphism in the patients with methamphetamine dependence were not significantly different from those of the control subjects. However, the Met72 carriers were associated with the emotional problems of methamphetamine dependence. The patients with the Met72 allele were more depressed and anxious than the homozygous patients with the wild Leu72 allele. The present study suggests that the ghrelin precursor polymorphism may not confer a susceptibility to the development of methamphetamine dependence in the Korean population. However, the Leu72Met polymorphism could have a potential role in the emotional problems that are associated with this disease.

Functional electrical stimulation-facilitated proliferation and regeneration of neural precursor cells in the brains of rats with cerebral infarction

PubMed Central

Xiang, Yun; Liu, Huihua; Yan, Tiebin; Zhuang, Zhiqiang; Jin, Dongmei; Peng, Yuan

2014-01-01

Previous studies have shown that proliferation of endogenous neural precursor cells cannot alone compensate for the damage to neurons and axons. From the perspective of neural plasticity, we observed the effects of functional electrical stimulation treatment on endogenous neural precursor cell proliferation and expression of basic fibroblast growth factor and epidermal growth factor in the rat brain on the infarct side. Functional electrical stimulation was performed in rat models of acute middle cerebral artery occlusion. Simultaneously, we set up a placebo stimulation group and a sham-operated group. Immunohistochemical staining showed that, at 7 and 14 days, compared with the placebo group, the numbers of nestin (a neural precursor cell marker)-positive cells in the subgranular zone and subventricular zone were increased in the functional electrical stimulation treatment group. Western blot assays and reverse-transcription PCR showed that total protein levels and gene expression of epidermal growth factor and basic fibroblast growth factor were also upregulated on the infarct side. Prehensile traction test results showed that, at 14 days, prehension function of rats in the functional electrical stimulation group was significantly better than in the placebo group. These results suggest that functional electrical stimulation can promote endogenous neural precursor cell proliferation in the brains of acute cerebral infarction rats, enhance expression of basic fibroblast growth factor and epidermal growth factor, and improve the motor function of rats. PMID:25206808

The construction and partial characterization of plasmids containing complementary DNA sequences to human calcitonin precursor polyprotein.

PubMed Central

Allison, J; Hall, L; MacIntyre, I; Craig, R K

1981-01-01

(1) Total poly(A)-containing RNA isolated from human thyroid medullary carcinoma tissue was shown to direct the synthesis in the wheat germ cell-free system of a major (Mr 21000) and several minor forms of human calcitonin precursor polyproteins. Evidence for processing of these precursor(s) by the wheat germ cell-free system is also presented. (2) A small complementary DNA (cDNA) plasmid library has been constructed in the PstI site of the plasmid pAT153, using total human thyroid medullary carcinoma poly(A)-containing RNA as the starting material. (3) Plasmids containing abundant cDNA sequences were selected by hybridization in situ, and two of these (ph T-B3 and phT-B6) were characterized by hybridization--translation and restriction analysis. Each was shown to contain human calcitonin precursor polyprotein cDNA sequences. (4) RNA blotting techniques demonstrate that the human calcitonin precursor polyprotein is encoded within a mRNA containing 1000 bases. (5) The results demonstrate that human calcitonin is synthesized as a precursor polyprotein. Images Fig. 1. Fig. 2. Fig. 3. PMID:6896146

Efficient production of reactive oxygen species in neural precursor cells after exposure to 250 MeV protons.

PubMed

Giedzinski, Erich; Rola, Radoslaw; Fike, John R; Limoli, Charles L

2005-10-01

The space radiation environment is composed of highly energetic ions, dominated by protons, that pose a range of potential health risks to astronauts. Traversals of these particles through certain tissues may compromise the viability and/or function of sensitive cells, including neural precursors found within the dentate subgranular zone of the hippocampus. Irradiation has been shown to deplete these cells in vivo, and reductions of these critical cells are believed to impair neurogenesis and cognition. To more fully understand the mechanisms underlying the behavior of these precursor cells after irradiation, we have developed an in vitro neural precursor cell system and used it to assess acute (0-48 h) changes in ROS and mitochondrial end points after exposure to Bragg-peak protons of 250 MeV. Relative ROS levels were increased at nearly all doses (1-10 Gy) and postirradiation times (6-24 h) compared to unirradiated controls. The increase in ROS after proton irradiation was more rapid than that observed with X rays and showed a well-defined dose response at 6 and 24 h, increasing approximately 10% and 3% per gray, respectively. However, by 48 h postirradiation, ROS levels fell below controls and coincided with minor reductions in mitochondrial content. Use of the antioxidant alpha-lipoic acid (before or after irradiation) was shown to eliminate the radiation-induced rise in ROS levels. Our results corroborate earlier studies using X rays and provide further evidence that elevated ROS are integral to the radioresponse of neural precursor cells.

3D characterization of EMT cell density in developing cardiac cushions using optical coherence tomography (Conference Presentation)

NASA Astrophysics Data System (ADS)

Yu, Siyao; Gu, Shi; Zhao, Xiaowei; Liu, Yehe; Jenkins, Michael W.; Watanabe, Michiko; Rollins, Andrew M.

2017-02-01

Congenital heart defects (CHDs) are the most common birth defect, affecting between 4 and 75 per 1,000 live births depending on the inclusion criteria. Many of these defects can be traced to defects of cardiac cushions, critical structures during development that serve as precursors to many structures in the mature heart, including the atrial and ventricular septa, and all four sets of cardiac valves. Epithelial-mesenchymal transition (EMT) is the process through which cardiac cushions become populated with cells. Altered cushion size or altered cushion cell density has been linked to many forms of CHDs, however, quantitation of cell density in the complex 3D cushion structure poses a significant challenge to conventional histology. Optical coherence tomography (OCT) is a technique capable of 3D imaging of the developing heart, but typically lacks the resolution to differentiate individual cells. Our goal is to develop an algorithm to quantitatively characterize the density of cells in the developing cushion using 3D OCT imaging. First, in a heart volume, the atrioventricular (AV) cushions were manually segmented. Next, all voxel values in the region of interest were pooled together to generate a histogram. Finally, two populations of voxels were classified using either K-means classification, or a Gaussian mixture model (GMM). The voxel population with higher values represents cells in the cushion. To test the algorithm, we imaged and evaluated avian embryonic hearts at looping stages. As expected, our result suggested that the cell density increases with developmental stages. We validated the technique against scoring by expert readers.

NG2-expressing cells as oligodendrocyte progenitors in the normal and demyelinated adult central nervous system

PubMed Central

Polito, Annabella; Reynolds, Richard

2005-01-01

The mammalian adult central nervous system (CNS) is known to respond rapidly to demyelinating insults by regenerating oligodendrocytes for remyelination from a dividing precursor population. A widespread population of cells exists within the adult CNS that is thought to belong to the oligodendrocyte lineage, but which do not express proteins characteristic of mature myelinating oligodendrocytes, such as myelin basic protein (MBP) and 2,3-cyclic nucleotide 3-phosphodiesterase (CNP). Instead, these cells have phenotypic characteristics of a more immature stage of the oligodendrocyte lineage. They express the NG2 chondroitin sulphate proteoglycan, in addition to O4 and the platelet-derived growth factor α-receptor, all widely accepted as markers for oligodendrocyte progenitor cells (OPCs) throughout development. However, NG2+ cells residing in the adult CNS do not resemble embryonic or neonatal NG2+ cells in terms of their morphology or proliferation characteristics, but instead represent a unique type of glial cell that has the ability to react rapidly to CNS damage. In this review, we present the evidence that adult NG2+ cells are part of the oligodendrocyte lineage and are capable of giving rise to new oligodendrocytes under both normal and demyelinating conditions. We also review the literature that these cells may have multiple functional roles within the adult CNS, notwithstanding their primary role as OPCs. PMID:16367798

A. cantoniensis inhibits the proliferation of murine leukemia WEHI-3 cells in vivo and promotes immunoresponses in vivo.

PubMed

Tan, Tzu-Wei; Lin, Yuh-Tzy; Yang, Jai-Sing; Lu, Chi-Cheng; Chiang, Jo-Hua; Wu, Chang-Lin; Lin, Jing-Pin; Tang, Nou-Ying; Yeh, Chin-Chung; Fan, Ming-Jen; Chung, Jing-Gung

2009-01-01

Ampelopsis cantoniensis (AC) has been used as a folk medicine for reducing pain in the Taiwanese population. Our previous studies have shown that the crude extract of AC induced apoptosis in human promyelocytic leukemia HL-60 cells. In this study, the in vivo effects of AC on leukemia WEHI-3 cells and immune responses such as phagocytosis and natural killer (NK) cell activity were investigated. The weights of the livers and spleens were decreased in the AC-treated groups compared to the control groups. The AC treatment increased the percentage of CD3 and CD19 marker cells in WEHI-3-injected mice, indicating that the precursors of T and B cells were inhibited. The AC treatment promoted the activity of macrophage phagocytosis in the peripheral blood mononuclear cells (PBMC) and peritoneal cells. It was found that the NK cells from mice after treatment with AC can kill the YAC-1 target cells. Therefore, the AC treatment increased NK cell activity. In conclusion, AC can affect WEHI-3 cells in vivo and promote macrophage and NK cell activities.

Single-cell RNA sequencing highlights transcription activity of autophagy-related genes during hematopoietic stem cell formation in mouse embryos.

PubMed

Hu, Yongfei; Huang, Yan; Yi, Ying; Wang, Hongwei; Liu, Bing; Yu, Jia; Wang, Dong

2017-04-03

Accumulating evidence has demonstrated that macroautophagy/autophagy plays an essential role in self-renewal and differentiation in embryonic hematopoiesis. Here, according to the RNA sequencing data sets of 5 population cells related to hematopoietic stem cell (HSC) formation during mouse embryogenesis (endothelial cells, PTPRC/CD45 - and PTPRC/CD45 + pre-HSCs in the E11 aorta-gonad-mesonephros (AGM) region, mature HSCs in E12 and E14 fetal liver), we explored the dynamic expression of mouse autophagy-related genes in this course at the single-cell level. Our results revealed that the transcription activity of autophagy-related genes had a substantial increase when endothelial cells (ECs) specified into pre-HSCs, and the upregulation of autophagy-essential genes correlated with reduced NOTCH signaling in pre-HSCs, suggesting the autophagy activity may be greatly enhanced during pre-HSC specification from endothelial precursors. In summary, our results presented strong evidence that autophagy plays a critical role in HSC emergence during mouse midgestation.

Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells

PubMed Central

Miura, Hirohito; Scott, Jennifer K.; Harada, Shuitsu; Barlow, Linda A.

2014-01-01

Background Taste buds contain ~60 elongate cells and several basal cells. Elongate cells comprise three functional taste cell types: I - glial cells, II - bitter/sweet/umami receptor cells, and III - sour detectors. Although taste cells are continuously renewed, lineage relationships among cell types are ill-defined. Basal cells have been proposed as taste bud stem cells, a subset of which express Sonic hedgehog (Shh). However, Shh+ basal cells turnover rapidly suggesting that Shh+ cells are precursors of some or all taste cell types. Results To fate map Shh-expressing cells, mice carrying ShhCreERT2 and a high (CAG-CAT-EGFP) or low (R26RLacZ) efficiency reporter allele were given tamoxifen to activate Cre in Shh+ cells. Using R26RLacZ, lineage-labeled cells occur singly within buds, supporting a post-mitotic state for Shh+ cells. Using either reporter, we show that Shh+ cells differentiate into all three taste cell types, in proportions reflecting cell type ratios in taste buds (I > II > III). Conclusions Shh+ cells are not stem cells, but are post-mitotic, immediate precursors of taste cells. Shh+ cells differentiate into each of the three taste cell types, and the choice of a specific taste cell fate is regulated to maintain the proper ratio within buds. PMID:24590958

Tumor suppressors BTG1 and IKZF1 cooperate during mouse leukemia development and increase relapse risk in B-cell precursor acute lymphoblastic leukemia patients.

PubMed

Scheijen, Blanca; Boer, Judith M; Marke, René; Tijchon, Esther; van Ingen Schenau, Dorette; Waanders, Esmé; van Emst, Liesbeth; van der Meer, Laurens T; Pieters, Rob; Escherich, Gabriele; Horstmann, Martin A; Sonneveld, Edwin; Venn, Nicola; Sutton, Rosemary; Dalla-Pozza, Luciano; Kuiper, Roland P; Hoogerbrugge, Peter M; den Boer, Monique L; van Leeuwen, Frank N

2017-03-01

Deletions and mutations affecting lymphoid transcription factor IKZF1 (IKAROS) are associated with an increased relapse risk and poor outcome in B-cell precursor acute lymphoblastic leukemia. However, additional genetic events may either enhance or negate the effects of IKZF1 deletions on prognosis. In a large discovery cohort of 533 childhood B-cell precursor acute lymphoblastic leukemia patients, we observed that single-copy losses of BTG1 were significantly enriched in IKZF1 -deleted B-cell precursor acute lymphoblastic leukemia ( P =0.007). While BTG1 deletions alone had no impact on prognosis, the combined presence of BTG1 and IKZF1 deletions was associated with a significantly lower 5-year event-free survival ( P =0.0003) and a higher 5-year cumulative incidence of relapse ( P =0.005), when compared with IKZF1 -deleted cases without BTG1 aberrations. In contrast, other copy number losses commonly observed in B-cell precursor acute lymphoblastic leukemia, such as CDKN2A/B, PAX5, EBF1 or RB1 , did not affect the outcome of IKZF1 -deleted acute lymphoblastic leukemia patients. To establish whether the combined loss of IKZF1 and BTG1 function cooperate in leukemogenesis, Btg1 -deficient mice were crossed onto an Ikzf1 heterozygous background. We observed that loss of Btg1 increased the tumor incidence of Ikzf1 +/- mice in a dose-dependent manner. Moreover, murine B cells deficient for Btg1 and Ikzf1 +/- displayed increased resistance to glucocorticoids, but not to other chemotherapeutic drugs. Together, our results identify BTG1 as a tumor suppressor in leukemia that, when deleted, strongly enhances the risk of relapse in IKZF1 -deleted B-cell precursor acute lymphoblastic leukemia, and augments the glucocorticoid resistance phenotype mediated by the loss of IKZF1 function. Copyright© Ferrata Storti Foundation.

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

PubMed Central

Zediak, Valerie P.; Maillard, Ivan

2007-01-01

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

Pure erythroid leukemia following precursor B-cell lymphoblastic leukemia.

PubMed

Xu, Min; Finn, Laura S; Tsuchiya, Karen D; Thomson, Blythe; Pollard, Jessica; Rutledge, Joe

2012-01-01

Therapy-related acute myeloid leukemia is an unfortunate sequel to current multimodal intensive chemotherapy. The patient described was diagnosed with pure erythroleukemia, AML-M6b, during therapy for precursor B-cell acute lymphoblastic leukemia. To the best of our knowledge, this is the first report of this unusual association.

Reversible immortalization of Nestin-positive precursor cells from pancreas and differentiation into insulin-secreting cells

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

Wei, Pei; Li, Li; Qi, Hui

2012-02-10

Highlights: Black-Right-Pointing-Pointer The NPPCs from mouse pancreas were isolated. Black-Right-Pointing-Pointer Tet-on system for SV40 large in NPPCs was used to get RINPPCs. Black-Right-Pointing-Pointer The RINPPCs can undergo at least 80 population doublings without senescence. Black-Right-Pointing-Pointer The RINPPCs can be induced to differentiate into insulin-producing cells. Black-Right-Pointing-Pointer The combination of GLP-1 and sodium butyrate promoted the differentiation process. -- Abstract: Pancreatic stem cells or progenitor cells posses the ability of directed differentiation into pancreatic {beta} cells. However, these cells usually have limited proliferative capacity and finite lifespan in vitro. In the present study, Nestin-positive progenitor cells (NPPCs) from mouse pancreas thatmore » expressed the pancreatic stem cells or progenitor cell marker Nestin were isolated to obtain a sufficient number of differentiated pancreatic {beta} cells. Tet-on system for SV40 large T-antigen expression in NPPCs was used to achieve reversible immortalization. The reversible immortal Nestin-positive progenitor cells (RINPPCs) can undergo at least 80 population doublings without senescence in vitro while maintaining their biological and genetic characteristics. RINPPCs can be efficiently induced to differentiate into insulin-producing cells that contain a combination of glucagon-like peptide-1 (GLP-1) and sodium butyrate. The results of the present study can be used to explore transplantation therapy of type I diabetes mellitus.« less

Merkel cells are long-lived cells whose production is stimulated by skin injury✰

PubMed Central

Wright, Margaret C.; Logan, Gregory J.; Bolock, Alexa M.; Kubicki, Adam C.; Hemphill, Julie A.; Sanders, Timothy A.; Maricich, Stephen M.

2017-01-01

Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. PMID:27998808

Merkel cells are long-lived cells whose production is stimulated by skin injury.

PubMed

Wright, Margaret C; Logan, Gregory J; Bolock, Alexa M; Kubicki, Adam C; Hemphill, Julie A; Sanders, Timothy A; Maricich, Stephen M

2017-02-01

Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluation of Accessory Lacrimal Gland in Muller’s Muscle Conjunctival Resection Specimens for Precursor Cell Markers and Biological Markers of Dry Eye Disease

PubMed Central

Ali, Marwan; Shah, Dhara; Pasha, Zeeshan; Jassim, Sarmad H.; Jaboori, Assraa Jassim; Setabutr, Pete; Aakalu, Vinay K.

2017-01-01

Purpose The accessory lacrimal glands (ALG) are an understudied component of the tear functional unit, even though they are important in the development of dry eye syndrome (DES). To advance our understanding of aging changes, regenerative potential and histologic correlates to human characteristics, we investigated human ALG tissue from surgical samples to determine the presence or absence of progenitor cell markers and lacrimal epithelial markers and to correlate marker expression to relevant patient characteristics. Materials and Methods ALG tissues obtained from Muller’s Muscle Conjunctival Resection (MMCR) specimens were created using tissue microarrays (TMAs). Immunofluorescence staining of MMCR sections was performed using primary antibodies specific to cell protein markers. Cell marker localization in TMAs was then assessed by two blinded observers using a standardized scoring system. Patient characteristics including age, race, and status of ocular surface health were then compared against expression of stem cell markers. Results Human ALG expressed a number of epithelial markers, and in particular, histatin-1 was well correlated with the expression of epithelial markers and was present in most acini. In addition, we noted the presence of precursor cell markers nestin, ABCG2 and CD90 in ALG tissue. There was a decrease in precursor cell marker expression with increasing age. Finally, we noted that a negative association was present between histatin-1 expression and DES. Conclusions Thus, we report for the first time that human ALG tissues contain precursor marker positive cells and that this marker expression may decrease with increasing age. Moreover, histatin-1 expression may be decreased in DES. Future studies will be performed to use these cell markers to isolate and culture lacrimal epithelial cells from heterogeneous tissues, determine the relevance of histatin-1 expression to DES and isolate candidate precursor cells from ALG tissue. PMID:27612554

Commensal bacterial–derived signals regulate basophil hematopoiesis and allergic inflammation

PubMed Central

Hill, David A.; Siracusa, Mark C.; Abt, Michael C.; Kim, Brian S.; Kobuley, Dmytro; Kubo, Masato; Kambayashi, Taku; LaRosa, David F.; Renner, Ellen D.; Orange, Jordan S.; Bushman, Frederic D.; Artis, David

2012-01-01

Commensal bacteria that colonize mammalian barrier surfaces are reported to influence T helper type 2 (TH2) cytokine–dependent inflammation and susceptibility to allergic disease, although the mechanisms that underlie these observations are poorly understood. In this report, we identify that deliberate alteration of commensal bacterial populations via oral antibiotic treatment resulted in elevated serum immunoglobulin E (IgE) levels, increased steady–state circulating basophil populations, and exaggerated basophil–mediated TH2 cell responses and allergic inflammation. Elevated serum IgE levels correlated with increased circulating basophil populations in mice and subjects with hyperimmunoglobulinemia E syndrome. Furthermore, B cell–intrinsic expression of MyD88 was required to limit serum IgE levels and circulating basophil populations in mice. Commensal–derived signals were found to influence basophil development by limiting proliferation of bone marrow–resident precursor populations. Collectively, these results identify a previously unrecognized pathway through which commensal–derived signals influence basophil hematopoiesis and susceptibility to TH2 cytokine–dependent inflammation and allergic disease. PMID:22447074

Whole-cell fungal transformation of precursors into dyes

PubMed Central

2010-01-01

Background Chemical methods of producing dyes involve extreme temperatures and unsafe toxic compounds. Application of oxidizing enzymes obtained from fungal species, for example laccase, is an alternative to chemical synthesis of dyes. Laccase can be replaced by fungal biomass acting as a whole-cell biocatalyst with properties comparable to the isolated form of the enzyme. The application of the whole-cell system simplifies the transformation process and reduces the time required for its completion. In the present work, four fungal strains with a well-known ability to produce laccase were tested for oxidation of 17 phenolic and non-phenolic precursors into stable and non-toxic dyes. Results An agar-plate screening test of the organic precursors was carried out using four fungal strains: Trametes versicolor, Fomes fomentarius, Abortiporus biennis, and Cerrena unicolor. Out of 17 precursors, nine were transformed into coloured substances in the presence of actively growing fungal mycelium. The immobilized fungal biomass catalyzed the transformation of 1 mM benzene and naphthalene derivatives in liquid cultures yielding stable and non-toxic products with good dyeing properties. The type of fungal strain had a large influence on the absorbance of the coloured products obtained after 48-hour transformation of the selected precursors, and the most effective was Fomes fomentarius (FF25). Whole-cell transformation of AHBS (3-amino-4-hydroxybenzenesulfonic acid) into a phenoxazinone dye was carried out in four different systems: in aqueous media comprising low amounts of carbon and nitrogen source, in buffer, and in distilled water. Conclusions This study demonstrated the ability of four fungal strains belonging to the ecological type of white rot fungi to transform precursors into dyes. This paper highlights the potential of fungal biomass for replacing isolated enzymes as a cheaper industrial-grade biocatalyst for the synthesis of dyes and other commercially important products. The use of immobilized fungal biomass limits free migration of cells and facilitates their reuse in a continuous system for precursor transformation. PMID:20598166

NG2 glia are required for vessel network formation during embryonic development

PubMed Central

Minocha, Shilpi; Valloton, Delphine; Brunet, Isabelle; Eichmann, Anne

2015-01-01

The NG2+ glia, also known as polydendrocytes or oligodendrocyte precursor cells, represent a new entity among glial cell populations in the central nervous system. However, the complete repertoire of their roles is not yet identified. The embryonic NG2+ glia originate from the Nkx2.1+ progenitors of the ventral telencephalon. Our analysis unravels that, beginning from E12.5 until E16.5, the NG2+ glia populate the entire dorsal telencephalon. Interestingly, their appearance temporally coincides with the establishment of blood vessel network in the embryonic brain. NG2+ glia are closely apposed to developing cerebral vessels by being either positioned at the sprouting tip cells or tethered along the vessel walls. Absence of NG2+ glia drastically affects the vascular development leading to severe reduction of ramifications and connections by E18.5. By revealing a novel and fundamental role for NG2+ glia, our study brings new perspectives to mechanisms underlying proper vessels network formation in embryonic brains. DOI: http://dx.doi.org/10.7554/eLife.09102.001 PMID:26651999

The union of somatic gonad precursors and primordial germ cells during C. elegans embryogenesis

PubMed Central

Rohrschneider, Monica R.; Nance, Jeremy

2013-01-01

Somatic gonadal niche cells control the survival, differentiation, and proliferation of germline stem cells. The establishment of this niche-stem cell relationship is critical, and yet the precursors to these two cell types are often born at a distance from one another. The simple C. elegans gonadal primordium, which contains two somatic gonad precursors (SGPs) and two primordial germ cells (PGCs), provides an accessible model for determining how stem cell and niche cell precursors first assemble during development. To visualize the morphogenetic events that lead to formation of the gonadal primordium, we generated transgenic strains to label the cell membranes of the SGPs and PGCs and captured time-lapse movies as the gonadal primordium formed. We identify three distinct phases of SGP behavior: posterior migration along the endoderm towards the PGCs, extension of a single long projection around the adjacent PGC, and a dramatic wrapping over the PGC surfaces. We show that the endoderm and PGCs are dispensable for SGP posterior migration and initiation of projections. However, both tissues are required for the final positioning of the SGPs and the morphology of their projections, and PGCs are absolutely required for SGP wrapping behaviors. Finally, we demonstrate that the basement membrane component laminin, which localizes adjacent to the developing gonadal primordium, is required to prevent the SGPs from over-extending past the PGCs. Our findings provide a foundation for understanding the cellular and molecular regulation of the establishment of a niche-stem cell relationship. PMID:23562590

Pancreatic Cancer

PubMed Central

Maitra, Anirban; Hruban, Ralph H.

2009-01-01

The past two decades have witnessed an explosion in our understanding of pancreatic cancer, and it is now clear that pancreatic cancer is a disease of inherited (germ-line) and somatic gene mutations. The genes mutated in pancreatic cancer include KRAS2, p16/CDKN2A, TP53, and SMAD4/DPC4, and these are accompanied by a substantial compendium of genomic and transcriptomic alterations that facilitate cell cycle deregulation, cell survival, invasion, and metastases. Pancreatic cancers do not arise de novo, and three distinct precursor lesions have been identified. Experimental models of pancreatic cancer have been developed in genetically engineered mice, which recapitulate the multistep progression of the cognate human disease. Although the putative cell of origin for pancreatic cancer remains elusive, minor populations of cells with stem-like properties have been identified that appear responsible for tumor initiation, metastases, and resistance of pancreatic cancer to conventional therapies. PMID:18039136

Epigenetics in myeloid derived suppressor cells: a sheathed sword towards cancer

PubMed Central

Zhang, Chao; Wang, Shuo; Liu, Yufeng; Yang, Cheng

2016-01-01

Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of cells composed of progenitors and precursors to myeloid cells, are deemed to participate in the development of tumor-favoring immunosuppressive microenvironment. Thus, the regulatory strategies targeting MDSCs' expansion, differentiation, accumulation and function could possibly be effective “weapons” in anti-tumor immunotherapies. Epigenetic mechanisms, which involve DNA modification, covalent histone modification and RNA interference, result in the heritable down-regulation or silencing of gene expression without a change in DNA sequences. Epigenetic modification of MDSC's functional plasticity leads to the remodeling of its characteristics, therefore reframing the microenvironment towards countering tumor growth and metastasis. This review summarized the pertinent findings on the DNA methylation, covalent histone modification, microRNAs and small interfering RNAs targeting MDSC in cancer genesis, progression and metastasis. The potentials as well as possible obstacles in translating into anti-cancer therapeutics were also discussed. PMID:27458169

Complex interactions in EML cell stimulation by stem cell factor and IL-3.

PubMed

Ye, Zhi-jia; Gulcicek, Erol; Stone, Kathryn; Lam, Tukiet; Schulz, Vincent; Weissman, Sherman M

2011-03-22

Erythroid myeloid lymphoid (EML) cells are an established multipotent hematopoietic precursor cell line that can be maintained in medium including stem cell factor (SCF). EML cultures contain a heterogeneous mixture of cells, including a lineage-negative, CD34+ subset of cells that propagate rapidly in SCF and can clonally regenerate the mixed population. A second major subset of EML cells consists of lineage-negative. CD34- cells that can be propagated in IL-3 but grow slowly, if at all, in SCF, although they express the SCF receptor (c-kit). The response of these cells to IL-3 is stimulated synergistically by SCF, and we present evidence that both the synergy and the inhibition of c-kit responses may be mediated by direct interaction with IL-3 receptor. Further, the relative level of tyrosine phosphorylation of various substrates by either cytokine alone differs from that produced by the combination of the two cytokines, suggesting that cell signaling by the combination of the two cytokines differs from that produced by either alone.

Complex interactions in EML cell stimulation by stem cell factor and IL-3

PubMed Central

Ye, Zhi-jia; Gulcicek, Erol; Stone, Kathryn; Lam, Tukiet; Schulz, Vincent; Weissman, Sherman M.

2011-01-01

Erythroid myeloid lymphoid (EML) cells are an established multipotent hematopoietic precursor cell line that can be maintained in medium including stem cell factor (SCF). EML cultures contain a heterogeneous mixture of cells, including a lineage-negative, CD34+ subset of cells that propagate rapidly in SCF and can clonally regenerate the mixed population. A second major subset of EML cells consists of lineage-negative. CD34− cells that can be propagated in IL-3 but grow slowly, if at all, in SCF, although they express the SCF receptor (c-kit). The response of these cells to IL-3 is stimulated synergistically by SCF, and we present evidence that both the synergy and the inhibition of c-kit responses may be mediated by direct interaction with IL-3 receptor. Further, the relative level of tyrosine phosphorylation of various substrates by either cytokine alone differs from that produced by the combination of the two cytokines, suggesting that cell signaling by the combination of the two cytokines differs from that produced by either alone. PMID:21383156

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB

PubMed Central

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D.; Garner, Ethan C.; Walker, Suzanne

2014-01-01

Summary The bacterial actin homolog MreB, which is critical for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids, but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis. PMID:25402772

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.

PubMed

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D; Garner, Ethan C; Walker, Suzanne

2015-01-01

The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

Thymic lymphocytes. III. Cooperative phenomenon in the proliferation of thymocytes under Con A stimulation.

PubMed

Papiernik, M; Jacobson, J B

1986-01-01

In the present paper, the response of thymocytes to Con A is analyzed in terms of a cooperative phenomenon between medullary thymocytes, cortical thymocytes, thymic accessory cells, and interleukin 2. Medullary thymocytes respond spontaneously to Con A and produce IL-2. The addition of exogenously produced IL-2 enhances their proliferation. Small numbers of cortical (PNA+) thymocytes do not respond to Con A, even in the presence of IL-2-containing supernatant. By increasing the number of PNA+ cells per well, sensitivity to Con A and IL-2 appears. This response may be linked either to the increase in a minor PNA+-responding population and/or to the enhanced contamination by medullary thymocytes and macrophages in non-responding PNA+ thymocyte population. In this hypothesis, either the contaminating cells respond by themselves and/or cooperate with PNA+ cells to induce their proliferation. Coculture of non-responding low numbers of PNA+ thymocytes with Con A- and IL-2-containing supernatant in the presence of PNA- cells containing thymic medullary thymocytes and macrophages always produces a higher response than that of each individual population. These results show that a cooperative phenomenon occurs in the cocultures of PNA+ and PNA- thymic cells. We can show using PNA+ and PNA- thymocytes with different Thy 1 alleles, that indeed both PNA+ and populations participate PNA-thymocytes with different Thy 1 alleles, that indeed both PNA+ and PNA- populations participate in the generation of proliferating cells. We can demonstrate, by lysis experiments with monoclonal antibodies and complement that at the end of coculture, most of the proliferating cells are Lyt 1+, and part are Lyt 2+ or L3T4+. We discuss the fact that the phenotype of the cells after activation does not allow us to deduce the phenotype of their precursors. Lysis of Ia+ cells prior to coculture, reduces the level of the proliferative response but does not modify the percentage of cooperation produced by the coculture. Cooperation with medullary mature thymocytes or the presence of active Ia- accessory cells possibly able to convert to Ia expression during coculture experiments may account for these results.

Increased neuronal beta-amyloid precursor protein expression in human temporal lobe epilepsy: association with interleukin-1 alpha immunoreactivity.

PubMed

Sheng, J G; Boop, F A; Mrak, R E; Griffin, W S

1994-11-01

Levels of immunoreactive beta-amyloid precursor protein and interleukin-1 alpha were found to be elevated in surgically resected human temporal lobe tissue from patients with intractable epilepsy compared with postmortem tissue from neurologically unaffected patients (controls). In tissue from epileptics, the levels of the 135-kDa beta-amyloid precursor protein isoform were elevated to fourfold (p < 0.05) those of controls and those of the 130-kDa isoform to threefold (p < 0.05), whereas those of the 120-kDa isoform (p > 0.05) were not different from control values. beta-Amyloid precursor protein-immunoreactive neurons were 16 times more numerous, and their cytoplasm and proximal processes were more intensely immunoreactive in tissue sections from epileptics than controls (133 +/- 12 vs. 8 +/- 3/mm2; p < 0.001). However, neither beta-amyloid precursor protein-immunoreactive dystrophic neurites nor beta-amyloid deposits were found in this tissue. Interleukin-1 alpha-immunoreactive cells (microglia) were three times more numerous in epileptics than in controls (80 +/- 8 vs. 25 +/- 5/mm2; p < 0.001), and these cells were often found adjacent to beta-amyloid precursor protein-immunoreactive neuronal cell bodies. Our findings, together with functions established in vitro for interleukin-1, suggest that increased expression of this protein contributes to the increased levels of beta-amyloid precursor protein in epileptics, thus indicating a potential role for both of these proteins in the neuronal dysfunctions, e.g., hyperexcitability, characteristic of epilepsy.

Derivation of Skeletal Myogenic Precursors from Human Pluripotent Stem Cells Using Conditional Expression of PAX7.

PubMed

Darabi, Radbod; Perlingeiro, Rita C R

2016-01-01

Cell-based therapies are considered as one of the most promising approaches for the treatment of degenerating pathologies including muscle disorders and dystrophies. Advances in the approach of reprogramming somatic cells into induced pluripotent stem (iPS) cells allow for the possibility of using the patient's own pluripotent cells to generate specific tissues for autologous transplantation. In addition, patient-specific tissue derivatives have been shown to represent valuable material for disease modeling and drug discovery. Nevertheless, directed differentiation of pluripotent stem cells into a specific lineage is not a trivial task especially in the case of skeletal myogenesis, which is generally poorly recapitulated during the in vitro differentiation of pluripotent stem cells.Here, we describe a practical and efficient method for the derivation of skeletal myogenic precursors from differentiating human pluripotent stem cells using controlled expression of PAX7. Flow cytometry (FACS) purified myogenic precursors can be expanded exponentially and differentiated in vitro into myotubes, enabling researchers to use these cells for disease modeling as well as therapeutic purposes.

CCR6 Defines Memory B Cell Precursors in Mouse and Human Germinal Centers, Revealing Light-Zone Location and Predominant Low Antigen Affinity.

PubMed

Suan, Dan; Kräutler, Nike J; Maag, Jesper L V; Butt, Danyal; Bourne, Katherine; Hermes, Jana R; Avery, Danielle T; Young, Clara; Statham, Aaron; Elliott, Michael; Dinger, Marcel E; Basten, Antony; Tangye, Stuart G; Brink, Robert

2017-12-19

Memory B cells (MBCs) and plasma cells (PCs) constitute the two cellular outputs of germinal center (GC) responses that together facilitate long-term humoral immunity. Although expression of the transcription factor BLIMP-1 identifies cells undergoing PC differentiation, no such marker exists for cells committed to the MBC lineage. Here, we report that the chemokine receptor CCR6 uniquely marks MBC precursors in both mouse and human GCs. CCR6 + GC B cells were highly enriched within the GC light zone (LZ), were the most quiescent of all GC B cells, exhibited a cell-surface phenotype and gene expression signature indicative of an MBC transition, and possessed the augmented response characteristics of MBCs. MBC precursors within the GC LZ predominantly possessed a low affinity for antigen but also included cells from within the high-affinity pool. These data indicate a fundamental dichotomy between the processes that drive MBC and PC differentiation during GC responses. Copyright © 2017 Elsevier Inc. All rights reserved.

Involvement of suppressors of cytokine signaling in toll-like receptor-mediated block of dendritic cell differentiation.

PubMed

Bartz, Holger; Avalos, Nicole M; Baetz, Andrea; Heeg, Klaus; Dalpke, Alexander H

2006-12-15

Dendritic cells (DCs) are important sentinels within innate immunity, monitoring the presence of infectious microorganisms. They operate in 2 different maturation stages, with transition from immature to mature DCs being induced by activation of toll-like receptors (TLRs). However, TLRs are also expressed on precursor cells of DCs. Here we analyzed the effects of TLR stimulation during the process of granulocyte-macrophage-colony-stimulating factor (GM-CSF)-mediated in vitro generation of immature DCs from precursor cells. We show that TLR triggering deviated phenotypic and functional differentiation from CD14+ monocytes to CD1a+ DCs. Similar results were obtained when differentiation of murine myeloid DCs from bone marrow cells was analyzed. The inhibitory effects were independent of soluble factors. TLR stimulation in DC precursor cells induced proteins of the suppressor of cytokine signaling family (SOCS), which correlated with loss of sensitivity to GM-CSF. Overexpression of SOCS-1 abolished GM-CSF signal transduction. Moreover, forced SOCS-1 expression in DC precursors mimicked the inhibitory effects on DC generation observed for TLR stimulation. The results indicate that TLR stimulation during the period of DC generation interferes with and deviates DC differentiation and that these effects are mediated particularly by SOCS-1.

Use of polysialic acid in repair of the central nervous system

PubMed Central

El Maarouf, Abderrahman; Petridis, Athanasios K.; Rutishauser, Urs

2006-01-01

Polysialic acid (PSA), a large cell-surface carbohydrate that regulates cell interactions, is used during vertebrate development to promote precursor cell migration and axon path-finding. The induction of PSA expression in damaged adult CNS tissues could help them to rebuild by creating conditions permissive for architectural remodeling. This possibility has been explored in two contexts, the regeneration of axons and the recruitment of endogenous neural precursors to a lesion. Glial scars that form at CNS injury sites block axon regeneration. It has been found that transfection of scar astrocytes by a viral vector encoding polysialyltransferase leads to sustained expression of high levels of PSA. With this treatment, a substantial portion of severed corticospinal tract axon processes were able to grow through a spinal injury site. In the studies of precursor cell migration to a cortical lesion, it was found that induced PSA expression in a path extending from the subventricular zone to a lesion near the cortical surface increased recruitment of BrdU/nestin-positive cells along the path and into the injury site. These displaced precursors were able to differentiate in a regionally appropriate manner. These findings suggest that induced PSA expression can be used as a strategy for promoting tissue repair involving both replacement of cells and rebuilding of neural connections. PMID:17075041

Brain-Derived Neurotrophic Factor Induces Cell Survival and the Migration of Murine Adult Hippocampal Precursor Cells During Differentiation In Vitro.

PubMed

Ortiz-López, Leonardo; Vega-Rivera, Nelly Maritza; Babu, Harish; Ramírez-Rodríguez, Gerardo Bernabé

2017-01-01

The generation of new neurons during adulthood involves local precursor cell migration and terminal differentiation in the dentate gyrus. These events are influenced by the hippocampal microenvironment. Brain-derived neurotrophic factor (BDNF) is relevant for hippocampal neuronal development and behavior. Interestingly, studies that have been performed in controlled in vitro systems that involve isolated precursor cells that were derived from the dentate gyrus (AHPCs) have shown that BDNF induces the activation of the TrkB receptor and, consequentially, might activate signaling pathways that favor survival and neuronal differentiation. Based on the fact that the cellular events of AHPCs that are induced by single factors can be studied in this controlled in vitro system, we investigated the ability of BDNF and the involvement of protein kinase C (PKC), as one of the TrkB-downstream activated signaling proteins, in the regulation of migration, here reflected by motility, of AHPCs. Precursor cells were cultured following a concentration-response curve (1-640 ng/ml) for 24 or 96 h. We found that BDNF favored cell survival without altering the viability under culture proliferative conditions of the AHPCs. Concomitantly, glial- and neuronal-differentiated precursor cells increased as a consequence of survival promoted by BDNF. Additionally, pharmacological approaches showed that BDNF (40 ng/ml)-induced migration of AHPCs was blocked with the compounds K252a and GF109203x, which prevent the activation of TrkB and PKC, respectively. The results indicate that in the in vitro migration of differentiated AHPCs it is involved the BDNF and TrkB cascade. Our results provide additional information about the mechanism by which BDNF impacts adult neurogenesis in the hippocampus.

Book lung development in juveniles and adults of the cobweb spider, Parasteatoda tepidariorum C. L. Koch, 1841 (Araneomorphae, Theridiidae).

PubMed

Farley, Roger D

2018-03-01

Light and transmission electron microscopy were used to study the development of new book lung lamellae in juvenile and adult spiders (Parasteatoda tepidariorum). As hypothesized earlier in a study of embryos, mesenchyme cells dispersed throughout the opisthosoma (EMT) are a likely source of precursor epithelial cells (MET) for the new lamellae. The precursor cells in juveniles and adults continue many of the complex activities observed in embryos, e.g., migration, alignment, lumen formation, thinning, elongation, and secretion of the cuticle of air channel walls and trabeculae. The apicobasal polarity of precursor cells for new channels is apparently induced by the polarity pattern of precursor cells of channels produced earlier. Thus, new air and hemolymph channels extend and continue the alternating pattern of older channels. At sites more distant from the spiracle and atrium, new channels are usually produced by the mode II process (intracellular alignment and merging of vesicles). These air channels have bridging trabeculae and are quite stable in size throughout their length. At sites closer to the spiracle and atrium, new channels may be produced by mode I (coalescence of merocrine vesicle secretion). This raises the hypothesis that structural and functional differences in mode I and II channels and differing oxygen and fluid conditions with distance from the spiracle and atrium determine the mode of formation of new channels. Observations herein support an earlier hypothesis that there is some intercellular apical/apical and basal/basal affinity among the opposed surfaces of aligned precursor cells. This results in the alternating pattern of air channels at the apical and hemolymph channels at the basal cell surfaces. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Brain injury expands the numbers of neural stem cells and progenitors in the SVZ by enhancing their responsiveness to EGF

PubMed Central

Alagappan, Dhivyaa; Lazzarino, Deborah A; Felling, Ryan J; Balan, Murugabaskar; Kotenko, Sergei V; Levison, Steven W

2009-01-01

There is an increase in the numbers of neural precursors in the SVZ (subventricular zone) after moderate ischaemic injuries, but the extent of stem cell expansion and the resultant cell regeneration is modest. Therefore our studies have focused on understanding the signals that regulate these processes towards achieving a more robust amplification of the stem/progenitor cell pool. The goal of the present study was to evaluate the role of the EGFR [EGF (epidermal growth factor) receptor] in the regenerative response of the neonatal SVZ to hypoxic/ischaemic injury. We show that injury recruits quiescent cells in the SVZ to proliferate, that they divide more rapidly and that there is increased EGFR expression on both putative stem cells and progenitors. With the amplification of the precursors in the SVZ after injury there is enhanced sensitivity to EGF, but not to FGF (fibroblast growth factor)-2. EGF-dependent SVZ precursor expansion, as measured using the neurosphere assay, is lost when the EGFR is pharmacologically inhibited, and forced expression of a constitutively active EGFR is sufficient to recapitulate the exaggerated proliferation of the neural stem/progenitors that is induced by hypoxic/ischaemic brain injury. Cumulatively, our results reveal that increased EGFR signalling precedes that increase in the abundance of the putative neural stem cells and our studies implicate the EGFR as a key regulator of the expansion of SVZ precursors in response to brain injury. Thus modulating EGFR signalling represents a potential target for therapies to enhance brain repair from endogenous neural precursors following hypoxic/ischaemic and other brain injuries. PMID:19570028

Recent thymic emigrants and mature naive T cells exhibit differential DNA methylation at key cytokine loci.

PubMed

Berkley, Amy M; Hendricks, Deborah W; Simmons, Kalynn B; Fink, Pamela J

2013-06-15

Recent thymic emigrants (RTEs) are the youngest T cells in the lymphoid periphery and exhibit phenotypic and functional characteristics distinct from those of their more mature counterparts in the naive peripheral T cell pool. We show in this study that the Il2 and Il4 promoter regions of naive CD4(+) RTEs are characterized by site-specific hypermethylation compared with those of both mature naive (MN) T cells and the thymocyte precursors of RTEs. Thus, RTEs do not merely occupy a midpoint between the thymus and the mature T cell pool, but represent a distinct transitional T cell population. Furthermore, RTEs and MN T cells exhibit distinct CpG DNA methylation patterns both before and after activation. Compared with MN T cells, RTEs express higher levels of several enzymes that modify DNA methylation, and inhibiting methylation during culture allows RTEs to reach MN T cell levels of cytokine production. Collectively, these data suggest that the functional differences that distinguish RTEs from MN T cells are influenced by epigenetic mechanisms and provide clues to a mechanistic basis for postthymic maturation.

Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

PubMed Central

Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; Pan, Gaofeng; Fu, Zhiping

2012-01-01

PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25–35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein. PMID:25745458

Effect of cooling rate on human and murine hemopoietic precursor cell recovery

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

Niskanen, E.; Pirsch, G.

1983-08-01

The effect of cooling rate on recovery of human and murine hemopoietic precursor cells was studied. In the presence of 10% Me2SO, a cooling rate of 7 degrees C/min from -4 to -30 degrees C was optimal for recovery of both human and murine precursor cells which give rise to colonies in diffusion chambers implanted in mice (CFU-DG). Cooling of human marrow at a rate between 3 and 7 degrees C/min resulted in the best CFU-C recovery, although no good correlation between the cooling rate and murine CFU-C recovery was demonstrated. These data suggest that recovery of the primitive hemopoieticmore » precursor cells can be improved by changing the standard cryopreservation programs used presently. However, improved recovery of CFU-DG does not necessarily translate into faster reconstitution of hemopoiesis. No significant difference was observed in overall recovery of bone marrow cellularity in lethally irradiated mice following injection of untreated marrow and marrow cooled at a rate of 1 and 7 degrees C/min.« less

Minute perivascular epithelioid cell (PEC) nests in the abdominal lymph nodes--a putative precursor of PEComa.

PubMed

Nagasaka, Toru; Murakami, Yoshiko; Sasaki, Eiichi; Hosoda, Waki; Nakanishi, Toru; Yatabe, Yasushi

2015-04-01

A perivascular epithelioid cell tumor (PEComa) is a peculiar growth defined as a mesenchymal tumor composed of histologically and immunohistochemically distinct perivascular epithelioid cells (PECs). Because neither normal counterparts nor precursor lesions of PEComa have been identified, we examined minute PEC nests, ranged from 0.8 mm to 10 mm, to investigate the possible origin of the PEComa. We examined a total of 80 677 para-aortic and pelvic lymph nodes that were systematically dissected from 1656 patients for gynecological malignancies. The identified lesions were confirmed immunohistochemically with multiple PEC markers, including smooth muscle actin, HMB45, melan-A, MiTF, ER and PgR. A total of 66 minute PEC nests were found in 21 patients (1.3% of the total population) with an average frequency of 3.1 lesions per patient. In cases of multiple involvement, 11 of 13 nests were located at the same level of multiple lymph node or on continuous levels. The lesions were preferentially distributed at the level of para-aortic and high pelvic lymph nodes. All nests were positive for actin and HMB45, whereas the other markers were positive with varying frequencies. The minute PEC nests may be associated with the possible normal counterpart of PEComas. © 2015 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

Defining disease with laser precision: laser capture microdissection in gastroenterology

PubMed Central

Blatt, Richard; Srinivasan, Shanthi

2013-01-01

Laser capture microdissection (LCM) is an efficient and precise method for obtaining pure cell populations or specific cells of interest from a given tissue sample. LCM has been applied to animal and human gastroenterology research in analyzing the protein, DNA and RNA from all organs of the gastrointestinal system. There are numerous potential applications for this technology in gastroenterology research including malignancies of the esophagus, stomach, colon, biliary tract and liver. This technology can also be used to study gastrointestinal infections, inflammatory bowel disease, pancreatitis, motility, malabsorption and radiation enteropathy. LCM has multiple advantages when compared to conventional methods of microdissection, and this technology can be exploited to identify precursors to disease, diagnostic biomarkers, and therapeutic interventions. PMID:18619446

Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo.

PubMed

Rezania, Alireza; Bruin, Jennifer E; Xu, Jean; Narayan, Kavitha; Fox, Jessica K; O'Neil, John J; Kieffer, Timothy J

2013-11-01

Human embryonic stem cells (hESCs) are considered a potential alternative to cadaveric islets as a source of transplantable cells for treating patients with diabetes. We previously described a differentiation protocol to generate pancreatic progenitor cells from hESCs, composed of mainly pancreatic endoderm (PDX1/NKX6.1-positive), endocrine precursors (NKX2.2/synaptophysin-positive, hormone/NKX6.1-negative), and polyhormonal cells (insulin/glucagon-positive, NKX6.1-negative). However, the relative contributions of NKX6.1-negative versus NKX6.1-positive cell fractions to the maturation of functional β-cells remained unclear. To address this question, we generated two distinct pancreatic progenitor cell populations using modified differentiation protocols. Prior to transplant, both populations contained a high proportion of PDX1-expressing cells (~85%-90%) but were distinguished by their relatively high (~80%) or low (~25%) expression of NKX6.1. NKX6.1-high and NKX6.1-low progenitor populations were transplanted subcutaneously within macroencapsulation devices into diabetic mice. Mice transplanted with NKX6.1-low cells remained hyperglycemic throughout the 5-month post-transplant period whereas diabetes was reversed in NKX6.1-high recipients within 3 months. Fasting human C-peptide levels were similar between groups throughout the study, but only NKX6.1-high grafts displayed robust meal-, glucose- and arginine-responsive insulin secretion as early as 3 months post-transplant. NKX6.1-low recipients displayed elevated fasting glucagon levels. Theracyte devices from both groups contained almost exclusively pancreatic endocrine tissue, but NKX6.1-high grafts contained a greater proportion of insulin-positive and somatostatin-positive cells, whereas NKX6.1-low grafts contained mainly glucagon-expressing cells. Insulin-positive cells in NKX6.1-high, but not NKX6.1-low grafts expressed nuclear MAFA. Collectively, this study demonstrates that a pancreatic endoderm-enriched population can mature into highly functional β-cells with only a minor contribution from the endocrine subpopulation. © AlphaMed Press.

A novel population of α-smooth muscle actin-positive cells activated in a rat model of stroke: an analysis of the spatio-temporal distribution in response to ischemia.

PubMed

Sharma, Varun; Ling, Tina W; Rewell, Sarah S; Hare, David L; Howells, David W; Kourakis, Angela; Wookey, Peter J

2012-11-01

In a rat model of stroke, the spatio-temporal distribution of α-smooth muscle actin-positive, (αSMA+) cells was investigated in the infarcted hemisphere (ipsilateral) and compared with the contralateral hemisphere. At day 3 postischemia, αSMA+ cells were concentrated in two main loci within the ipsilateral hemisphere (Area A) in the medial corpus callosum and (Area B) midway through the striatum adjacent to the lateral ventricle. By day 7 and further by day 14, fewer αSMA+ cells remained in Areas A and B but a steady increase in the peri-infarct was observed. αSMA+ cells also expressed glial acidic fibrillary protein [GFAP: αSMA+/GFAP+ (29%); αSMA+/GFAP- (71%) phenotypes] and feline leukemia virus C receptor 2 (FLVCR2), but not ED1(microglia) and established markers of pericytes normally located in vascular wall. αSMA+ cells were also located close to the subventricular zones (SVZ) adjacent to Areas A and B. In conclusion, αSMA+ cells have been identified in a spatial and temporal sequence from the SVZ, at intermediate loci and in the vicinity of the peri-infarct. It is hypothesized that novel populations of αSMA+ precursors of pericytes are born on the SVZ, migrate into the peri-infarct region and are incorporated into new vessels of the peri-infarct regions.

The protocol for the isolation and cryopreservation of osteoclast precursors from mouse bone marrow and spleen.

PubMed

Boraschi-Diaz, Iris; Komarova, Svetlana V

2016-01-01

Osteoclasts are responsible for physiological bone remodeling as well as pathological bone destruction in osteoporosis, periodontitis and rheumatoid arthritis, and thus represent a pharmacological target for drug development. We aimed to characterize and compare the cytokine-induced osteoclastogenesis of bone marrow and spleen precursors. Established protocols used to generate osteoclasts from bone marrow were modified to examine osteoclastogenesis of the spleen cells of healthy mice. Osteoclast formation was successfully induced from spleen precursors using receptor activator of nuclear factor κB ligand (50 ng/ml) and macrophage colony stimulating factor (50 ng/ml). Compared to bone marrow cultures, differentiation from spleen required a longer cultivation time (9 days for spleen, as compared to 5 days for marrow cultures) and a higher plating density of non-adherent cells (75,000/cm(2) for spleen, as compared to 50,000/cm(2) for bone marrow). Osteoclasts generated from spleen precursors expressed osteoclast marker genes calcitonin receptor, cathepsin K and matrix metalloproteinase 9 and were capable of resorbing hydroxyapatite. The differentiation capacity of spleen and bone marrow precursors was comparable for BALB/c, C57BL/6 and FVB mice. We also developed and tested a cryopreservation protocol for the osteoclast precursors. While 70-80 % of cells were lost during the first week of freezing, during the subsequent 5 weeks the losses were within 2-5 % per week. Osteoclastogenesis from the recovered bone marrow precursors was successful up to 5 weeks after freezing. Spleen precursors retained their osteoclastogenic capacity for 1 week after freezing, but not thereafter. The described protocol is useful for the studies of genetically modified animals as well as for screening new osteoclast-targeting therapeutics.

CD44-positive cells are candidates for astrocyte precursor cells in developing mouse cerebellum.

PubMed

Cai, Na; Kurachi, Masashi; Shibasaki, Koji; Okano-Uchida, Takayuki; Ishizaki, Yasuki

2012-03-01

Neural stem cells are generally considered to be committed to becoming precursor cells before terminally differentiating into either neurons or glial cells during neural development. Neuronal and oligodendrocyte precursor cells have been identified in several areas in the murine central nervous system. The presence of astrocyte precursor cells (APCs) is not so well understood. The present study provides several lines of evidence that CD44-positive cells are APCs in the early postnatal mouse cerebellum. In developing mouse cerebellum, CD44-positive cells, mostly located in the white matter, were positive for the markers of the astrocyte lineage, but negative for the markers of mature astrocytes. CD44-positive cells were purified from postnatal cerebellum by fluorescence-activated cell sorting and characterized in vitro. In the absence of any signaling molecule, many cells died by apoptosis. The surviving cells gradually expressed glial fibrillary acidic protein, a marker for mature astrocytes, indicating that differentiation into mature astrocytes is the default program for these cells. The cells produced no neurospheres nor neurons nor oligodendrocytes under any condition examined, indicating these cells are not neural stem cells. Leukemia inhibitory factor greatly promoted astrocytic differentiation of CD44-positive cells, whereas bone morphogenetic protein 4 (BMP4) did not. Fibroblast growth factor-2 was a potent mitogen for these cells, but was insufficient for survival. BMP4 inhibited activation of caspase-3 and greatly promoted survival, suggesting a novel role for BMP4 in the control of development of astrocytes in cerebellum. We isolated and characterized only CD44 strongly positive large cells and discarded small and/or CD44 weakly positive cells in this study. Further studies are necessary to characterize these cells to help determine whether CD44 is a selective and specific marker for APCs in the developing mouse cerebellum. In conclusion, we succeeded in preparing APC candidates from developing mouse cerebellum, characterized them in vitro, and found that BMPs are survival factors for these cells.

Evolutionarily conserved morphogenetic movements at the vertebrate head-trunk interface coordinate the transport and assembly of hypopharyngeal structures.

PubMed

Lours-Calet, Corinne; Alvares, Lucia E; El-Hanfy, Amira S; Gandesha, Saniel; Walters, Esther H; Sobreira, Débora Rodrigues; Wotton, Karl R; Jorge, Erika C; Lawson, Jennifer A; Kelsey Lewis, A; Tada, Masazumi; Sharpe, Colin; Kardon, Gabrielle; Dietrich, Susanne

2014-06-15

The vertebrate head-trunk interface (occipital region) has been heavily remodelled during evolution, and its development is still poorly understood. In extant jawed vertebrates, this region provides muscle precursors for the throat and tongue (hypopharyngeal/hypobranchial/hypoglossal muscle precursors, HMP) that take a stereotype path rostrally along the pharynx and are thought to reach their target sites via active migration. Yet, this projection pattern emerged in jawless vertebrates before the evolution of migratory muscle precursors. This suggests that a so far elusive, more basic transport mechanism must have existed and may still be traceable today. Here we show for the first time that all occipital tissues participate in well-conserved cell movements. These cell movements are spearheaded by the occipital lateral mesoderm and ectoderm that split into two streams. The rostrally directed stream projects along the floor of the pharynx and reaches as far rostrally as the floor of the mandibular arch and outflow tract of the heart. Notably, this stream leads and engulfs the later emerging HMP, neural crest cells and hypoglossal nerve. When we (i) attempted to redirect hypobranchial/hypoglossal muscle precursors towards various attractants, (ii) placed non-migratory muscle precursors into the occipital environment or (iii) molecularly or (iv) genetically rendered muscle precursors non-migratory, they still followed the trajectory set by the occipital lateral mesoderm and ectoderm. Thus, we have discovered evolutionarily conserved morphogenetic movements, driven by the occipital lateral mesoderm and ectoderm, that ensure cell transport and organ assembly at the head-trunk interface. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Evolutionarily conserved morphogenetic movements at the vertebrate head–trunk interface coordinate the transport and assembly of hypopharyngeal structures

PubMed Central

Lours-Calet, Corinne; Alvares, Lucia E.; El-Hanfy, Amira S.; Gandesha, Saniel; Walters, Esther H.; Sobreira, Débora Rodrigues; Wotton, Karl R.; Jorge, Erika C.; Lawson, Jennifer A.; Kelsey Lewis, A.; Tada, Masazumi; Sharpe, Colin; Kardon, Gabrielle; Dietrich, Susanne

2014-01-01

The vertebrate head–trunk interface (occipital region) has been heavily remodelled during evolution, and its development is still poorly understood. In extant jawed vertebrates, this region provides muscle precursors for the throat and tongue (hypopharyngeal/hypobranchial/hypoglossal muscle precursors, HMP) that take a stereotype path rostrally along the pharynx and are thought to reach their target sites via active migration. Yet, this projection pattern emerged in jawless vertebrates before the evolution of migratory muscle precursors. This suggests that a so far elusive, more basic transport mechanism must have existed and may still be traceable today. Here we show for the first time that all occipital tissues participate in well-conserved cell movements. These cell movements are spearheaded by the occipital lateral mesoderm and ectoderm that split into two streams. The rostrally directed stream projects along the floor of the pharynx and reaches as far rostrally as the floor of the mandibular arch and outflow tract of the heart. Notably, this stream leads and engulfs the later emerging HMP, neural crest cells and hypoglossal nerve. When we (i) attempted to redirect hypobranchial/hypoglossal muscle precursors towards various attractants, (ii) placed non-migratory muscle precursors into the occipital environment or (iii) molecularly or (iv) genetically rendered muscle precursors non-migratory, they still followed the trajectory set by the occipital lateral mesoderm and ectoderm. Thus, we have discovered evolutionarily conserved morphogenetic movements, driven by the occipital lateral mesoderm and ectoderm, that ensure cell transport and organ assembly at the head–trunk interface. PMID:24662046

Cell-type-specific enrichment of risk-associated regulatory elements at ovarian cancer susceptibility loci.

PubMed

Coetzee, Simon G; Shen, Howard C; Hazelett, Dennis J; Lawrenson, Kate; Kuchenbaecker, Karoline; Tyrer, Jonathan; Rhie, Suhn K; Levanon, Keren; Karst, Alison; Drapkin, Ronny; Ramus, Susan J; Couch, Fergus J; Offit, Kenneth; Chenevix-Trench, Georgia; Monteiro, Alvaro N A; Antoniou, Antonis; Freedman, Matthew; Coetzee, Gerhard A; Pharoah, Paul D P; Noushmehr, Houtan; Gayther, Simon A

2015-07-01

Understanding the regulatory landscape of the human genome is a central question in complex trait genetics. Most single-nucleotide polymorphisms (SNPs) associated with cancer risk lie in non-protein-coding regions, implicating regulatory DNA elements as functional targets of susceptibility variants. Here, we describe genome-wide annotation of regions of open chromatin and histone modification in fallopian tube and ovarian surface epithelial cells (FTSECs, OSECs), the debated cellular origins of high-grade serous ovarian cancers (HGSOCs) and in endometriosis epithelial cells (EECs), the likely precursor of clear cell ovarian carcinomas (CCOCs). The regulatory architecture of these cell types was compared with normal human mammary epithelial cells and LNCaP prostate cancer cells. We observed similar positional patterns of global enhancer signatures across the three different ovarian cancer precursor cell types, and evidence of tissue-specific regulatory signatures compared to non-gynecological cell types. We found significant enrichment for risk-associated SNPs intersecting regulatory biofeatures at 17 known HGSOC susceptibility loci in FTSECs (P = 3.8 × 10(-30)), OSECs (P = 2.4 × 10(-23)) and HMECs (P = 6.7 × 10(-15)) but not for EECs (P = 0.45) or LNCaP cells (P = 0.88). Hierarchical clustering of risk SNPs conditioned on the six different cell types indicates FTSECs and OSECs are highly related (96% of samples using multi-scale bootstrapping) suggesting both cell types may be precursors of HGSOC. These data represent the first description of regulatory catalogues of normal precursor cells for different ovarian cancer subtypes, and provide unique insights into the tissue specific regulatory variation with respect to the likely functional targets of germline genetic susceptibility variants for ovarian cancer. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Chromatin remodeling and histone modification in the conversion of oligodendrocyte precursors to neural stem cells

PubMed Central

Kondo, Toru; Raff, Martin

2004-01-01

We showed previously that purified rat oligodendrocyte precursor cells (OPCs) can be induced by extracellular signals to convert to multipotent neural stem-like cells (NSLCs), which can then generate both neurons and glial cells. Because the conversion of precursor cells to stem-like cells is of both intellectual and practical interest, it is important to understand its molecular basis. We show here that the conversion of OPCs to NSLCs depends on the reactivation of the sox2 gene, which in turn depends on the recruitment of the tumor suppressor protein Brca1 and the chromatin-remodeling protein Brahma (Brm) to an enhancer in the sox2 promoter. Moreover, we show that the conversion is associated with the modification of Lys 4 and Lys 9 of histone H3 at the same enhancer. Our findings suggest that the conversion of OPCs to NSLCs depends on progressive chromatin remodeling, mediated in part by Brca1 and Brm. PMID:15574597

Dual-Responsive Metabolic Precursor and Light-Up AIEgen for Cancer Cell Bio-orthogonal Labeling and Precise Ablation.

PubMed

Hu, Fang; Yuan, Youyong; Wu, Wenbo; Mao, Duo; Liu, Bin

2018-06-05

Metabolic glycoengineering of unnatural glycans with bio-orthogonal chemical groups and a subsequent click reaction with fluorescent probes have been widely used in monitoring various bioprocesses. Herein, we developed a dual-responsive metabolic precursor that could specifically generate unnatural glycans with azide groups on the membrane of targeted cancer cells with high selectivity. Moreover, a water-soluble fluorescent light-up probe with aggregation-induced emission (AIE) was synthesized, which turned its fluorescence on upon a click reaction with azide groups on the cancer cell surface, enabling special cancer cell imaging with low background signal. Furthermore, the probe can generate 1 O 2 upon light irradiation, fulfilling its dual role as an imaging and therapeutic agent for cancer cells. Therefore, the concepts of the cancer-cell-specific metabolic precursor cRGD-S-Ac 3 ManNAz and the AIE light-up probe are promising in bio-orthogonal labeling and cancer-specific imaging and therapy.

Evolving gene regulation networks into cellular networks guiding adaptive behavior: an outline how single cells could have evolved into a centralized neurosensory system

PubMed Central

Fritzsch, Bernd; Jahan, Israt; Pan, Ning; Elliott, Karen L.

2014-01-01

Understanding the evolution of the neurosensory system of man, able to reflect on its own origin, is one of the major goals of comparative neurobiology. Details of the origin of neurosensory cells, their aggregation into central nervous systems and associated sensory organs, their localized patterning into remarkably different cell types aggregated into variably sized parts of the central nervous system begin to emerge. Insights at the cellular and molecular level begin to shed some light on the evolution of neurosensory cells, partially covered in this review. Molecular evidence suggests that high mobility group (HMG) proteins of pre-metazoans evolved into the definitive Sox [SRY (sex determining region Y)-box] genes used for neurosensory precursor specification in metazoans. Likewise, pre-metazoan basic helix-loop-helix (bHLH) genes evolved in metazoans into the group A bHLH genes dedicated to neurosensory differentiation in bilaterians. Available evidence suggests that the Sox and bHLH genes evolved a cross-regulatory network able to synchronize expansion of precursor populations and their subsequent differentiation into novel parts of the brain or sensory organs. Molecular evidence suggests metazoans evolved patterning gene networks early and not dedicated to neuronal development. Only later in evolution were these patterning gene networks tied into the increasing complexity of diffusible factors, many of which were already present in pre-metazoans, to drive local patterning events. It appears that the evolving molecular basis of neurosensory cell development may have led, in interaction with differentially expressed patterning genes, to local network modifications guiding unique specializations of neurosensory cells into sensory organs and various areas of the central nervous system. PMID:25416504

Evolving gene regulatory networks into cellular networks guiding adaptive behavior: an outline how single cells could have evolved into a centralized neurosensory system.

PubMed

Fritzsch, Bernd; Jahan, Israt; Pan, Ning; Elliott, Karen L

2015-01-01

Understanding the evolution of the neurosensory system of man, able to reflect on its own origin, is one of the major goals of comparative neurobiology. Details of the origin of neurosensory cells, their aggregation into central nervous systems and associated sensory organs and their localized patterning leading to remarkably different cell types aggregated into variably sized parts of the central nervous system have begun to emerge. Insights at the cellular and molecular level have begun to shed some light on the evolution of neurosensory cells, partially covered in this review. Molecular evidence suggests that high mobility group (HMG) proteins of pre-metazoans evolved into the definitive Sox [SRY (sex determining region Y)-box] genes used for neurosensory precursor specification in metazoans. Likewise, pre-metazoan basic helix-loop-helix (bHLH) genes evolved in metazoans into the group A bHLH genes dedicated to neurosensory differentiation in bilaterians. Available evidence suggests that the Sox and bHLH genes evolved a cross-regulatory network able to synchronize expansion of precursor populations and their subsequent differentiation into novel parts of the brain or sensory organs. Molecular evidence suggests metazoans evolved patterning gene networks early, which were not dedicated to neuronal development. Only later in evolution were these patterning gene networks tied into the increasing complexity of diffusible factors, many of which were already present in pre-metazoans, to drive local patterning events. It appears that the evolving molecular basis of neurosensory cell development may have led, in interaction with differentially expressed patterning genes, to local network modifications guiding unique specializations of neurosensory cells into sensory organs and various areas of the central nervous system.

Effect of bariatric surgery on peripheral blood lymphocyte subsets in women.

PubMed

Merhi, Zaher O; Durkin, Helen G; Feldman, Joseph; Macura, Jerzy; Rodriguez, Carlos; Minkoff, Howard

2009-01-01

The use of bariatric surgery to treat refractory obesity is increasingly common. The great weight loss that can result from these procedures has been shown to ameliorate certain deleterious effects of obesity. However, the effect of surgery on immune status is unclear. We investigated the relationship between surgical weight loss and peripheral blood lymphocyte percentages in women. Women (n=20, age range 25-59 years, body mass index [BMI] range 36.4-68.2 kg/m2) who had undergone either gastric banding (n=14) or gastric bypass (n=6) were enrolled in a prospective study to determine the percentages of their peripheral blood T cells (CD3+, CD4+, and CD8+), CD19+ B cells, and CD3-/CD16+CD56+ natural killer precursor cells before and 85+/-7 days (3 months) postoperatively using flow cytometry. The data are expressed as the percentage of total lymphocytes+/-the standard error of the mean. A decrease in the BMI at 3 months postoperatively was 12% in the overall study population and 8% and 20% in the banding and bypass groups, respectively. No significant changes were found in the CD4+ or CD8+ T cells (P=.9 and P=.5, respectively), CD19+ B cells (P=.6), or natural killer precursor cells (P=.25) in the overall population or among the patients when stratified by surgical procedure (gastric banding or bypass). The change in CD3+ T cells approached significance (P=.06). A "same direction" (negative) correlation was found between the decrease in BMI and changes in the CD4+ T cell percentages between the pre- and postoperative levels in all the participants, and in the bypass and banding groups separately. However, it only reached statistical significance in the bypass group (r=-.96, P=.002). When studying the correlation between the decrease in BMI and the changes in CD3+ T cell percentages between the pre- and postoperative levels, a borderline significant negative correlation was found for all participants (r=-.44, P=.05) and in the bypass group (r=-.76, P=.08). The rate of change in the CD4+ and CD3+ T cells was greatest among those with the least weight loss and decreased with greater weight loss. An inverse relationship exists between the change in certain T cells (CD4+ and CD3+) and the amount of weight lost after bariatric surgery, mainly gastric bypass surgery. The greater the decrease in BMI, the lower the change in these T cells.

Thrombopoietin inhibits murine mast cell differentiation

PubMed Central

Martelli, Fabrizio; Ghinassi, Barbara; Lorenzini, Rodolfo; Vannucchi, Alessandro M; Rana, Rosa Alba; Nishikawa, Mitsuo; Partamian, Sandra; Migliaccio, Giovanni; Migliaccio, Anna Rita

2009-01-01

We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin, or addition of this growth factor to bone marrow-derived mast cell cultures, severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target anti-apoptotic gene Bcl2. PMID:18276801

Polymer/Nanocrystal Hybrid Solar Cells: Influence of Molecular Precursor Design on Film Nanomorphology, Charge Generation and Device Performance

PubMed Central

MacLachlan, Andrew J; Rath, Thomas; Cappel, Ute B; Dowland, Simon A; Amenitsch, Heinz; Knall, Astrid-Caroline; Buchmaier, Christine; Trimmel, Gregor; Nelson, Jenny; Haque, Saif A

2015-01-01

In this work, molecular tuning of metal xanthate precursors is shown to have a marked effect on the heterojunction morphology of hybrid poly(3-hexylthiophene-2,5-diyl) (P3HT)/CdS blends and, as a result, the photochemical processes and overall performance of in situ fabricated hybrid solar cells. A series of cadmium xanthate complexes is synthesized for use as in situ precursors to cadmium sulfide nanoparticles in hybrid P3HT/CdS solar cells. The formation of CdS domains is studied by simultaneous GIWAXS (grazing incidence wide-angle X-ray scattering) and GISAXS (grazing incidence small-angle X-ray scattering), revealing knowledge about crystal growth and the formation of different morphologies observed using TEM (transmission electron microscopy). These measurements show that there is a strong relationship between precursor structure and heterojunction nanomorphology. A combination of TAS (transient absorption spectroscopy) and photovoltaic device performance measurements is used to show the intricate balance required between charge photogeneration and percolated domains in order to effectively extract charges to maximize device power conversion efficiencies. This study presents a strong case for xanthate complexes as a useful route to designing optimal heterojunction morphologies for use in the emerging field of hybrid organic/inorganic solar cells, due to the fact that the nanomorphology can be tuned via careful design of these precursor materials. PMID:25866496

Region-specific DNA synthesis in brains of F344 rats following a six-day bromodeoxyuridine infusion.

PubMed

Bolon, B; Dunn, C; Goldsworthy, T L

1996-09-01

Prolonged exposure to certain alkylating chemicals induces glial and meningeal tumours in rats, probably resulting from DNA damage to dividing neural cells. The present work evaluated DNA synthesis in the brains of untreated, young adult male F344 rats in order to define a BrdUrd infusion protocol to more adequately assess proliferation in slowly dividing neural cell populations. BrdUrd (2.5 to 160 mg/ml) was administered for 6 days via subcutaneous osmotic pumps. Clinical toxicity was not observed at any dose. The labelling index (LI; % of cells per brain area that incorporated BrdUrd) and unit length labelling index (ULLI; % of cells per meningeal length that incorporated BrdUrd) were calculated for selected regions by counting labelled neural cells in defined areas of the right hemisphere in coronal brain sections. Intensely stained cells were numerous in the cerebral subependymal layer (LI = 35.8%); scattered in cerebral white matter tracts (e.g. corpus callosum and internal capsule; LI = 6.2%) as well as cerebral (ULLI = 4.2%) and cerebellar (ULLI = 3.6%) meninges; and rare in the hippocampus (LI > 0.1%). Mildy stained cells were dispersed in the pons (LI = 2.1%), deep cerebral (LI = 1.8%) and cerebellar (LI = 1.0%) grey matter, and thalamus (LI = 0.3%). Phenotypically, BrdUrd-positive cells in neuropil were glial cell precursors and their progeny, while those associated with meninges were usually located in the superficial subarachnoid space and appeared to be fibrocytes. Using BrdUrd infusion, LI for glial precursors at these sites ranged from two- to 10-fold higher than those reported previously after a brief parenteral pulse dose. These data indicate that continuous BrdUrd infusion for 6 days by subcutaneous osmotic pump is an efficient means of labelling neural cells throughout the brain.

The Cdk4-E2f1 pathway regulates early pancreas development by targeting Pdx1+ progenitors and Ngn3+ endocrine precursors

PubMed Central

Kim, So Yoon; Rane, Sushil G.

2011-01-01

Cell division and cell differentiation are intricately regulated processes vital to organ development. Cyclin-dependent kinases (Cdks) are master regulators of the cell cycle that orchestrate the cell division and differentiation programs. Cdk1 is essential to drive cell division and is required for the first embryonic divisions, whereas Cdks 2, 4 and 6 are dispensable for organogenesis but vital for tissue-specific cell development. Here, we illustrate an important role for Cdk4 in regulating early pancreas development. Pancreatic development involves extensive morphogenesis, proliferation and differentiation of the epithelium to give rise to the distinct cell lineages of the adult pancreas. The cell cycle molecules that specify lineage commitment within the early pancreas are unknown. We show that Cdk4 and its downstream transcription factor E2f1 regulate mouse pancreas development prior to and during the secondary transition. Cdk4 deficiency reduces embryonic pancreas size owing to impaired mesenchyme development and fewer Pdx1+ pancreatic progenitor cells. Expression of activated Cdk4R24C kinase leads to increased Nkx2.2+ and Nkx6.1+ cells and a rise in the number and proliferation of Ngn3+ endocrine precursors, resulting in expansion of the β cell lineage. We show that E2f1 binds and activates the Ngn3 promoter to modulate Ngn3 expression levels in the embryonic pancreas in a Cdk4-dependent manner. These results suggest that Cdk4 promotes β cell development by directing E2f1-mediated activation of Ngn3 and increasing the pool of endocrine precursors, and identify Cdk4 as an important regulator of early pancreas development that modulates the proliferation potential of pancreatic progenitors and endocrine precursors. PMID:21490060

Pediatric precursor B acute lymphoblastic leukemia: are T helper cells the missing link in the infectious etiology theory?

PubMed

Bürgler, Simone; Nadal, David

2017-12-01

Precursor B acute lymphoblastic leukemia (BCP-ALL), the most common childhood malignancy, arises from an expansion of malignant B cell precursors in the bone marrow. Epidemiological studies suggest that infections or immune responses to infections may promote such an expansion and thus BCP-ALL development. Nevertheless, a specific pathogen responsible for this process has not been identified. BCP-ALL cells critically depend on interactions with the bone marrow microenvironment. The bone marrow is also home to memory T helper (Th) cells that have previously expanded during an immune response in the periphery. In secondary lymphoid organs, Th cells can interact with malignant cells of mature B cell origin, while such interactions between Th cells and malignant immature B cell in the bone marrow have not been described yet. Nevertheless, literature supports a model where Th cells-expanded during an infection in early childhood-migrate to the bone marrow and support BCP-ALL cells as they support normal B cells. Further research is required to mechanistically confirm this model and to elucidate the interaction pathways between leukemia cells and cells of the tumor microenvironment. As benefit, targeting these interactions could be included in current treatment regimens to increase therapeutic efficiency and to reduce relapses.

Distal regeneration involves the age dependent activity of branchial sac stem cells in the ascidian Ciona intestinalis

PubMed Central

2014-01-01

Abstract Tunicates have high capacities for regeneration but the underlying mechanisms and their relationship to life cycle progression are not well understood. Here we investigate the regeneration of distal structures in the ascidian tunicate Ciona intestinalis. Analysis of regenerative potential along the proximal−distal body axis indicated that distal organs, such as the siphons, their pigmented sensory organs, and the neural complex, could only be replaced from body fragments containing the branchial sac. Distal regeneration involves the formation of a blastema composed of cells that undergo cell proliferation prior to differentiation and cells that differentiate without cell proliferation. Both cell types originate in the branchial sac and appear in the blastema at different times after distal injury. Whereas the branchial sac stem cells are present in young animals, they are depleted in old animals that have lost their regeneration capacity. Thus Ciona adults contain a population of age‐related stem cells located in the branchial sac that are a source of precursors for distal body regeneration. PMID:25893097

Developmental changes in electrophysiological characteristics of human-induced pluripotent stem cell-derived cardiomyocytes.

PubMed

Ben-Ari, Meital; Naor, Shulamit; Zeevi-Levin, Naama; Schick, Revital; Ben Jehuda, Ronen; Reiter, Irina; Raveh, Amit; Grijnevitch, Inna; Barak, Omri; Rosen, Michael R; Weissman, Amir; Binah, Ofer

2016-12-01

Previous studies proposed that throughout differentiation of human induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs), only 3 types of action potentials (APs) exist: nodal-, atrial-, and ventricular-like. To investigate whether there are precisely 3 phenotypes or a continuum exists among them, we tested 2 hypotheses: (1) During culture development a cardiac precursor cell is present that-depending on age-can evolve into the 3 phenotypes. (2) The predominant pattern is early prevalence of a nodal phenotype, transient appearance of an atrial phenotype, evolution to a ventricular phenotype, and persistence of transitional phenotypes. To test these hypotheses, we (1) performed fluorescence-activated cell sorting analysis of nodal, atrial, and ventricular markers; (2) recorded APs from 280 7- to 95-day-old iPSC-CMs; and (3) analyzed AP characteristics. The major findings were as follows: (1) fluorescence-activated cell sorting analysis of 30- and 60-day-old cultures showed that an iPSC-CMs population shifts from the nodal to the atrial/ventricular phenotype while including significant transitional populations; (2) the AP population did not consist of 3 phenotypes; (3) culture aging was associated with a shift from nodal to ventricular dominance, with a transient (57-70 days) appearance of the atrial phenotype; and (4) beat rate variability was more prominent in nodal than in ventricular cardiomyocytes, while pacemaker current density increased in older cultures. From the onset of development in culture, the iPSC-CMs population includes nodal, atrial, and ventricular APs and a broad spectrum of transitional phenotypes. The most readily distinguishable phenotype is atrial, which appears only transiently yet dominates at 57-70 days of evolution. Copyright © 2016 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

The Use of Variable Q1 Isolation Windows Improves Selectivity in LC-SWATH-MS Acquisition.

PubMed

Zhang, Ying; Bilbao, Aivett; Bruderer, Tobias; Luban, Jeremy; Strambio-De-Castillia, Caterina; Lisacek, Frédérique; Hopfgartner, Gérard; Varesio, Emmanuel

2015-10-02

As tryptic peptides and metabolites are not equally distributed along the mass range, the probability of cross fragment ion interference is higher in certain windows when fixed Q1 SWATH windows are applied. We evaluated the benefits of utilizing variable Q1 SWATH windows with regards to selectivity improvement. Variable windows based on equalizing the distribution of either the precursor ion population (PIP) or the total ion current (TIC) within each window were generated by an in-house software, swathTUNER. These two variable Q1 SWATH window strategies outperformed, with respect to quantification and identification, the basic approach using a fixed window width (FIX) for proteomic profiling of human monocyte-derived dendritic cells (MDDCs). Thus, 13.8 and 8.4% additional peptide precursors, which resulted in 13.1 and 10.0% more proteins, were confidently identified by SWATH using the strategy PIP and TIC, respectively, in the MDDC proteomic sample. On the basis of the spectral library purity score, some improvement warranted by variable Q1 windows was also observed, albeit to a lesser extent, in the metabolomic profiling of human urine. We show that the novel concept of "scheduled SWATH" proposed here, which incorporates (i) variable isolation windows and (ii) precursor retention time segmentation further improves both peptide and metabolite identifications.

A Common Origin for B-1a and B-2 Lymphocytes in Clonal Pre- Hematopoietic Stem Cells.

PubMed

Hadland, Brandon K; Varnum-Finney, Barbara; Mandal, Pankaj K; Rossi, Derrick J; Poulos, Michael G; Butler, Jason M; Rafii, Shahin; Yoder, Mervin C; Yoshimoto, Momoko; Bernstein, Irwin D

2017-06-06

Recent evidence points to the embryonic emergence of some tissue-resident innate immune cells, such as B-1a lymphocytes, prior to and independently of hematopoietic stem cells (HSCs). However, whether the full hematopoietic repertoire of embryonic HSCs initially includes these unique lineages of innate immune cells has been difficult to assess due to lack of clonal assays that identify and assess HSC precursor (pre-HSC) potential. Here, by combining index sorting of single embryonic hemogenic precursors with in vitro HSC maturation and transplantation assays, we analyze emerging pre-HSCs at the single-cell level, revealing their unique stage-specific properties and clonal lineage potential. Remarkably, clonal pre-HSCs detected between E9.5 and E11.5 contribute to the complete B cell repertoire, including B-1a lymphocytes, revealing a previously unappreciated common precursor for all B cell lineages at the pre-HSC stage and a second embryonic origin for B-1a lymphocytes. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

CD73 Protein as a Source of Extracellular Precursors for Sustained NAD+ Biosynthesis in FK866-treated Tumor Cells*

PubMed Central

Grozio, Alessia; Sociali, Giovanna; Sturla, Laura; Caffa, Irene; Soncini, Debora; Salis, Annalisa; Raffaelli, Nadia; De Flora, Antonio; Nencioni, Alessio; Bruzzone, Santina

2013-01-01

NAD+ is mainly synthesized in human cells via the “salvage” pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the “salvage” pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD+ or NAD+ precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD+ precursors for NAD+ biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD+ biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors. PMID:23880765

Toxicological effects of three types of silver nanoparticles and their salt precursors acting on human U-937 and HL-60 cells.

PubMed

Barbasz, Anna; Oćwieja, Magdalena; Walas, Stanisław

2017-01-01

The growing popularity of nanomaterials requires a systematic study of their effects on the human body. Silver nanoparticles (AgNPs), due to their antiseptic properties, are used in almost every area of life. The purpose of the study was to examine whether the precursor used for the synthesis of nanoparticles affects their bio-influence and modifies their impact on cells of the human immune system. To compare the effects of precursor silver salts (AgNO 3 , CH 3 COOAg and AgClO 4 ) and corresponding nanoparticles (TAN TAA and TAC) cytotoxicity study was conducted on two cell lines U-937 and HL-60. For both cell lines, silver salts are more toxic than the corresponding nanoparticles. Cell viability after treatment with the two forms of silver (salt/particle) is dependent on silver dose and degree of cells differentiation. Addition of the silver salt of doses greater than 5 mg/L results in decreased cell viability by over 60%, whereas nanoparticles' addition reduces cell viability on average by 30%. On the basis of the determined LD 50 values it can be stated that for the tested cells the most toxic are AgClO 4 and TAC. Production of nitric oxide, which is a mediator of inflammation, is the greatest after treatment of the cells by TAC. Different interactions of studied nanoparticles with albumin has been found and it was shown that addition of albumin to the cells treated by nanoparticles reduces their toxic effects. Obtained by us highly purified, mono-disperse AgNPs exhibit diverse effects relative to the biological systems, depending on the precursor salt used.

Genes commonly deleted in childhood B-cell precursor acute lymphoblastic leukemia: association with cytogenetics and clinical features

PubMed Central

Schwab, Claire J.; Chilton, Lucy; Morrison, Heather; Jones, Lisa; Al-Shehhi, Halima; Erhorn, Amy; Russell, Lisa J.; Moorman, Anthony V.; Harrison, Christine J.

2013-01-01

In childhood B-cell precursor acute lymphoblastic leukemia, cytogenetics is important in diagnosis and as an indicator of response to therapy, thus playing a key role in risk stratification of patients for treatment. Little is known of the relationship between different cytogenetic subtypes in B-cell precursor acute lymphoblastic leukemia and the recently reported copy number abnormalities affecting significant leukemia associated genes. In a consecutive series of 1427 childhood B-cell precursor acute lymphoblastic leukemia patients, we have determined the incidence and type of copy number abnormalities using multiplex ligation-dependent probe amplification. We have shown strong links between certain deletions and cytogenetic subtypes, including the novel association between RB1 deletions and intrachromosomal amplification of chromosome 21. In this study, we characterized the different copy number abnormalities and show heterogeneity of PAX5 and IKZF1 deletions and the recurrent nature of RB1 deletions. Whole gene losses are often indicative of larger deletions, visible by conventional cytogenetics. An increased number of copy number abnormalities is associated with NCI high risk, specifically deletions of IKZF1 and CDKN2A/B, which occur more frequently among these patients. IKZF1 deletions and rearrangements of CRLF2 among patients with undefined karyotypes may point to the poor risk BCR-ABL1-like group. In conclusion, this study has demonstrated in a large representative cohort of children with B-cell precursor acute lymphoblastic leukemia that the pattern of copy number abnormalities is highly variable according to the primary genetic abnormality. PMID:23508010

The neurotoxicant, cuprizone, retards the differentiation of oligodendrocytes in vitro.

PubMed

Cammer, W

1999-10-15

The effects of oxalyldihydrazone (cuprizone) on weanling rodents provided an early protocol for toxic demyelination in vivo, in which degeneration of oligodendrocytes preceded disruption of the myelin sheath, and in which remyelination could take place. We administered cuprizone to oligodendrocyte-enriched glial-cell cultures and to mixed glial-cell cultures from neonatal rat brains. The cultures were treated with cuprizone for 1 h and allowed to continue differentiating on subsequent days. Treated cultures and respective control cultures were fixed with 4% paraformaldehyde (w/v) and immunostained with double immunofluorescence. MAbO4 was used to mark precursors and mature oligodendrocytes, and anti-myelin basic protein (MBP) to mark mature oligodendrocytes (O4+/MBP+), as distinguished from precursors, which were O4+/MBP-. Cell counts suggested that cuprizone inhibited the maturation of oligodendrocytes without diminishing the numbers of precursors, and appeared to affect the mitochondria in those cells.

Cellular compartmentalization of secondary metabolism

PubMed Central

Kistler, H. Corby; Broz, Karen

2015-01-01

Fungal secondary metabolism is often considered apart from the essential housekeeping functions of the cell. However, there are clear links between fundamental cellular metabolism and the biochemical pathways leading to secondary metabolite synthesis. Besides utilizing key biochemical precursors shared with the most essential processes of the cell (e.g., amino acids, acetyl CoA, NADPH), enzymes for secondary metabolite synthesis are compartmentalized at conserved subcellular sites that position pathway enzymes to use these common biochemical precursors. Co-compartmentalization of secondary metabolism pathway enzymes also may function to channel precursors, promote pathway efficiency and sequester pathway intermediates and products from the rest of the cell. In this review we discuss the compartmentalization of three well-studied fungal secondary metabolite biosynthetic pathways for penicillin G, aflatoxin and deoxynivalenol, and summarize evidence used to infer subcellular localization. We also discuss how these metabolites potentially are trafficked within the cell and may be exported. PMID:25709603

The effects of vitamin D binding protein-macrophage activating factor and colony-stimulating factor-1 on hematopoietic cells in normal and osteopetrotic rats.

PubMed

Benis, K A; Schneider, G B

1996-10-15

Osteopetrosis is a heterogeneous group of bone disorders characterized by the failure of osteoclasts to resorb bone and by several immunological defects including macrophage dysfunction. Two compounds, colony-stimulating factor-1 (CSF-1) and vitamin D-binding protein-macrophage activating factor (DBP-MAF) were used in the present study to evaluate their effects on the peritoneal population of cells and on cells within the bone marrow microenvironment in normal and incisors absent (ia) osteopetrotic rats. Previous studies in this laboratory have demonstrated that administration of DBP-MAF to newborn ia animals results in a substantial increase in bone marrow cavity size due to upregulated osteoclast function. To study the effects of these compounds on the macrophage/osteoclast precursors, DBP-MAF, CSF-1, and the combination of these compounds were given to newborn ia and normal littermate animals. Both the normal and mutant phenotypes responded similarly when treated with these compounds. Rats exhibited a profound shift toward the macrophage lineage from the neutrophil lineage when compared with vehicle-treated control animals after treatment with these compounds. In the in vivo peritoneal lavage study, animals received injections of CSF-1, DBP-MAF or DBP-MAF/CSF-1 over a 4-week period. The various types of cells in the peritoneal cavity were then enumerated. The in vitro study consisted of cells isolated from the bone marrow microenvironment and cultured on feeder layers of CSF-1, DBP-MAF, or DBP-MAF/CSF-1 for colony enumeration. The increase in macrophage numbers at the expense of neutrophil numbers could be seen in both the in vivo and in vitro experiments. The macrophage/osteoclast and neutrophil lineages have a common precursor, the granulocyte/macrophage colony-forming cell (GM-CFC). With the addition of CSF-1, the GM-CFC precursor may be induced into the macrophage/osteoclast lineage rather than the granulocyte lineage. This increased pool of cells in the macrophage/osteoclast lineage can be functionally upregulated with the subsequent addition of DBP-MAF to perform the activities of phagocytosis and bone resorption. The in vitro data also showed that DBP-MAF did not support colony development as in CSF-1 or the combination treatment. The recruitment and activation of cells into the macrophage/ osteoclast lineage may help to correct the bone and immune defects found in diseases demonstrating a significant lack of myeloid cells, as well as neutrophilia disorders and the disease, osteopetrosis.

piRNA biogenesis during adult spermatogenesis in mice is independent of the ping-pong mechanism.

PubMed

Beyret, Ergin; Liu, Na; Lin, Haifan

2012-10-01

piRNAs, a class of small non-coding RNAs associated with PIWI proteins, have broad functions in germline development, transposon silencing, and epigenetic regulation. In diverse organisms, a subset of piRNAs derived from repeat sequences are produced via the interplay between two PIWI proteins. This mechanism, termed "ping-pong" cycle, operates among the PIWI proteins of the primordial mouse testis; however, its involvement in postnatal testes remains elusive. Here we show that adult testicular piRNAs are produced independent of the ping-pong mechanism. We identified and characterized large populations of piRNAs in the adult and postnatal developing testes associated with MILI and MIWI, the only PIWI proteins detectable in these testes. No interaction between MILI and MIWI or sequence feature for the ping-pong mechanism among their piRNAs was detected in the adult testis. The majority of MILI- and MIWI-associated piRNAs originate from the same DNA strands within the same loci. Both populations of piRNAs are biased for 5' Uracil but not for Adenine on the 10th nucleotide position, and display no complementarity. Furthermore, in Miwi mutants, MILI-associated piRNAs are not downregulated, but instead upregulated. These results indicate that the adult testicular piRNAs are predominantly, if not exclusively, produced by a primary processing mechanism instead of the ping-pong mechanism. In this primary pathway, biogenesis of MILI- and MIWI-associated piRNAs may compete for the same precursors; the types of piRNAs produced tend to be non-selectively dictated by the available precursors in the cell; and precursors with introns tend to be spliced before processed into piRNAs.

TGFbeta regulation of membrane mucin Muc4 via proteosome degradation.

PubMed

Lomako, Wieslawa M; Lomako, Joseph; Soto, Pedro; Carraway, Coralie A Carothers; Carraway, Kermit L

2009-07-01

Muc4 is a heterodimeric membrane mucin implicated in epithelial differentiation and tumor progression. It is expressed from a single gene as a 300 kDa precursor protein which is cleaved in the endoplasmic reticulum to its two subunits. Our previous work has shown that Muc4 is regulated by TGFbeta, which represses the precursor cleavage. Working with Muc4-transfected A375 tumor cells, we now show that Muc4 undergoes proteosomal degradation. Proteosome inhibitors prolong the life of the precursor, shunt the Muc4 into cytoplasmic aggresomes, increase the level of Muc4 associated with the endoplasmic reticulum chaperones calnexin and calreticulin and increase the levels of ubiquitinated Muc4. Most importantly, proteosome inhibitors repress the TGFbeta inhibition of Muc4 expression. These results suggest a model in which TGFbeta inhibits precursor cleavage, shunting the precursor into the proteosomal degradation pathway. Thus, the cells have evolved a mechanism to use the quality control pathway for glycoproteins to control the quantity of the protein produced. 2009 Wiley-Liss, Inc.

Development of advanced test methods for the improvement of production standards for ceramic powders used in solid oxide fuel cells

NASA Astrophysics Data System (ADS)

Ward, Brian

Solid oxide fuel cells (SOFCs) are energy conversion devices that use ceramic powders as a precursor material for their electrodes. Presently, powder manufacturers are encountering complications producing consistent precursor powders. Through various thermal, chemical and physical tests, such as DSC and XRD, a preliminary production standard will be developed.

Evaluation of T-lymphocyte subpopulations in actinic keratosis, in situ and invasive squamous cell carcinoma of the skin.

PubMed

Stravodimou, Aristea; Tzelepi, Vassiliki; Papadaki, Helen; Mouzaki, Athanasia; Georgiou, Sophia; Melachrinou, Maria; Kourea, Eleni P

2018-05-01

Tumor infiltrating lymphocytes (TILs) represent important regulators of carcinogenesis. Cutaneous invasive squamous cell carcinoma (inSCC) develops through precursor lesions, namely in situ squamous cell carcinoma (isSCC) and actinic keratosis (AK), representing a natural model of carcinogenesis. The study evaluates TIL subpopulations in inSCC and its precursors by comparing 2 semiquantitative scoring systems, and assesses the presence of regulatory T-cells (Tregs) in these lesions. Paraffin sections from 33 cases of AK, 19 isSCCs and 34 inSCCs with adjacent precursor lesions or normal skin (NS) were immunostained for CD3, CD4, CD8 and Foxp3. TIL subgroups were evaluated by the semiquantitative Klintrup-Mäkinen (K-M) score, and by a more detailed modification of this system. Treg counts were assessed by image analysis quantification. An increase of all TIL subpolulations from precursor lesions toward inSCC was shown by both scoring systems. Treg counts progressively increased from NS to AK and isSCC, but decreased in inSCC. Tregs were more numerous in pT2 and around indolent inSCCs compared to T1 and aggressive subtypes. T-cells and cytotoxic T-cells progressively increase in cutaneous squamous cell carcinogenesis, while Treg counts diminish in inSCC. The K-M score is an appropriate, easily applicable TIL scoring system in cutaneous inSCC. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

β-Catenin signaling regulates temporally discrete phases of anterior taste bud development

PubMed Central

Thirumangalathu, Shoba; Barlow, Linda A.

2015-01-01

The sense of taste is mediated by multicellular taste buds located within taste papillae on the tongue. In mice, individual taste buds reside in fungiform papillae, which develop at mid-gestation as epithelial placodes in the anterior tongue. Taste placodes comprise taste bud precursor cells, which express the secreted factor sonic hedgehog (Shh) and give rise to taste bud cells that differentiate around birth. We showed previously that epithelial activation of β-catenin is the primary inductive signal for taste placode formation, followed by taste papilla morphogenesis and taste bud differentiation, but the degree to which these later elements were direct or indirect consequences of β-catenin signaling was not explored. Here, we define discrete spatiotemporal functions of β-catenin in fungiform taste bud development. Specifically, we show that early epithelial activation of β-catenin, before taste placodes form, diverts lingual epithelial cells from a taste bud fate. By contrast, β-catenin activation a day later within Shh+ placodes, expands taste bud precursors directly, but enlarges papillae indirectly. Further, placodal activation of β-catenin drives precocious differentiation of Type I glial-like taste cells, but not other taste cell types. Later activation of β-catenin within Shh+ precursors during papilla morphogenesis also expands taste bud precursors and accelerates Type I cell differentiation, but papilla size is no longer enhanced. Finally, although Shh regulates taste placode patterning, we find that it is dispensable for the accelerated Type I cell differentiation induced by β-catenin. PMID:26525674

Tissue Motion and Assembly During Early Cardiovascular Morphogenesis

NASA Astrophysics Data System (ADS)

Rongish, Brenda

2010-03-01

Conventional dogma in the field of cardiovascular developmental biology suggests that cardiac precursor cells migrate to the embryonic midline to form a tubular heart. These progenitors are believed to move relative to their extracellular matrix (ECM); responding to stimulatory and inhibitory cues in their environment. The tubular heart that is formed by 30 hours post fertilization is comprised of two concentric layers: the muscular myocardium and the endothelial-like endocardium, which are separated by a thick layer of ECM believed to be secreted predominantly by the myocardial cells. Here we describe the origin and motility of fluorescently tagged endocardial precursors in transgenic (Tie1-YFP) quail embryos (R. Lansford, Caltech) using epifluorescence time-lapse imaging. To visualize the environment of migrating endocardial progenitors, we labeled two ECM components, fibronectin and fibrillin-2, via in vivo microinjection of fluorochrome-conjugated monoclonal antibodies. Dynamic imaging was performed at stages encompassing tubular heart assembly and early looping. We established the motion of endocardial precursor cells and presumptive cardiac ECM fibrils using both object tracking and particle image velocimetry (image cross correlation). We determined the relative importance of directed cell autonomous motility versus passive tissue movements in endocardial morphogenesis. The data show presumptive endocardial cells and cardiac ECM fibrils are swept passively into the anterior and posterior poles of the elongating tubular heart. These quantitative data indicate the contribution of cell autonomous motility displayed by endocardial precursors is limited. Thus, tissue motion drives most of the cell displacements during endocardial morphogenesis.

Modeling variability in air pollution-related health damages from individual airport emissions.

PubMed

Penn, Stefani L; Boone, Scott T; Harvey, Brian C; Heiger-Bernays, Wendy; Tripodis, Yorghos; Arunachalam, Sarav; Levy, Jonathan I

2017-07-01

In this study, we modeled concentrations of fine particulate matter (PM 2.5 ) and ozone (O 3 ) attributable to precursor emissions from individual airports in the United States, developing airport-specific health damage functions (deaths per 1000t of precursor emissions) and physically-interpretable regression models to explain variability in these functions. We applied the Community Multiscale Air Quality model using the Decoupled Direct Method to isolate PM 2.5 - or O 3 -related contributions from precursor pollutants emitted by 66 individual airports. We linked airport- and pollutant-specific concentrations with population data and literature-based concentration-response functions to create health damage functions. Deaths per 1000t of primary PM 2.5 emissions ranged from 3 to 160 across airports, with variability explained by population patterns within 500km of the airport. Deaths per 1000t of precursors for secondary PM 2.5 varied across airports from 0.1 to 2.7 for NOx, 0.06 to 2.9 for SO 2 , and 0.06 to 11 for VOCs, with variability explained by population patterns and ambient concentrations influencing particle formation. Deaths per 1000t of O 3 precursors ranged from -0.004 to 1.0 for NOx and 0.03 to 1.5 for VOCs, with strong seasonality and influence of ambient concentrations. Our findings reinforce the importance of location- and source-specific health damage functions in design of health-maximizing emissions control policies. Copyright © 2017 Elsevier Inc. All rights reserved.

Quercetin prevents protein nitration and glycolytic block of proliferation in hydrogen peroxide insulted cultured neuronal precursor cells (NPCs): Implications on CNS regeneration.

PubMed

Sajad, Mir; Zargan, Jamil; Zargar, Mohammad Afzal; Sharma, Jyoti; Umar, Sadiq; Arora, Rajesh; Khan, Haider A

2013-05-01

Survival along with optimal proliferation of neuronal precursors determines the outcomes of the endogenous cellular repair in CNS. Cellular-oxidation based cell death has been described in several neurodegenerative disorders. Therefore, this study was aimed at the identification of the potent targets of oxidative damage to the neuronal precursors and its effective prevention by a natural flavonoid, Quercetin. Neuronal precursor cells (NPCs), Nestin+ and GFAP (Glial fibrillary acidic protein)+ were isolated and cultured from adult rat SVZ (subventricular zone). These cells were challenged with a single dose of H2O2 (50μM) and/or pre-treated with different concentrations of Quercetin. H2O2 severely limited the cellular viability and expansion of the neurospheres. Cellular-oxidation studies revealed reduction in glutathione dependent redox buffering along with depletion of enzymatic cellular antioxidants that might potentiate the nitrite (NO2(-)) and superoxide anion (O2(-)) mediated peroxynitrite (ONOO(-)) formation and irreversible protein nitration. We identified depleted PK-M2 (M2 isoform of pyruvate kinase) activity and apoptosis of NPCs revealed by the genomic DNA fragmentation and elevated PARP (poly ADP ribose polymerase) activity along with increased Caspase activity initiated by severely depolarised mitochondrial membranes. However, the pre-treatment of Quercetin in a dose-response manner prevented these changes and restored the expansion of neurospheres preferably by neutralizing the oxidative conditions and thereby reducing peroxynitrite formation, protein nitration and PK-M2 depletion. Our results unravel the potential interactions of oxidative environment and respiration in the survival and activation of precursors and offer a promise shown by a natural flavonoid in the protective strategy for neuronal precursors of adult brain. Copyright © 2013 Elsevier Inc. All rights reserved.

Acute lymphoblastic leukemia: a comprehensive review and 2017 update

PubMed Central

Terwilliger, T; Abdul-Hay, M

2017-01-01

Acute lymphoblastic leukemia (ALL) is the second most common acute leukemia in adults, with an incidence of over 6500 cases per year in the United States alone. The hallmark of ALL is chromosomal abnormalities and genetic alterations involved in differentiation and proliferation of lymphoid precursor cells. In adults, 75% of cases develop from precursors of the B-cell lineage, with the remainder of cases consisting of malignant T-cell precursors. Traditionally, risk stratification has been based on clinical factors such age, white blood cell count and response to chemotherapy; however, the identification of recurrent genetic alterations has helped refine individual prognosis and guide management. Despite advances in management, the backbone of therapy remains multi-agent chemotherapy with vincristine, corticosteroids and an anthracycline with allogeneic stem cell transplantation for eligible candidates. Elderly patients are often unable to tolerate such regimens and carry a particularly poor prognosis. Here, we review the major recent advances in the treatment of ALL. PMID:28665419

Mathematical modeling provides kinetic details of the human immune response to vaccination

PubMed Central

Le, Dustin; Miller, Joseph D.; Ganusov, Vitaly V.

2015-01-01

With major advances in experimental techniques to track antigen-specific immune responses many basic questions on the kinetics of virus-specific immunity in humans remain unanswered. To gain insights into kinetics of T and B cell responses in human volunteers we combined mathematical models and experimental data from recent studies employing vaccines against yellow fever and smallpox. Yellow fever virus-specific CD8 T cell population expanded slowly with the average doubling time of 2 days peaking 2.5 weeks post immunization. Interestingly, we found that the peak of the yellow fever-specific CD8 T cell response was determined by the rate of T cell proliferation and not by the precursor frequency of antigen-specific cells as has been suggested in several studies in mice. We also found that while the frequency of virus-specific T cells increased slowly, the slow increase could still accurately explain clearance of yellow fever virus in the blood. Our additional mathematical model described well the kinetics of virus-specific antibody-secreting cell and antibody response to vaccinia virus in vaccinated individuals suggesting that most of antibodies in 3 months post immunization were derived from the population of circulating antibody-secreting cells. Taken together, our analysis provided novel insights into mechanisms by which live vaccines induce immunity to viral infections and highlighted challenges of applying methods of mathematical modeling to the current, state-of-the-art yet limited immunological data. PMID:25621280

Mathematical modeling provides kinetic details of the human immune response to vaccination.

PubMed

Le, Dustin; Miller, Joseph D; Ganusov, Vitaly V

2014-01-01

With major advances in experimental techniques to track antigen-specific immune responses many basic questions on the kinetics of virus-specific immunity in humans remain unanswered. To gain insights into kinetics of T and B cell responses in human volunteers we combined mathematical models and experimental data from recent studies employing vaccines against yellow fever and smallpox. Yellow fever virus-specific CD8 T cell population expanded slowly with the average doubling time of 2 days peaking 2.5 weeks post immunization. Interestingly, we found that the peak of the yellow fever-specific CD8 T cell response was determined by the rate of T cell proliferation and not by the precursor frequency of antigen-specific cells as has been suggested in several studies in mice. We also found that while the frequency of virus-specific T cells increased slowly, the slow increase could still accurately explain clearance of yellow fever virus in the blood. Our additional mathematical model described well the kinetics of virus-specific antibody-secreting cell and antibody response to vaccinia virus in vaccinated individuals suggesting that most of antibodies in 3 months post immunization were derived from the population of circulating antibody-secreting cells. Taken together, our analysis provided novel insights into mechanisms by which live vaccines induce immunity to viral infections and highlighted challenges of applying methods of mathematical modeling to the current, state-of-the-art yet limited immunological data.

Converging roads: evidence for an adult hemangioblast.

PubMed

Bailey, Alexis S; Fleming, William H

2003-11-01

Classical studies of the developing embryo first suggested the existence of the hemangioblast, a precursor cell with the potential to differentiate into both blood and blood vessels. Several lines of investigation demonstrated that many of the genes activated during early hematopoietic development are also expressed in the vascular endothelium. Gene-targeting studies using embryonic stem cells have identified Flk-1, SCL, and Runx-1 as important regulatory molecules that specify both hematopoietic and vascular outcomes. Although it was anticipated that the hemangioblast would be present only during the earliest stages of vascular development in the yolk sac, accumulating evidence now indicates that hematopoietic cells with hemangioblast activity persist into adulthood. In the adult, bone marrow-derived, circulating endothelial progenitors contribute to postnatal neovascularization and enhance vascular repair following ischemic injury. Highly purified populations of hematopoietic stem cells from humans and mice can differentiate into both blood cells and vascular tissue at the single cell level. These recent findings suggest that bone marrow-derived hematopoietic stem cells or their progeny may contribute to the maintenance and repair of both the hematopoietic and the vascular systems during adult life.

Concise review: preleukemic stem cells: molecular biology and clinical implications of the precursors to leukemia stem cells.

PubMed

Pandolfi, Ashley; Barreyro, Laura; Steidl, Ulrich

2013-02-01

Recent experimental evidence has shown that acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) arise from transformed immature hematopoietic cells following the accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells and committed progenitors. The series of transforming events initially gives rise to preleukemic stem cells (pre-LSC), preceding the formation of fully transformed leukemia stem cells (LSC). Despite the established use of poly-chemotherapy, relapse continues to be the most common cause of death in AML and MDS. The therapeutic elimination of all LSC, as well as pre-LSC, which provide a silent reservoir for the re-formation of LSC, will be essential for achieving lasting cures. Conventional sequencing and next-generation genome sequencing have allowed us to describe many of the recurrent mutations in the bulk cell populations in AML and MDS, and recent work has also focused on identifying the initial molecular changes contributing to leukemogenesis. Here we review recent and ongoing advances in understanding the roles of pre-LSC, and the aberrations that lead to pre-LSC formation and subsequent LSC transformation.

Dopant ink composition and method of fabricating a solar cell there from

DOEpatents

Loscutoff, Paul; Wu, Kahn; Molesa, Steven Edward

2017-10-25

Dopant ink compositions and methods of fabricating solar cells there from are described. A dopant ink composition may include a cross-linkable matrix precursor, a bound dopant species, and a solvent. A method of fabricating a solar cell may include delivering a dopant ink composition to a region above a substrate. The dopant ink composition includes a cross-linkable matrix precursor, a bound dopant species, and a solvent. The method also includes baking the dopant ink composition to remove a substantial portion of the solvent of the dopant ink composition, curing the baked dopant ink composition to cross-link a substantial portion of the cross-linkable matrix precursor of the dopant ink composition, and driving dopants from the cured dopant ink composition toward the substrate.

Dopant ink composition and method of fabricating a solar cell there from

DOEpatents

Loscutoff, Paul; Wu, Kahn; Molesa, Steven Edward

2015-03-31

Dopant ink compositions and methods of fabricating solar cells there from are described. A dopant ink composition may include a cross-linkable matrix precursor, a bound dopant species, and a solvent. A method of fabricating a solar cell may include delivering a dopant ink composition to a region above a substrate. The dopant ink composition includes a cross-linkable matrix precursor, a bound dopant species, and a solvent. The method also includes baking the dopant ink composition to remove a substantial portion of the solvent of the dopant ink composition, curing the baked dopant ink composition to cross-link a substantial portion of the cross-linkable matrix precursor of the dopant ink composition, and driving dopants from the cured dopant ink composition toward the substrate.

Co-occurrence of biphenotypic acute leukaemia, glucose 6-phosphate dehydrogenase deficiency and haemoglobin E trait in a single child.

PubMed

Mallick, Debkrishna; Thapa, Rajoo; Biswas, Biswajit

2016-02-01

Acute leukaemias occur as the result of clonal expansion subsequent to transformation and arrest at a normal differentiation stage of haematopoietic precursors, which commit to a single lineage, such as myeloid or B-lymphoid or T-lymphoid cells. Biphenotypic acute leukaemia (BAL) constitutes a biologically different group of leukaemia arising from a precursor stem cell and co-expressing more than one lineage specific marker. The present report describes a child with unusual co-occurrence of biphenotypic (B-precursor cell and Myeloid) acute leukaemia, haemoglobin E trait and glucose 6-phosphate dehydrogenase (G6-PD) deficiency. To the best of our knowledge, this constellation of haematological conditions in a single child has never been described before. 2016 BMJ Publishing Group Ltd.

Navigating novel mechanisms of cellular plasticity with the NAD+ precursor and nutrient nicotinamide.

PubMed

Li, Faqi; Chong, Zhao Zhong; Maiese, Kenneth

2004-09-01

Interest in neuroprotectants for the central nervous system continues to garner significant attention. Nicotinamide, the amide form of niacin (vitamin B3), is the precursor for the coenzyme beta-nicotinamide adenine dinucleotide (NAD+) and is considered to be necessary for cellular function and metabolism. However, recent work has focused on the development of nicotinamide as a novel agent that is critical for modulating cellular plasticity, longevity, and inflammatory microglial function. The ability of nicotinamide to preserve both neuronal and vascular cell populations in the brain during injury is intriguing, but further knowledge of the specific cellular mechanisms that determine protection by this agent is required. The capacity of nicotinamide to govern not only intrinsic cellular integrity, but also extrinsic cellular inflammation rests with the modulation of a host of cellular targets that involve protein kinase B, glycogen synthase kinase-3 beta (GSK-3 beta), Forkhead transcription factors, mitochondrial dysfunction, poly(ADP-ribose) polymerase, cysteine proteases, and microglial activation. Intimately tied to the cytoprotection of nicotinamide is the modulation of an early and late phase of apoptotic injury that is triggered by the loss of membrane asymmetry. Identifying robust cytoprotective agents as nicotinamide in conjunction with the elucidation of the cellular mechanisms responsible for cell survival will continue to solidify the development of therapeutic strategies against neurodegenerative diseases

Large Population-Based Study Reveals Disparities in Myeloma Precursor Disease | Center for Cancer Research

Cancer.gov

Multiple myeloma (MM) is a cancer of plasma cells, which are antibody-producing white blood cells. Patients with MM have a characteristic excess of monoclonal antibodies, so called M proteins, in their serum, urine, or both and plasma cell infiltration into their bone marrow at multiple sites. African Americans are more than twice as likely as whites to develop MM, but the reason for this higher prevalence is not entirely clear. Since MM is nearly always preceded by the premalignant condition monoclonal gammopathy of undetermined significance (MGUS), Ola Landgren, M.D., Ph.D., a Senior Investigator in CCR’s Lymphoid Malignancies Branch, and colleagues from NCI’s Division of Cancer Epidemiology and Genetics, the Mayo Clinic, and the Centers for Disease Control and Prevention (CDC), wanted to determine whether there were also disparities in MGUS prevalence or in biomarkers associated with a high risk of MGUS progression to MM.

The compartmentation of phosphorylated thiamine derivatives in cultured neuroblastoma cells.

PubMed

Bettendorff, L

1994-05-26

Thiamine transport in cultured neuroblastoma cells is mediated by a high-affinity carrier (KM = 40 nM). In contrast, the uptake of the more hydrophobic sulbutiamine (isobutyrylthiamine disulfide) is unsaturable and its initial transport rate is 20-times faster than for thiamine. In the cytoplasm, sulbutiamine is rapidly hydrolyzed and reduced to free thiamine, the overall process resulting in a rapid and concentrative thiamine accumulation. Incorporation of radioactivity from [14C]thiamine or [14C]sulbutiamine into intracellular thiamine diphosphate is slow in both cases. Despite the fact that the diphosphate is probably the direct precursor for both thiamine monophosphate and triphosphate, the specific radioactivity increased much faster for the latter two compounds than for thiamine diphosphate. This suggests the existence of two pools of thiamine diphosphate, the larger one having a very slow turnover (about 17 h); a much smaller, rapidly turning over pool would be the precursor of thiamine mono- and triphosphate. The turnover time for thiamine triphosphate could be estimated to be 1-2 h. When preloading the cells with [14C]sulbutiamine was followed by a chase with the same concentration of the unlabeled compound, the specific radioactivities of thiamine and thiamine monophosphate decreased exponentially as expected, but labeling of the diphosphate continued to increase slowly. Specific radioactivity of thiamine triphosphate increased first, but after 30 min it began to slowly decrease. These results show for the first time the existence of distinct thiamine diphosphate pools in the same homogeneous cell population. They also suggest a complex compartmentation of thiamine metabolism.

Competition among gene regulatory networks imposes order within the eye-antennal disc of Drosophila

PubMed Central

Weasner, Bonnie M.; Kumar, Justin P.

2013-01-01

The eye-antennal disc of Drosophila gives rise to numerous adult tissues, including the compound eyes, ocelli, antennae, maxillary palps and surrounding head capsule. The fate of each tissue is governed by the activity of unique gene regulatory networks (GRNs). The fate of the eye, for example, is controlled by a set of fourteen interlocking genes called the retinal determination (RD) network. Mutations within network members lead to replacement of the eyes with head capsule. Several studies have suggested that in these instances all retinal progenitor and precursor cells are eliminated via apoptosis and as a result the surrounding head capsule proliferates to compensate for retinal tissue loss. This model implies that the sole responsibility of the RD network is to promote the fate of the eye. We have re-analyzed eyes absent mutant discs and propose an alternative model. Our data suggests that in addition to promoting an eye fate the RD network simultaneously functions to actively repress GRNs that are responsible for directing antennal and head capsule fates. Compromising the RD network leads to the inappropriate expression of several head capsule selector genes such as cut, Lim1 and wingless. Instead of undergoing apoptosis, a population of mutant retinal progenitors and precursor cells adopt a head capsule fate. This transformation is accompanied by an adjustment of cell proliferation rates such that just enough head capsule is generated to produce an intact adult head. We propose that GRNs simultaneously promote primary fates, inhibit alternative fates and establish cell proliferation states. PMID:23222441

Development of polyimide foams with blowing agents

NASA Technical Reports Server (NTRS)

Gagliani, John (Inventor); Sorathia, Usman A. K. (Inventor); Lee, Raymond (Inventor)

1985-01-01

A method of preparing a polyimide foam which includes the steps of: preparing, foaming, and curing a precursor containing at least one alkyl ester of 3,3'4,4'-benzophenonetetracarboxylic acid; a meta- or para-substituted aromatic diamine; a heterocyclic diamine; an aliphatic diamine; and a solid blowing agent. The blowing agent is added to said precursor in a concentration which is sufficient to effect at least one of the following attributes of the foam: cell size, proportion of open cells, cell density, and indentation load deflection.

Molecular basis of autosomal dominant neurohypophyseal diabetes insipidus. Cellular toxicity caused by the accumulation of mutant vasopressin precursors within the endoplasmic reticulum.

PubMed Central

Ito, M; Jameson, J L; Ito, M

1997-01-01

Mutations in the arginine vasopressin (AVP) gene cause autosomal dominant familial neurohypophyseal diabetes insipidus (FNDI). The dominant inheritance pattern has been postulated to reflect neuronal toxicity of the mutant proteins, but the mechanism for such cytotoxicity is unknown. In this study, wild-type or several different mutant AVP genes were stably expressed in neuro2A neuroblastoma cells. When cells were treated with valproic acid to induce neuronal differentiation, each of the mutants caused reduced viability. Metabolic labeling revealed diminished intracellular trafficking of mutant AVP precursors and confirmed inefficient secretion of immunoreactive AVP. Immunofluorescence studies demonstrated marked accumulation of mutant AVP precursors within the endoplasmic reticulum. These studies suggest that the cellular toxicity in FNDI may be caused by the intracellular accumulation of mutant precursor proteins. PMID:9109434

Subtypes of medulloblastoma have distinct developmental origins

PubMed Central

Gibson, Paul; Tong, Yiai; Robinson, Giles; Thompson, Margaret C.; Currle, D. Spencer; Eden, Christopher; Kranenburg, Tanya A.; Hogg, Twala; Poppleton, Helen; Martin, Julie; Finkelstein, David; Pounds, Stanley; Weiss, Aaron; Patay, Zoltan; Scoggins, Matthew; Ogg, Robert; Pei, Yanxin; Yang, Zeng-Jie; Brun, Sonja; Lee, Youngsoo; Zindy, Frederique; Lindsey, Janet C.; Taketo, Makoto M.; Boop, Frederick A.; Sanford, Robert A.; Gajjar, Amar; Clifford, Steven C.; Roussel, Martine F.; McKinnon, Peter J.; Gutmann, David H.; Ellison, David W.; Wechsler-Reya, Robert; Gilbertson, Richard J.

2010-01-01

Medulloblastoma encompasses a collection of clinically and molecularly diverse tumor subtypes that together comprise the most common malignant childhood brain tumor1–4. These tumors are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) following aberrant activation of the Sonic Hedgehog pathway (hereafter, SHH-subtype)3–8. The pathological processes that drive heterogeneity among the other medulloblastoma subtypes are not known, hindering the development of much needed new therapies. Here, we provide evidence that a discrete subtype of medulloblastoma that contains activating mutations in the WNT pathway effector CTNNB1 (hereafter, WNT-subtype)1,3,4, arises outside the cerebellum from cells of the dorsal brainstem. We found that genes marking human WNT-subtype medulloblastomas are more frequently expressed in the lower rhombic lip (LRL) and embryonic dorsal brainstem than in the upper rhombic lip (URL) and developing cerebellum. Magnetic resonance imaging (MRI) and intra-operative reports showed that human WNT-subtype tumors infiltrate the dorsal brainstem, while SHH-subtype tumors are located within the cerebellar hemispheres. Activating mutations in Ctnnb1 had little impact on progenitor cell populations in the cerebellum, but caused the abnormal accumulation of cells on the embryonic dorsal brainstem that included aberrantly proliferating Zic1+ precursor cells. These lesions persisted in all mutant adult mice and in 15% of cases in which Tp53 was concurrently deleted, progressed to form medulloblastomas that recapitulated the anatomy and gene expression profiles of human WNT-subtype medulloblastoma. We provide the first evidence that subtypes of medulloblastoma have distinct cellular origins. Our data provide an explanation for the marked molecular and clinical differences between SHH and WNT-subtype medulloblastomas and have profound implications for future research and treatment of this important childhood cancer. PMID:21150899

Gamma Radiation Induces Micronucleated Reticulocytes in 3-D Bone Marrow Bioreactors in Vitro

PubMed Central

Sun, Hongliang; Dertinger, Stephen D.; Hyrien, Ollivier; David Wu, J. H.; Chen, Yuhchyau

2009-01-01

Radiation injury to the bone marrow is potentially lethal due to the potent DNA-damaging effects on cells of the hematopoietic system, including bone marrow stem cell, progenitor, and the precursor cell populations. Investigation of radiation genotoxic effects on bone marrow progenitor/precursor cells has been challenged by the lack of optimal in vitro surrogate organ culture systems, and the overall difficulty to sustain lineage-specific proliferation and differentiation of hematopoiesis in vitro. We report the investigation of radiation genotoxic effects in bone marrow cultures of C57Bl/6 mice established in 3-D bioreactors, which sustain long-term bone marrow cultures. For these studies, genotoxicity is measured by the induction of micronucleated reticulocytes (MN-RET). The kinetics and dose-response relationship of MN-RET induction in response to gamma-radiation of bioreactor-maintained bone marrow cultures are presented. Our data showed that 3-D long-term bone marrow cultures had sustained erythropoiesis capable of generating reticulocytes up to 8 weeks. The peak time-interval of viable cell output and percentage of reticulocytes increased steadily and reached the initial peak between the 14th to 21st days after inoculations. This was followed by a rebound or staying relatively constant until week 8. The percentage of MN-RET reached the maximum between 24 and 32 hours post 1 Gy gamma-ray. There was a near linear MN-RET induction by gamma radiation from 0 Gy to 1.0 Gy, followed by an attenuated increase to 1.5 – 2.0 Gy. The MN-RET response showed a downtrend beyond 2 Gy. Our data suggest that bone marrow culture in the 3-D bioreactor may be a useful organ culture system for the investigation of radiation genotoxic effect in vitro. PMID:19786117

ABCA1-dependent sterol release: sterol molecule specificity and potential membrane domain for HDL biogenesis

PubMed Central

Yamauchi, Yoshio; Yokoyama, Shinji; Chang, Ta-Yuan

2016-01-01

Mammalian cells synthesize various sterol molecules, including the C30 sterol, lanosterol, as cholesterol precursors in the endoplasmic reticulum. The build-up of precursor sterols, including lanosterol, displays cellular toxicity. Precursor sterols are found in plasma HDL. How these structurally different sterols are released from cells is poorly understood. Here, we show that newly synthesized precursor sterols arriving at the plasma membrane (PM) are removed by extracellular apoA-I in a manner dependent on ABCA1, a key macromolecule for HDL biogenesis. Analysis of sterol molecules by GC-MS and tracing the fate of radiolabeled acetate-derived sterols in normal and mutant Niemann-Pick type C cells reveal that ABCA1 prefers newly synthesized sterols, especially lanosterol, as the substrates before they are internalized from the PM. We also show that ABCA1 resides in a cholesterol-rich membrane domain resistant to the mild detergent, Brij 98. Blocking ACAT activity increases the cholesterol contents of this domain. Newly synthesized C29/C30 sterols are transiently enriched within this domain, but rapidly disappear from this domain with a half-life of less than 1 h. Our work shows that substantial amounts of precursor sterols are transported to a certain PM domain and are removed by the ABCA1-dependent pathway. PMID:26497474

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

PubMed Central

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

2013-01-01

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

Bioreactor Expansion of Skin-Derived Precursor Schwann Cells.

PubMed

Walsh, Tylor; Biernaskie, Jeff; Midha, Rajiv; Kallos, Michael S

2016-01-01

Scaling up the production of cells in a culture process is a critical step when trying to develop cell-based regenerative therapies. Static cultures often cannot be easily scaled up to clinically relevant cell numbers. Alternatively, bioreactors offer a highly valuable means to develop a clinical-ready process. To culture adherent cells in suspension, such as skin-derived precursor Schwann cells (SKP-SCs), microcarriers need to be used. Microcarriers are small spherical beads suspended within the vessel that allow for higher growth surface area to volume ratio. Here we describe the procedure of combining microcarriers with the controllability of bioreactors to generate higher cell densities in smaller reactor volumes leading to a more efficient and cost-effective cell production for applications in regenerative medicine.

Rhomboid Enhancer Activity Defines a Subset of Drosophila Neural Precursors Required for Proper Feeding, Growth and Viability

PubMed Central

Gresser, Amy L.; Gutzwiller, Lisa M.; Gauck, Mackenzie K.; Hartenstein, Volker; Cook, Tiffany A.; Gebelein, Brian

2015-01-01

Organismal growth regulation requires the interaction of multiple metabolic, hormonal and neuronal pathways. While the molecular basis for many of these are well characterized, less is known about the developmental origins of growth regulatory structures and the mechanisms governing control of feeding and satiety. For these reasons, new tools and approaches are needed to link the specification and maturation of discrete cell populations with their subsequent regulatory roles. In this study, we characterize a rhomboid enhancer element that selectively labels four Drosophila embryonic neural precursors. These precursors give rise to the hypopharyngeal sensory organ of the peripheral nervous system and a subset of neurons in the deutocerebral region of the embryonic central nervous system. Post embryogenesis, the rhomboid enhancer is active in a subset of cells within the larval pharyngeal epithelium. Enhancer-targeted toxin expression alters the morphology of the sense organ and results in impaired larval growth, developmental delay, defective anterior spiracle eversion and lethality. Limiting the duration of toxin expression reveals differences in the critical periods for these effects. Embryonic expression causes developmental defects and partially penetrant pre-pupal lethality. Survivors of embryonic expression, however, ultimately become viable adults. In contrast, post-embryonic toxin expression results in fully penetrant lethality. To better define the larval growth defect, we used a variety of assays to demonstrate that toxin-targeted larvae are capable of locating, ingesting and clearing food and they exhibit normal food search behaviors. Strikingly, however, following food exposure these larvae show a rapid decrease in consumption suggesting a satiety-like phenomenon that correlates with the period of impaired larval growth. Together, these data suggest a critical role for these enhancer-defined lineages in regulating feeding, growth and viability. PMID:26252385

Immaturity associated antigens are lost during induction for T cell lymphoblastic leukemia: implications for minimal residual disease detection

PubMed Central

Roshal, Mikhail; Fromm, Jonathan R; Winter, Stuart; Dunsmore, Kimberly; Wood, Brent

2011-01-01

Background Induction chemotherapy for acute leukemia often leads to antigenic shifts in residual abnormal blast populations. Studies in precursor B cell ALL (B-ALL) and AML have demonstrated that chemotherapy commonly results in the loss of antigens associated with immaturity, limiting their utility for minimal residual disease (MRD) detection. Little information is available about the stability of these antigens in precursor T cell ALL (T-ALL) though it is presumed that CD99 and TdT are highly informative based on limited studies. Methods In a longitudinal investigation, we explored patterns of lineage specific and immaturity associated antigens in T-ALL in a large cohort of patients treated under the multicenter Children's Oncology Group (COG) protocol. All samples were analyzed using multicolor flow cytometry in a standardized fashion at a single institution. Results We report that markers of immaturity particularly, TdT and CD99 dramatically decline on leukemic blasts during therapy. CD34 and CD10 expression is confined to a minority of pre-treatment samples and is also not stable. In contrast, lineage associated markers including CD2, CD3, CD4, CD5, CD7 and CD8 failed to show significant trends. Conclusions Our study strongly argues for expansion of immunophenotyping panels for T-ALL MRD to decrease reliance on immature antigens. This study represents the first demonstration of consistent immunophenotypic shifts in T-ALL. PMID:20155852

Perovskite ink with wide processing window for scalable high-efficiency solar cells

DOE PAGES

Yang, Mengjin; Li, Zhen; Reese, Matthew O.; ...

2017-03-20

Perovskite solar cells have made tremendous progress using laboratory-scale spin-coating methods in the past few years owing to advances in controls of perovskite film deposition. However, devices made via scalable methods are still lagging behind state-of-the-art spin-coated devices because of the complicated nature of perovskite crystallization from a precursor state. Here we demonstrate a chlorine-containing methylammonium lead iodide precursor formulation along with solvent tuning to enable a wide precursor-processing window (up to ~8 min) and a rapid grain growth rate (as short as ~1 min). Coupled with antisolvent extraction, this precursor ink delivers high-quality perovskite films with large-scale uniformity. Themore » ink can be used by both spin-coating and blade-coating methods with indistinguishable film morphology and device performance. Using a blade-coated absorber, devices with 0.12-cm 2 and 1.2-cm 2 areas yield average efficiencies of 18.55% and 17.33%, respectively. As a result, we further demonstrate a 12.6-cm 2 four-cell module (88% geometric fill factor) with 13.3% stabilized active-area efficiency output.« less

Amyloid-like aggregation of provasopressin in diabetes insipidus and secretory granule sorting.

PubMed

Beuret, Nicole; Hasler, Franziska; Prescianotto-Baschong, Cristina; Birk, Julia; Rutishauser, Jonas; Spiess, Martin

2017-01-26

Aggregation of peptide hormone precursors in the trans-Golgi network is an essential process in the biogenesis of secretory granules in endocrine cells. It has recently been proposed that this aggregation corresponds to the formation of functional amyloids. Our previous finding that dominant mutations in provasopressin, which cause cell degeneration and diabetes insipidus, prevent native folding and produce fibrillar aggregates in the endoplasmic reticulum (ER) might thus reflect mislocalized amyloid formation by sequences that evolved to mediate granule sorting. Here we identified two sequences responsible for fibrillar aggregation of mutant precursors in the ER: the N-terminal vasopressin nonapeptide and the C-terminal glycopeptide. To test their role in granule sorting, the glycopeptide was deleted and/or vasopressin mutated to inactivate ER aggregation while still permitting precursor folding and ER exit. These mutations strongly reduced sorting into granules and regulated secretion in endocrine AtT20 cells. The same sequences - vasopressin and the glycopeptide - mediate physiological aggregation of the wild-type hormone precursor into secretory granules and the pathological fibrillar aggregation of disease mutants in the ER. These findings support the amyloid hypothesis for secretory granule biogenesis.

Protein secretion and surface display in Gram-positive bacteria

PubMed Central

Schneewind, Olaf; Missiakas, Dominique M.

2012-01-01

The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions. PMID:22411983

Ca-P spots modified zirconia by liquid precursor infiltration and the effect on osteoblast-like cell responses.

PubMed

Li, Yongmei; Liu, Yan; Zhang, Zutai; Zhuge, Ruishen; Ding, Ning; Tian, Yueming

2018-01-26

Ca-P spots modified zirconia by liquid precursor infiltration and the cell responses were investigated. Pre-sintered zirconia specimens were immersed in Ca-P precursor solution. After dense sintering, scanning electron microscopy showed Ca-P spots were formed on the zirconia and anchored with zirconia substrates. The distribution density was increased with the extension of immersion time. Energy dispersive spectrometer confirmed the stoichiometric Ca/P ratio was about 1.67. After hydrothermal treatment, Ca-P spots turned into rod crystals where diffraction peaks of tricalcium phosphate and hydroxyapatite were detected by X-ray diffraction, and Ca 2+ and PO 4 3- release decreased slightly (p>0.05). There was no significant decrease on three-point bending strength (p>0.05). Osteoblast-like MC3T3-E1 cells attached and spread well and showed higher proliferation on Ca-P spots modified zirconia (p<0.05), though its initial alkaline phosphatase activity was not significant high (p>0.05). In conclusion, Ca-P liquid precursor infiltration is a potential method to modify the zirconia ceramics for improving bioactivity.

Perovskite ink with wide processing window for scalable high-efficiency solar cells

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

Yang, Mengjin; Li, Zhen; Reese, Matthew O.

Perovskite solar cells have made tremendous progress using laboratory-scale spin-coating methods in the past few years owing to advances in controls of perovskite film deposition. However, devices made via scalable methods are still lagging behind state-of-the-art spin-coated devices because of the complicated nature of perovskite crystallization from a precursor state. Here we demonstrate a chlorine-containing methylammonium lead iodide precursor formulation along with solvent tuning to enable a wide precursor-processing window (up to ~8 min) and a rapid grain growth rate (as short as ~1 min). Coupled with antisolvent extraction, this precursor ink delivers high-quality perovskite films with large-scale uniformity. Themore » ink can be used by both spin-coating and blade-coating methods with indistinguishable film morphology and device performance. Using a blade-coated absorber, devices with 0.12-cm 2 and 1.2-cm 2 areas yield average efficiencies of 18.55% and 17.33%, respectively. As a result, we further demonstrate a 12.6-cm 2 four-cell module (88% geometric fill factor) with 13.3% stabilized active-area efficiency output.« less

Recent thymic emigrants and mature naïve T cells exhibit differential DNA methylation at key cytokine loci

PubMed Central

Berkley, Amy M.; Hendricks, Deborah W.; Simmons, Kalynn B.; Fink, Pamela J.

2013-01-01

Recent thymic emigrants (RTEs) are the youngest T cells in the lymphoid periphery, and exhibit phenotypic and functional characteristics distinct from those of their more mature counterparts in the naïve peripheral T cell pool. We show here that the Il2 and Il4 promoter regions of naïve CD4+ RTEs are characterized by site-specific hypermethylation compared to those of both mature naïve (MN) T cells and the thymocyte precursors of RTEs. Thus, RTEs do not merely occupy a midpoint between the thymus and the mature T cell pool, but represent a distinct transitional T cell population. Furthermore, RTEs and MN T cells exhibit distinct CpG DNA methylation patterns both before and after activation. Compared to MN T cells, RTEs express higher levels of several enzymes that modify DNA methylation, and inhibiting methylation during culture allows RTEs to reach MN T cell levels of cytokine production. Collectively, these data suggest that the functional differences that distinguish RTEs from MN T cells are influenced by epigenetic mechanisms and provide clues to a mechanistic basis for post-thymic maturation. PMID:23686491

Pathways to Mathematics: Longitudinal Predictors of Performance

ERIC Educational Resources Information Center

LeFevre, Jo-Anne; Fast, Lisa; Skwarchuk, Sheri-Lynn; Smith-Chant, Brenda L.; Bisanz, Jeffrey; Kamawar, Deepthi; Penner-Wilger, Marcie

2010-01-01

A model of the relations among cognitive precursors, early numeracy skill, and mathematical outcomes was tested for 182 children from 4.5 to 7.5 years of age. The model integrates research from neuroimaging, clinical populations, and normal development in children and adults. It includes 3 precursor pathways: quantitative, linguistic, and spatial…

Myc and Fgf Are Required for Zebrafish Neuromast Hair Cell Regeneration.

PubMed

Lee, Sang Goo; Huang, Mingqian; Obholzer, Nikolaus D; Sun, Shan; Li, Wenyan; Petrillo, Marco; Dai, Pu; Zhou, Yi; Cotanche, Douglas A; Megason, Sean G; Li, Huawei; Chen, Zheng-Yi

2016-01-01

Unlike mammals, the non-mammalian vertebrate inner ear can regenerate the sensory cells, hair cells, either spontaneously or through induction after hair cell loss, leading to hearing recovery. The mechanisms underlying the regeneration are poorly understood. By microarray analysis on a chick model, we show that chick hair cell regeneration involves the activation of proliferation genes and downregulation of differentiation genes. Both MYC and FGF are activated in chick hair cell regeneration. Using a zebrafish lateral line neuromast hair cell regeneration model, we show that the specific inhibition of Myc or Fgf suppresses hair cell regeneration, demonstrating that both pathways are essential to the process. Rapid upregulation of Myc and delayed Fgf activation during regeneration suggest a role of Myc in proliferation and Fgf in differentiation. The dorsal-ventral pattern of fgfr1a in the neuromasts overlaps with the distribution of hair cell precursors. By laser ablation, we show that the fgfr1a-positive supporting cells are likely the hair cell precursors that directly give rise to new hair cells; whereas the anterior-posterior fgfr1a-negative supporting cells have heightened proliferation capacity, likely to serve as more primitive progenitor cells to replenish lost precursors after hair cell loss. Thus fgfr1a is likely to mark compartmentalized supporting cell subtypes with different capacities in renewal proliferation and hair cell regeneration. Manipulation of c-MYC and FGF pathways could be explored for mammalian hair cell regeneration.

Myc and Fgf Are Required for Zebrafish Neuromast Hair Cell Regeneration

PubMed Central

Obholzer, Nikolaus D.; Sun, Shan; Li, Wenyan; Petrillo, Marco; Dai, Pu; Zhou, Yi; Cotanche, Douglas A.; Megason, Sean G.; Li, Huawei; Chen, Zheng-Yi

2016-01-01

Unlike mammals, the non-mammalian vertebrate inner ear can regenerate the sensory cells, hair cells, either spontaneously or through induction after hair cell loss, leading to hearing recovery. The mechanisms underlying the regeneration are poorly understood. By microarray analysis on a chick model, we show that chick hair cell regeneration involves the activation of proliferation genes and downregulation of differentiation genes. Both MYC and FGF are activated in chick hair cell regeneration. Using a zebrafish lateral line neuromast hair cell regeneration model, we show that the specific inhibition of Myc or Fgf suppresses hair cell regeneration, demonstrating that both pathways are essential to the process. Rapid upregulation of Myc and delayed Fgf activation during regeneration suggest a role of Myc in proliferation and Fgf in differentiation. The dorsal-ventral pattern of fgfr1a in the neuromasts overlaps with the distribution of hair cell precursors. By laser ablation, we show that the fgfr1a-positive supporting cells are likely the hair cell precursors that directly give rise to new hair cells; whereas the anterior-posterior fgfr1a-negative supporting cells have heightened proliferation capacity, likely to serve as more primitive progenitor cells to replenish lost precursors after hair cell loss. Thus fgfr1a is likely to mark compartmentalized supporting cell subtypes with different capacities in renewal proliferation and hair cell regeneration. Manipulation of c-MYC and FGF pathways could be explored for mammalian hair cell regeneration. PMID:27351484

A Major Population of Functional KLRG1- ILC2s in Female Lungs Contributes to a Sex Bias in ILC2 Numbers.

PubMed

Kadel, Sapana; Ainsua-Enrich, Erola; Hatipoglu, Ibrahim; Turner, Sean; Singh, Simar; Khan, Sohaib; Kovats, Susan

2018-02-01

Humans show significant sex differences in the incidence and severity of respiratory diseases, including asthma and virus infection. Sex hormones contribute to the female sex bias in type 2 inflammation associated with respiratory diseases, consistent with recent reports that female lungs harbor greater numbers of GATA-3-dependent group 2 innate lymphoid cells (ILC2s). In this study, we determined whether sex hormone levels govern sex differences in the numbers, phenotype, and function of ILC2s in the murine lung and bone marrow (BM). Our data show that lungs of female mice harbor significantly greater ILC2 numbers in homeostasis, in part due to a major subset of ILC2s lacking killer-cell lectin like receptor G1 (KLRG1), a population largely absent in male lungs. The KLRG1 - ILC2s were capable of type 2 cytokine production and increased with age after sexual maturity, suggesting that a unique functional subset exists in females. Experiments with gonadectomized mice or mice bearing either global or lymphocyte restricted estrogen receptor α ( Esr1 ) deficiency showed that androgens rather than estrogens regulated numbers of the KLRG1 - ILC2 subset and ILC2 functional capacity in the lung and BM, as well as levels of GATA-3 expression in BM ILC2s. Furthermore, the frequency of BM PLZF + ILC precursors was higher in males and increased by excess androgens, suggesting that androgens act to inhibit the transition of ILC precursors to ILC2s. Taken together, these data show that a functional subset of KLRG1 - ILC2s in females contributes to the sex bias in lung ILC2s that is observed after reproductive age.

Nano-sized metabolic precursors for heterogeneous tumor-targeting strategy using bioorthogonal click chemistry in vivo.

PubMed

Lee, Sangmin; Jung, Seulhee; Koo, Heebeom; Na, Jin Hee; Yoon, Hong Yeol; Shim, Man Kyu; Park, Jooho; Kim, Jong-Ho; Lee, Seulki; Pomper, Martin G; Kwon, Ick Chan; Ahn, Cheol-Hee; Kim, Kwangmeyung

2017-12-01

Herein, we developed nano-sized metabolic precursors (Nano-MPs) for new tumor-targeting strategy to overcome the intrinsic limitations of biological ligands such as the limited number of biological receptors and the heterogeneity in tumor tissues. We conjugated the azide group-containing metabolic precursors, triacetylated N-azidoacetyl-d-mannosamine to generation 4 poly(amidoamine) dendrimer backbone. The nano-sized dendrimer of Nano-MPs could generate azide groups on the surface of tumor cells homogeneously regardless of cell types via metabolic glycoengineering. Importantly, these exogenously generated 'artificial chemical receptors' containing azide groups could be used for bioorthogonal click chemistry, regardless of phenotypes of different tumor cells. Furthermore, in tumor-bearing mice models, Nano-MPs could be mainly localized at the target tumor tissues by the enhanced permeation and retention (EPR) effect, and they successfully generated azide groups on tumor cells in vivo after an intravenous injection. Finally, we showed that these azide groups on tumor tissues could be used as 'artificial chemical receptors' that were conjugated to bioorthogonal chemical group-containing liposomes via in vivo click chemistry in heterogeneous tumor-bearing mice. Therefore, overall results demonstrated that our nano-sized metabolic precursors could be extensively applied to new alternative tumor-targeting technique for molecular imaging and drug delivery system, regardless of the phenotype of heterogeneous tumor cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Spermine oxidase, a polyamine catabolic enzyme that links Helicobacter pylori CagA and gastric cancer risk

PubMed Central

Chaturvedi, Rupesh; de Sablet, Thibaut; Peek, Richard M.; Wilson, Keith T.

2012-01-01

We have recently reported that Helicobacter pylori strains expressing the virulence factor cytotoxin-associated gene A (CagA) stimulate increased levels of spermine oxidase (SMO) in gastric epithelial cells, while cagA– strains did not. SMO catabolizes the polyamine spermine and produces H2O2 that results in both apoptosis and DNA damage. Exogenous overexpression of CagA confirmed these findings, and knockdown or inhibition of SMO blocked CagA-mediated apoptosis and DNA damage. The strong association of SMO, apoptosis, and DNA damage was also demonstrated in humans infected with cagA+, but not cagA– strains. In infected gerbils and mice, DNA damage was CagA-dependent and only present in epithelial cells that expressed SMO. We also discovered SMOhigh gastric epithelial cells from infected animals with dysplasia that are resistant to apoptosis despite high levels of DNA damage. Inhibition of polyamine synthesis or SMO could abrogate the development of this cell population that may represent precursors for neoplastic transformation. PMID:22555547

Trained Memory of Human Uterine NK Cells Enhances Their Function in Subsequent Pregnancies.

PubMed

Gamliel, Moriya; Goldman-Wohl, Debra; Isaacson, Batya; Gur, Chamutal; Stein, Natan; Yamin, Rachel; Berger, Michael; Grunewald, Myriam; Keshet, Eli; Rais, Yoach; Bornstein, Chamutal; David, Eyal; Jelinski, Adam; Eisenberg, Iris; Greenfield, Caryn; Ben-David, Arbel; Imbar, Tal; Gilad, Ronit; Haimov-Kochman, Ronit; Mankuta, David; Elami-Suzin, Matan; Amit, Ido; Hanna, Jacob H; Yagel, Simcha; Mandelboim, Ofer

2018-05-15

Natural killer cells (NKs) are abundant in the human decidua, regulating trophoblast invasion and angiogenesis. Several diseases of poor placental development are associated with first pregnancies, so we thus looked to characterize differences in decidual NKs (dNKs) in first versus repeated pregnancies. We discovered a population found in repeated pregnancies, which has a unique transcriptome and epigenetic signature, and is characterized by high expression of the receptors NKG2C and LILRB1. We named these cells Pregnancy Trained decidual NK cells (PTdNKs). PTdNKs have open chromatin around the enhancers of IFNG and VEGFA. Activation of PTdNKs led to increased production and secretion of IFN-γ and VEGFα, with the latter supporting vascular sprouting and tumor growth. The precursors of PTdNKs seem to be found in the endometrium. Because repeated pregnancies are associated with improved placentation, we propose that PTdNKs, which are present primarily in repeated pregnancies, might be involved in proper placentation. Copyright © 2018 Elsevier Inc. All rights reserved.

Expression of homeobox genes Msx-1 (Hox-7) and Msx-2 (Hox-8) during cardiac development in the chick.

PubMed

Chan-Thomas, P S; Thompson, R P; Robert, B; Yacoub, M H; Barton, P J

1993-07-01

The vertebrate homeobox genes Msx-1 and Msx-2 are related to the Drosophila msh gene and are expressed in a variety of tissues during embryogenesis. We have examined their expression by in situ hybridisation during critical stages of cardiac development in the chick from stages 15+ to 37. Msx-1 expression is apparent in a number of non-myocardial cell populations, including cells undergoing an epithelial to mesenchymal transformation in the atrioventricular and the outflow tract regions that play an integral role in heart septation and valve formation. Msx-2 expression is restricted to a distinct subpopulation of myocardial cells that, in later stages, coincides morphologically with the cardiac conduction system. The timing of Msx-2 expression suggests that it plays a role in conduction system tissue formation and that it identifies precursor cells of this specialised myocardium. The pattern of Msx-2 expression is discussed with reference to current models of conduction tissue development.

Multipotent versus differentiated cell fate selection in the developing Drosophila airways

PubMed Central

Matsuda, Ryo; Hosono, Chie; Samakovlis, Christos; Saigo, Kaoru

2015-01-01

Developmental potentials of cells are tightly controlled at multiple levels. The embryonic Drosophila airway tree is roughly subdivided into two types of cells with distinct developmental potentials: a proximally located group of multipotent adult precursor cells (P-fate) and a distally located population of more differentiated cells (D-fate). We show that the GATA-family transcription factor (TF) Grain promotes the P-fate and the POU-homeobox TF Ventral veinless (Vvl/Drifter/U-turned) stimulates the D-fate. Hedgehog and receptor tyrosine kinase (RTK) signaling cooperate with Vvl to drive the D-fate at the expense of the P-fate while negative regulators of either of these signaling pathways ensure P-fate specification. Local concentrations of Decapentaplegic/BMP, Wingless/Wnt, and Hedgehog signals differentially regulate the expression of D-factors and P-factors to transform an equipotent primordial field into a concentric pattern of radially different morphogenetic potentials, which gradually gives rise to the distal-proximal organization of distinct cell types in the mature airway. DOI: http://dx.doi.org/10.7554/eLife.09646.001 PMID:26633813

Oligodendrocyte Precursor Cells Synthesize Neuromodulatory Factors

PubMed Central

Sakry, Dominik; Yigit, Hatice; Dimou, Leda; Trotter, Jacqueline

2015-01-01

NG2 protein-expressing oligodendrocyte progenitor cells (OPC) are a persisting and major glial cell population in the adult mammalian brain. Direct synaptic innervation of OPC by neurons throughout the brain together with their ability to sense neuronal network activity raises the question of additional physiological roles of OPC, supplementary to generating myelinating oligodendrocytes. In this study we investigated whether OPC express neuromodulatory factors, typically synthesized by other CNS cell types. Our results show that OPC express two well-characterized neuromodulatory proteins: Prostaglandin D2 synthase (PTGDS) and neuronal Pentraxin 2 (Nptx2/Narp). Expression levels of the enzyme PTGDS are influenced in cultured OPC by the NG2 intracellular region which can be released by cleavage and localizes to glial nuclei upon transfection. Furthermore PTGDS mRNA levels are reduced in OPC from NG2-KO mouse brain compared to WT cells after isolation by cell sorting and direct analysis. These results show that OPC can contribute to the expression of these proteins within the CNS and suggest PTGDS expression as a downstream target of NG2 signaling. PMID:25966014

An adipoinductive role of inflammation in adipose tissue engineering: key factors in the early development of engineered soft tissues.

PubMed

Lilja, Heidi E; Morrison, Wayne A; Han, Xiao-Lian; Palmer, Jason; Taylor, Caroline; Tee, Richard; Möller, Andreas; Thompson, Erik W; Abberton, Keren M

2013-05-15

Tissue engineering and cell implantation therapies are gaining popularity because of their potential to repair and regenerate tissues and organs. To investigate the role of inflammatory cytokines in new tissue development in engineered tissues, we have characterized the nature and timing of cell populations forming new adipose tissue in a mouse tissue engineering chamber (TEC) and characterized the gene and protein expression of cytokines in the newly developing tissues. EGFP-labeled bone marrow transplant mice and MacGreen mice were implanted with TEC for periods ranging from 0.5 days to 6 weeks. Tissues were collected at various time points and assessed for cytokine expression through ELISA and mRNA analysis or labeled for specific cell populations in the TEC. Macrophage-derived factors, such as monocyte chemotactic protein-1 (MCP-1), appear to induce adipogenesis by recruiting macrophages and bone marrow-derived precursor cells to the TEC at early time points, with a second wave of nonbone marrow-derived progenitors. Gene expression analysis suggests that TNFα, LCN-2, and Interleukin 1β are important in early stages of neo-adipogenesis. Increasing platelet-derived growth factor and vascular endothelial cell growth factor expression at early time points correlates with preadipocyte proliferation and induction of angiogenesis. This study provides new information about key elements that are involved in early development of new adipose tissue.

β-Catenin signaling regulates temporally discrete phases of anterior taste bud development.

PubMed

Thirumangalathu, Shoba; Barlow, Linda A

2015-12-15

The sense of taste is mediated by multicellular taste buds located within taste papillae on the tongue. In mice, individual taste buds reside in fungiform papillae, which develop at mid-gestation as epithelial placodes in the anterior tongue. Taste placodes comprise taste bud precursor cells, which express the secreted factor sonic hedgehog (Shh) and give rise to taste bud cells that differentiate around birth. We showed previously that epithelial activation of β-catenin is the primary inductive signal for taste placode formation, followed by taste papilla morphogenesis and taste bud differentiation, but the degree to which these later elements were direct or indirect consequences of β-catenin signaling was not explored. Here, we define discrete spatiotemporal functions of β-catenin in fungiform taste bud development. Specifically, we show that early epithelial activation of β-catenin, before taste placodes form, diverts lingual epithelial cells from a taste bud fate. By contrast, β-catenin activation a day later within Shh(+) placodes, expands taste bud precursors directly, but enlarges papillae indirectly. Further, placodal activation of β-catenin drives precocious differentiation of Type I glial-like taste cells, but not other taste cell types. Later activation of β-catenin within Shh(+) precursors during papilla morphogenesis also expands taste bud precursors and accelerates Type I cell differentiation, but papilla size is no longer enhanced. Finally, although Shh regulates taste placode patterning, we find that it is dispensable for the accelerated Type I cell differentiation induced by β-catenin. © 2015. Published by The Company of Biologists Ltd.

Simultaneous and Sequential MS/MS Scan Combinations and Permutations in a Linear Quadrupole Ion Trap.

PubMed

Snyder, Dalton T; Szalwinski, Lucas J; Cooks, R Graham

2017-10-17

Methods of performing precursor ion scans as well as neutral loss scans in a single linear quadrupole ion trap have recently been described. In this paper we report methodology for performing permutations of MS/MS scan modes, that is, ordered combinations of precursor, product, and neutral loss scans following a single ion injection event. Only particular permutations are allowed; the sequences demonstrated here are (1) multiple precursor ion scans, (2) precursor ion scans followed by a single neutral loss scan, (3) precursor ion scans followed by product ion scans, and (4) segmented neutral loss scans. (5) The common product ion scan can be performed earlier in these sequences, under certain conditions. Simultaneous scans can also be performed. These include multiple precursor ion scans, precursor ion scans with an accompanying neutral loss scan, and multiple neutral loss scans. We argue that the new capability to perform complex simultaneous and sequential MS n operations on single ion populations represents a significant step in increasing the selectivity of mass spectrometry.

Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

PubMed

Nakayama, T; Munoz, L E; Sadaie, Y; Doi, R H

1978-09-01

Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.

The effect of porous lead iodide precursor film on perovskite film formation and its photovoltaic property after an effective pretreatment

NASA Astrophysics Data System (ADS)

Yan, Jian-Jun; Li, Yan; Chang, Yin; Jiang, Pan; Wang, Cheng-Wei

2016-06-01

An effective solvent sealed natural drying (SND) pretreatment was introduced for forming a satisfactory crystalline porous iodide (PbI2) precursor film, which could help to generate excellent CH3NH3PbI3 perovskite films for high performance of planar heterojunction perovskite solar cells. And the influence of SND pretreated time on the device performance was investigated in detail. We found that the PbI2 precursor film after 10 min pretreatment could make the perovskite device achieve the optimal power conversion efficiency (PCE) of 8.6%, significantly increased up to 95.5% and 28.4% compared to without pretreatment or traditional treatment. The results show that the time of SND pretreatment is critical to forming large grain size and good crystallinity for PbI2 precursor film, which would markedly improve the efficiency of planar heterojunction perovskite solar cells.

Endogenous peptide profile for elucidating biosynthetic processing of the ghrelin precursor.

PubMed

Tsuchiya, Takashi; Iwakura, Hiroshi; Minamino, Naoto; Kangawa, Kenji; Sasaki, Kazuki

2017-09-02

Ghrelin is an orexigenic peptide primarily produced by gastric endocrine cells. The biosynthetic cleavage site of ghrelin has been well documented, but how its downstream region undergoes proteolytic processing remains poorly explored. Here, we provide the first snapshot of endogenous peptides from the ghrelin precursor by profiling the secretopeptidome of cultured mouse ghrelin-producing cells during exocytosis. Mapping of MS/MS sequenced peptides to the precursor highlighted three atypical monobasic processing sites, including the established C-terminus of ghrelin and the N-terminal cleavage site for obestatin, a putative 23-amino-acid C-terminally amidated peptide. However, we found that mouse obestatin does not occur in the form originally reported, but that a different amidation site is used to generate a shorter peptide. These data can be extended to study and characterize the precursor-derived peptides located downstream of ghrelin in different biological contexts. Copyright © 2017 Elsevier Inc. All rights reserved.

Properties of skin stem cells and their potential clinical applications in modern dermatology.

PubMed

Niezgoda, Anna; Niezgoda, Piotr; Nowowiejska, Laura; Białecka, Agnieszka; Męcińska-Jundziłł, Kaja; Adamska, Urszula; Czajkowski, Rafał

2017-06-01

Stem cells play an important role in medical science, and scientists are investing large sums in order to perform sophisticated studies designed to establish potential clinical applications of stem cells. Growing experience has enabled researchers to determine the precise nature of stem cell division. Although the properties of this particular population of cells have been known and used for some time, mainly with regards to bone marrow-derived mesenchymal stem cell transplantation, we now face a significant challenge in implementing the practical use of skin-derived precursors, making it possible to avoid the necessity for patients to undergo invasive procedures in order to obtain stem cells from bone marrow. Multiple trials have so far been performed, bringing hope for the treatment of disorders previously considered untreatable. Patients suffering from a number of dermatological diseases, including malignant melanoma, systemic lupus erythematosus, vitiligo, alopecia or junctional epidermolysis bullosa, may benefit from treatment based on stem cells. The aim of this review is to summarize available data on stem cells and their potential applications in the treatment of dermatological disorders. The work described is based on data published up to the end of September 2016.

Developmental insights from early mammalian embryos and core signaling pathways that influence human pluripotent cell growth and differentiation.

PubMed

Chen, Kevin G; Mallon, Barbara S; Johnson, Kory R; Hamilton, Rebecca S; McKay, Ronald D G; Robey, Pamela G

2014-05-01

Human pluripotent stem cells (hPSCs) have two potentially attractive applications: cell replacement-based therapies and drug discovery. Both require the efficient generation of large quantities of clinical-grade stem cells that are free from harmful genomic alterations. The currently employed colony-type culture methods often result in low cell yields, unavoidably heterogeneous cell populations, and substantial chromosomal abnormalities. Here, we shed light on the structural relationship between hPSC colonies/embryoid bodies and early-stage embryos in order to optimize current culture methods based on the insights from developmental biology. We further highlight core signaling pathways that underlie multiple epithelial-to-mesenchymal transitions (EMTs), cellular heterogeneity, and chromosomal instability in hPSCs. We also analyze emerging methods such as non-colony type monolayer (NCM) and suspension culture, which provide alternative growth models for hPSC expansion and differentiation. Furthermore, based on the influence of cell-cell interactions and signaling pathways, we propose concepts, strategies, and solutions for production of clinical-grade hPSCs, stem cell precursors, and miniorganoids, which are pivotal steps needed for future clinical applications. Published by Elsevier B.V.

An efficient copper phthalocyanine additive of perovskite precursor for improving the photovoltaic performance of planar perovskite solar cells

NASA Astrophysics Data System (ADS)

Wu, Shufang; Liu, Qingwei; Zheng, Ya; Li, Renjie; Peng, Tianyou

2017-08-01

Solution processable planar heterojunction perovskite solar cell has drawn much attention as a promising low-cost photovoltaic device, and much effort has been made to improve its power conversion efficiency by choosing appropriate additives for the perovskite precursor solution. Different to those additives reported, a soluble and thermal stable tert-butyl substituted copper phthalocyanine (CuPc(tBu)4) as additive is first introduced into the perovskite precursor solution of a planar perovskite solar cell that is fabricated via the one-step solution process. It is found that the pristine device without CuPc(tBu)4 additive exhibits a power conversion efficiency of 15.3%, while an extremely low concentration (4.4 × 10-3 mM) of CuPc(tBu)4 in the precursor solution leads to the corresponding device achieving an enhanced power conversion efficiency of 17.3%. CuPc(tBu)4 as an additive can improve the quality of perovskite layer with higher crystallinity and surface coverage, then resulting in enhanced light absorption and reduced charge recombination, and thus the better power conversion efficiency. The finding presented here provides a new choice for improving the quality of perovskite layer and the photovoltaic performance of the planar heterojunction perovskite solar cells.

Lineage analysis of quiescent regenerative stem cells in the adult brain by genetic labelling reveals spatially restricted neurogenic niches in the olfactory bulb.

PubMed

Giachino, Claudio; Taylor, Verdon

2009-07-01

The subventricular zone (SVZ) of the lateral ventricles is the major neurogenic region in the adult mammalian brain, harbouring neural stem cells within defined niches. The identity of these stem cells and the factors regulating their fate are poorly understood. We have genetically mapped a population of Nestin-expressing cells during postnatal development to study their potential and fate in vivo. Taking advantage of the recombination characteristics of a nestin::CreER(T2) allele, we followed a subpopulation of neural stem cells and traced their fate in a largely unrecombined neurogenic niche. Perinatal nestin::CreER(T2)-expressing cells give rise to multiple glial cell types and neurons, as well as to stem cells of the adult SVZ. In the adult SVZ nestin::CreER(T2)-expressing neural stem cells give rise to several neuronal subtypes in the olfactory bulb (OB). We addressed whether the same population of neural stem cells play a role in SVZ regeneration. Following anti-mitotic treatment to eliminate rapidly dividing progenitors, relatively quiescent nestin::CreER(T2)-targeted cells are spared and contribute to SVZ regeneration, generating new proliferating precursors and neuroblasts. Finally, we have identified neurogenic progenitors clustered in ependymal-like niches within the rostral migratory stream (RMS) of the OB. These OB-RMS progenitors generate neuroblasts that, upon transplantation, graft, migrate and differentiate into granule and glomerular neurons. In summary, using conditional lineage tracing we have identified neonatal cells that are the source of neurogenic and regenerative neural stem cells in the adult SVZ and occupy a novel neurogenic niche in the OB.

Two-Photon Microscopy Imaging of thy1GFP-M Transgenic Mice: A Novel Animal Model to Investigate Brain Dendritic Cell Subsets In Vivo

PubMed Central

Laperchia, Claudia; Allegra Mascaro, Anna L.; Sacconi, Leonardo; Andrioli, Anna; Mattè, Alessandro; De Franceschi, Lucia; Grassi-Zucconi, Gigliola; Bentivoglio, Marina; Buffelli, Mario; Pavone, Francesco S.

2013-01-01

Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for in vivo brain studies with two-photon fluorescence (TPF) microscopy. Mice of the thy1GFP-M line have been engineered for selective expression of green fluorescent protein (GFP) in neuronal populations. Here, we report that TPF microscopy reveals, at the brain surface of these mice, also motile non-neuronal GFP+ cells. We have analyzed the behavior of these cells in vivo and characterized in brain sections their immunophenotype. With TPF imaging, motile GFP+ cells were found in the meninges, subarachnoid space and upper cortical layers. The striking feature of these cells was their ability to move across the brain parenchyma, exhibiting evident shape changes during their scanning-like motion. In brain sections, GFP+ cells were immunonegative to antigens recognizing motile cells such as migratory neuroblasts, neuronal and glial precursors, mast cells, and fibroblasts. GFP+ non-neuronal cells exhibited instead the characteristic features and immunophenotype (CD11c and major histocompatibility complex molecule class II immunopositivity) of dendritic cells (DCs), and were immunonegative to the microglial marker Iba-1. GFP+ cells were also identified in lymph nodes and blood of thy1GFP-M mice, supporting their identity as DCs. Thus, TPF microscopy has here allowed the visualization for the first time of the motile behavior of brain DCs in situ. The results indicate that the thy1GFP-M mouse line provides a novel animal model for the study of subsets of these professional antigen-presenting cells in the brain. Information on brain DCs is still very limited and imaging in thy1GFP-M mice has a great potential for analyses of DC-neuron interaction in normal and pathological conditions. PMID:23409142

Molecular dissection of prethymic progenitor entry into the T lymphocyte developmental pathway

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

Fung, Elizabeth-sharon

2008-01-01

Notch signaling activates T lineage differentiation from hemopoietic progenitors, but relatively few regulators that initiate this program have been identified, e.g., GATA3 and T cell factor-I (TCF-1) (gene name Tcli). To identify additional regulators of T cell specification, a cDNA libnlrY from mouse Pro-T cells was screened for genes that are specifically up-regulated in intrathymic T cell precursors as compared with myeloid progenitors. Over 90 genes of interest were identified, and 35 of 44 tested were confirmed to be more highly expressed in T lineage precursors relative to precursors of B and/or myeloid lineage. To a remarkable extent, however, expressionmore » of these T lineage-enriched genes, including zinc finger transcription factor, helicase, and signaling adaptor genes, was also shared by stem cells (Lin{sup -}Sca-1{sup +}Kit{sup +}CD27{sup -}) and multipotent progenitors (Lin{sup -}Sca-l{sup +}Kit{sup +}CD27{sup +}), although down-regulated in other lineages. Thus, a major fraction of these early T lineage genes are a regulatory legacy from stem cells. The few genes sharply up-regulated between multipotent progenitors and Pro-T cell stages included those encoding transcription factors Bclllb, TCF-I (Tcli), and HEBalt, Notch target Deltexl, Deltex3L, Fkbp5, Eval, and Tmem13l. Like GATA3 and Deltexl, Bclllb, Fkbp5, and Eval were dependent on Notch/Delta signaling for induction in fetal liver precursors, but only BcIlI band HEBalt were up-regulated between the first two stages of intrathymic T cell development (double negative I and double negative 2) corresponding to T lineage specification. Bclllb was uniquely T lineage restricted and induced by NotchlDelta signaling specifically upon entry into the T lineage differentiation pathway.« less

Ionizing Radiation Perturbs Cell Cycle Progression of Neural Precursors in the Subventricular Zone Without Affecting Their Long-Term Self-Renewal

PubMed Central

Chen, Hongxin; Goodus, Matthew T; de Toledo, Sonia M; Azzam, Edouard I; Levison, Steven W

2015-01-01

Damage to normal human brain cells from exposure to ionizing radiation may occur during the course of radiotherapy or from accidental exposure. Delayed effects may complicate the immediate effects resulting in neurodegeneration and cognitive decline. We examined cellular and molecular changes associated with exposure of neural stem/progenitor cells (NSPs) to 137Cs γ-ray doses in the range of 0 to 8 Gy. Subventricular zone NSPs isolated from newborn mouse pups were analyzed for proliferation, self-renewal, and differentiation, shortly after irradiation. Strikingly, there was no apparent increase in the fraction of dying cells after irradiation, and the number of single cells that formed neurospheres showed no significant change from control. Upon differentiation, irradiated neural precursors did not differ in their ability to generate neurons, astrocytes, and oligodendrocytes. By contrast, progression of NSPs through the cell cycle decreased dramatically after exposure to 8 Gy (p < .001). Mice at postnatal day 10 were exposed to 8 Gy of γ rays delivered to the whole body and NSPs of the subventricular zone were analyzed using a four-color flow cytometry panel combined with ethynyl deoxyuridine incorporation. Similar flow cytometric analyses were performed on NSPs cultured as neurospheres. These studies revealed that neither the percentage of neural stem cells nor their proliferation was affected. By contrast, γ-irradiation decreased the proliferation of two classes of multipotent cells and increased the proliferation of a specific glial-restricted precursor. Altogether, these results support the conclusion that primitive neural precursors are radioresistant, but their proliferation is slowed down as a consequence of γ-ray exposure. PMID:26056396

Characterization of glial-restricted precursors from rhesus monkey embryonic stem cells.

PubMed

Chen, Hongwei; Mao, Yu; Wang, Shufen; Li, Bin; Wang, Jinhuan; Li, Jian; Ma, Yuanye

2015-01-01

Glial-restricted precursor (GRP) cells, the earliest glial progenitors for both astrocytes and oligodendrocytes, have been derived from embryos and embryonic stem cells (ESC) in rodents. However, knowledge regarding the equivalent cell type in primates is limited due to restrictions imposed by ethics and resources. Here we report successful derivation and characterization of primate GRP cells from rhesus monkey ESC. The purified monkey GRP cells were A 2 B 5 -positive and FGF2-dependent for survival and proliferation. The differentiation assays indicated that they were tri-potential in vitro and bi-potential in vivo . These newly purified GRP cells will help to facilitate understanding of the molecular mechanism of glial development in primates as well as provide a source of therapeutic donor cells for use in neuroregenerative medicine.

Push-pull strategy in the regulation of postembryonic root development.

PubMed

Choe, Goh; Lee, Ji-Young

2017-02-01

Unlike animals, plants continue to grow throughout their lives. The stem cell niche, protected in meristems of shoots and roots, enables this process. In the root, stem cells produce precursors for highly organized cell types via asymmetric cell divisions. These precursors, which are "transit-amplifying cells," actively divide for several rounds before entering into differentiation programs. In this review, we highlight positive feedback regulation between shoot- and root-ward signals during the postembryonic root growth, which is reminiscent of a "push-pull strategy" in business parlance. This property of molecular networks underlies the regulation of stem cells and their organizer, the "quiescent center," as well as of the signaling between stem cell niche, transit-amplifying cells, and beyond. Copyright © 2016 Elsevier Ltd. All rights reserved.

Revascularization of ischemic limbs after transplantation of human bone marrow cells with high aldehyde dehydrogenase activity

PubMed Central

Capoccia, Benjamin J.; Robson, Debra L.; Levac, Krysta D.; Maxwell, Dustin J.; Hohm, Sarah A.; Neelamkavil, Marian J.; Bell, Gillian I.; Xenocostas, Anargyros; Link, Daniel C.; Piwnica-Worms, David; Nolta, Jan A.

2009-01-01

The development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDHhi) activity, a progenitor cell function conserved between several lineages. BM ALDHhi cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDHhi cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDHhi cells, mice transplanted with purified ALDHhi cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDHhi cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue, suggesting that transient ALDHhi cell engraftment stimulated endogenous revascularization. Thus, human BM ALDHhi cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans. PMID:19324906

Identification and Characterization of Distinct Apoptotic Pathways in Cancer Cells Activated in Response to Treatment with Different Anti-Cancer Agents

DTIC Science & Technology

2000-08-01

caspases precursors in different cell type induce apoptosis (Miura et al. 1993; Fernandes- 3 Alnemri et al. 1994; Kumar et al. 1994; Wang et al. 1994; Boldin ...association of ribosomes with mRNA. The lysates were sedimented through a linear sucrose gradient and fractions were collected. RNA was extracted from...precursors in different cell type induces apoptosis (Miura et al. 1993; Fernandes- Alnemri et al. 1994; Kumar et al. 1994; Wang et al. 1994; Boldin et al

Decreased Migration of Langerhans Precursor-Like Cells in Response to Human Keratinocytes Expressing HPV-16 E6/E7 is Related to Reduced Macrophage Inflammatory Protein-3Alpha Production

DTIC Science & Technology

2005-01-01

high-risk human papillomavirus ( HPV ) types, particularly type 16 and 18, contributes to 90% of cervical cancer cases. HPV infects cutaneous or mucosal...been implicated in cervical/ anogenital cancer and oral squamous cell carcinomas (41). The mucosal lesions caused by HPVs often resolve over time, and a...Decreased Migration of Langerhans Precursor-Like Cells in Response to Human Keratinocytes Expressing HPV -16 E6/E7 is Related to Reduced Macrophage

Generation and characterization of Tbx1-AmCyan1 transgenic reporter mouse line that selectively labels developing thymus primordium.

PubMed

Kimura, Wataru; Sharkar, Mohammad Tofael Kabir; Sultana, Nishat; Islam, Mohammod Johirul; Uezato, Tadayoshi; Miura, Naoyuki

2013-06-01

Thymus development is a complicated process that includes highly dynamic morphological changes and reciprocal tissue interactions between endoderm-derived epithelial cells of the anterior foregut and neural crest-derived mesenchymal cells. We generated and characterized a Tbx1-AmCyan1 reporter transgenic mouse to visualize thymus precursor cells during early embryonic development. In transgenic embryos, AmCyan1 fluorescence was specifically detected in the endoderm of the developing 3rd and 4th pharyngeal pouches and later in thymus epithelium until E14.5. Cells expressing AmCyan1 that were isolated based on AmCyan1 fluorescence expressed endodermal, thymic, and parathyroid markers, but they did not express neural crest or endothelial markers; these findings indicated that this transgenic mouse strain could be used to collect thymic or parathyroid precursor cells or both. We also showed that in nude mice, which exhibit defects in thymus development, the thymus precursors were clearly labeled with AmCyan1. In summary, these AmCyan1-fluorescent transgenic mice are useful for investigating early thymus development.

Changes of vessel-cells complex in zones of adaptive remodeling of the bone tissue under microgravity conditions

NASA Astrophysics Data System (ADS)

Rodionova, N.; Oganov, V.; Nosova, L.

The development and differentiation of osteogenic cells in organism happen in closely topographical and functional connection with blood capillaries. We formerly proofed, that small-differentiated cells, which are in the population of perivascular cells are osteogenic cells -precursors . At the present time it is actually to clear up, how these biostructures react on conditions of less of biomechanical load on skeleton bones. We researched peculiarities of blood-bed structure and perivascular cells in metaphises of thighbones and tibial bones in rats, which were onboard the American space station SLS-2 and in experiments of modeling hypokinesia. There were used methods of cytochemistry, histology and electron microscopy. We established, that under the support and functional load decreasing in zones of bones adaptive remodeling, comparatively to control, on histosections the own volume of sinusoid capillaries reduces. The small vessels prevail here. The spaces of sinusoid capillaries are limited by 1 2 cells of the endothelia. Endotheliocytes in- general have the typical ultrastructure. Basal membranes are expressed not-distinctly. Perivascular cells don't create the unbroken layer. The population of these cells is not-homogeneous. It includes enclosed to endothelia small-differentiated forms and separating cells with sings of fibroblastic differentiation (the own volume of rough endoplasmic reticulum in cytoplasm induces). The part of these cells reacts on the alkaline phosphatase (the marker of the osteogenic differentiation). Under the conditions of support load decreasing (especially under the microgravity) there is a tendency to reducing of separating osteogenic cells number. We noted the priority of differentiating fibroblasts. It leads to further development in zones of bone remodeling of hearths of fibrous tissue, that doesn't mineralize. The obtained data are seen as one of mechanisms of osteoporosis and osteopenia development under the deficite of support load.

Comparison of direct and indirect radiation effects on osteoclast formation from progenitor cells derived from different hemopoietic sources.

PubMed

Scheven, B A; Wassenaar, A M; Kawilarang-de Haas, E W; Nijweide, P J

1987-07-01

Hemopoietic stem and progenitor cells from different sources differ in radiosensitivity. Recently, we have demonstrated that the multinucleated cell responsible for bone resorption and marrow cavity formation, the osteoclast, is in fact of hemopoietic lineage. In this investigation we have studied the radiosensitivity of osteoclast formation from two different hemopoietic tissues: fetal liver and adult bone marrow. Development of osteoclasts from hemopoietic progenitors was induced by coculture of hemopoietic cell populations with fetal mouse long bones depleted of their own osteoclast precursor pool. During culture, osteoclasts developed from the exogenous cell population and invaded the calcified hypertrophic cartilage of the long bone model, thereby giving rise to the formation of a primitive marrow cavity. To analyze the radiosensitivity of osteoclast formation, either the hemopoietic cells or the bone rudiments were irradiated before coculture. Fetal liver cells were found to be less radiosensitive than bone marrow cells. The D0, Dq values and extrapolation numbers were 1.69 Gy, 5.30 Gy, and 24.40 for fetal liver cells and 1.01 Gy, 1.85 Gy, and 6.02 for bone marrow cells. Irradiation of the (pre)osteoclast-free long bone rudiments instead of the hemopoietic sources resulted in a significant inhibition of osteoclast formation at doses of 4 Gy or more. This indirect effect appeared to be more prominent in the cocultures with fetal than with adult hemopoietic cells. Furthermore, radiation doses of 8.0-10.0 Gy indirectly affected the appearance of other cell types (e.g., granulocytes) in the newly formed but underdeveloped marrow cavity. The results indicate that osteoclast progenitors from different hemopoietic sources exhibit a distinct sensitivity to ionizing irradiation. Radiation injury to long bone rudiments disturbs the osteoclast-forming capacity as well as the hemopoietic microenvironment.

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

PubMed

Koyama, T; Hughes, R C

1992-12-25

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.

ABCA1-dependent sterol release: sterol molecule specificity and potential membrane domain for HDL biogenesis.

PubMed

Yamauchi, Yoshio; Yokoyama, Shinji; Chang, Ta-Yuan

2016-01-01

Mammalian cells synthesize various sterol molecules, including the C30 sterol, lanosterol, as cholesterol precursors in the endoplasmic reticulum. The build-up of precursor sterols, including lanosterol, displays cellular toxicity. Precursor sterols are found in plasma HDL. How these structurally different sterols are released from cells is poorly understood. Here, we show that newly synthesized precursor sterols arriving at the plasma membrane (PM) are removed by extracellular apoA-I in a manner dependent on ABCA1, a key macromolecule for HDL biogenesis. Analysis of sterol molecules by GC-MS and tracing the fate of radiolabeled acetate-derived sterols in normal and mutant Niemann-Pick type C cells reveal that ABCA1 prefers newly synthesized sterols, especially lanosterol, as the substrates before they are internalized from the PM. We also show that ABCA1 resides in a cholesterol-rich membrane domain resistant to the mild detergent, Brij 98. Blocking ACAT activity increases the cholesterol contents of this domain. Newly synthesized C29/C30 sterols are transiently enriched within this domain, but rapidly disappear from this domain with a half-life of less than 1 h. Our work shows that substantial amounts of precursor sterols are transported to a certain PM domain and are removed by the ABCA1-dependent pathway. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

EPO Receptor Gain-of-Function Causes Hereditary Polycythemia, Alters CD34+ Cell Differentiation and Increases Circulating Endothelial Precursors

PubMed Central

Perrotta, Silverio; Cucciolla, Valeria; Ferraro, Marcella; Ronzoni, Luisa; Tramontano, Annunziata; Rossi, Francesca; Scudieri, Anna Chiara; Borriello, Adriana; Roberti, Domenico; Nobili, Bruno; Cappellini, Maria Domenica; Oliva, Adriana; Amendola, Giovanni; Migliaccio, Anna Rita; Mancuso, Patrizia; Martin-Padura, Ines; Bertolini, Francesco; Yoon, Donghoon; Prchal, Josef T.; Della Ragione, Fulvio

2010-01-01

Background Gain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined. Methodology/Principal Findings We describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G→T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34+ cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway. Conclusions/Significance Our data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis. PMID:20700488

Evidence for heterogeneity of astrocyte de-differentiation in vitro: astrocytes transform into intermediate precursor cells following induction of ACM from scratch-insulted astrocytes.

PubMed

Yang, Hao; Qian, Xin-Hong; Cong, Rui; Li, Jing-wen; Yao, Qin; Jiao, Xi-Ying; Ju, Gong; You, Si-Wei

2010-04-01

Our previous study definitely demonstrated that the mature astrocytes could undergo a de-differentiation process and further transform into pluripotential neural stem cells (NSCs), which might well arise from the effect of diffusible factors released from scratch-insulted astrocytes. However, these neurospheres passaged from one neurosphere-derived from de-differentiated astrocytes possessed a completely distinct characteristic in the differentiation behavior, namely heterogeneity of differentiation. The heterogeneity in cell differentiation has become a crucial but elusive issue. In this study, we show that purified astrocytes could de-differentiate into intermediate precursor cells (IPCs) with addition of scratch-insulted astrocyte-conditioned medium (ACM) to the culture, which can express NG2 and A2B5, the IPCs markers. Apart from the number of NG2(+) and A2B5(+) cells, the percentage of proliferative cells as labeled with BrdU progressively increased with prolonged culture period ranging from 1 to 10 days. Meanwhile, the protein level of A2B5 in cells also increased significantly. These results revealed that not all astrocytes could de-differentiate fully into NSCs directly when induced by ACM, rather they generated intermediate or more restricted precursor cells that might undergo progressive de-differentiation to generate NSCs.

A network model for the specification of vulval precursor cells and cell fusion control in Caenorhabditis elegans

PubMed Central

Weinstein, Nathan; Mendoza, Luis

2013-01-01

The vulva of Caenorhabditis elegans has been long used as an experimental model of cell differentiation and organogenesis. While it is known that the signaling cascades of Wnt, Ras/MAPK, and NOTCH interact to form a molecular network, there is no consensus regarding its precise topology and dynamical properties. We inferred the molecular network, and developed a multivalued synchronous discrete dynamic model to study its behavior. The model reproduces the patterns of activation reported for the following types of cell: vulval precursor, first fate, second fate, second fate with reversed polarity, third fate, and fusion fate. We simulated the fusion of cells, the determination of the first, second, and third fates, as well as the transition from the second to the first fate. We also used the model to simulate all possible single loss- and gain-of-function mutants, as well as some relevant double and triple mutants. Importantly, we associated most of these simulated mutants to multivulva, vulvaless, egg-laying defective, or defective polarity phenotypes. The model shows that it is necessary for RAL-1 to activate NOTCH signaling, since the repression of LIN-45 by RAL-1 would not suffice for a proper second fate determination in an environment lacking DSL ligands. We also found that the model requires the complex formed by LAG-1, LIN-12, and SEL-8 to inhibit the transcription of eff-1 in second fate cells. Our model is the largest reconstruction to date of the molecular network controlling the specification of vulval precursor cells and cell fusion control in C. elegans. According to our model, the process of fate determination in the vulval precursor cells is reversible, at least until either the cells fuse with the ventral hypoderm or divide, and therefore the cell fates must be maintained by the presence of extracellular signals. PMID:23785384

Chemo-sensitivity in a panel of B-cell precursor acute lymphoblastic leukemia cell lines, YCUB series, derived from children.

PubMed

Goto, Hiroaki; Naruto, Takuya; Tanoshima, Reo; Kato, Hiromi; Yokosuka, Tomoko; Yanagimachi, Masakatsu; Fujii, Hisaki; Yokota, Shumpei; Komine, Hiromi

2009-10-01

Sensitivity to 10 anticancer drugs was evaluated in 6 childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell lines. Authenticity of newly established cell lines was confirmed by genomic fingerprinting. The line YCUB-5R established at relapse was more resistant to 4-hydroperoxy-cyclophosphamide, cytarabine, L-asparaginase, topotecan, fludarabine, and etoposide than YCUB-5 from the same patient at diagnosis. Of the drugs tested, etoposide and SN-38 (irinotecan) showed highest efficacy in the panel, with 50% growth inhibition at 0.22-1.8 microg/ml and 0.57-3.6 ng/ml, respectively. This cell line panel offers an in vitro model for the development of new therapies for childhood BCP-ALL.

Cell proliferation in mammalian gastrulation: the ventral node and notochord are relatively quiescent.

PubMed

Bellomo, D; Lander, A; Harragan, I; Brown, N A

1996-04-01

During gastrulation, the node of the mammalian embryo appears to be an organising centre, homologous to Hensen's node in the chick and the dorsal lip of the amphibian blastopore. In addition, the node serves as a precursor population for the head process, notochord and foregut endoderm. We have studied node architecture and cell morphology by electron microscopy, and cell proliferation using bromodeoxyuridine incorporation and mitotic counts. The dorsal (ectodermal) and ventral (endodermal) components of the node are two distinct populations, separated by a basement membrane. The ventral node, contiguous with the head process, is characterised by a relatively low proliferation rate, with only approximately 10% of cells incorporating BrdU over 4 hr, compared to > 95% in surrounding mesodermal and ectodermal tissues. This is the case from the beginning of node formation, at the no-allantoic-bud stage, until the 7 somite stage, and is not compatible with the idea that the ventral node is a stem cell population. The dorsal node is highly proliferative, its rate of division being indistinguishable from the neurectoderm, with which it is contiguous. In the ventral node, two regions can be recognised: cells in the "pit" are columnar and all monociliated; around them lies a "crown" of cells arranged radially in a horseshoe shape and less often ciliated. Node derivatives share common features with the ventral node; the head process and the notochord are relatively quiescent; and some head process cells are also monociliated. Node and head process monocilia are immotile and appear to be associated with non-proliferation. We suggest that the ventral node contains all the properties of the organiser, while the dorsal node is indistinct from the surrounding epiblast. The cranial end of the foregut pouch, the thyroid diverticulum, and the promyocardium of early somite stage embryos are also areas of low cell division. All the described regions of relative quiescence are sites of expression of members of the TGF beta family, which may be involved in maintaining non-proliferation.

FOXO1 opposition of CD8+ T cell effector programming confers early memory properties and phenotypic diversity.

PubMed

Delpoux, Arnaud; Lai, Chen-Yen; Hedrick, Stephen M; Doedens, Andrew L

2017-10-17

The factors and steps controlling postinfection CD8 + T cell terminal effector versus memory differentiation are incompletely understood. Whereas we found that naive TCF7 (alias "Tcf-1") expression is FOXO1 independent, early postinfection we report bimodal, FOXO1-dependent expression of the memory-essential transcription factor TCF7 in pathogen-specific CD8 + T cells. We determined the early postinfection TCF7 high population is marked by low TIM3 expression and bears memory signature hallmarks before the appearance of established memory precursor marker CD127 (IL-7R). These cells exhibit diminished TBET, GZMB, mTOR signaling, and cell cycle progression. Day 5 postinfection, TCF7 high cells express higher memory-associated BCL2 and EOMES, as well as increased accumulation potential and capacity to differentiate into memory phenotype cells. TCF7 retroviral transduction opposes GZMB expression and the formation of KLRG1 pos phenotype cells, demonstrating an active role for TCF7 in extinguishing the effector program and forestalling terminal differentiation. Past the peak of the cellular immune response, we report a gradient of FOXO1 and TCF7 expression, which functions to oppose TBET and orchestrate a continuum of effector-to-memory phenotypes.

Megakaryocyte polyploidization is associated with decreased expression of polo-like kinase (PLK).

PubMed

Yagi, M; Roth, G J

2006-09-01

During differentiation, megakaryocytes (MK), the bone marrow precursors of circulating blood platelets, undergo polyploidization, repeated rounds of DNA replication without cell division. Mature normal MK may contain a DNA content of up to 128N, in contrast to normal diploid (2N) cells. The extent of polyploidy may influence the number of platelets produced by the MK. Therefore, understanding the molecular mechanisms regulating polyploidization could identify events involved in controlling both cell division and thrombopoiesis. We investigated the expression of several proteins involved in mitosis in cultured mouse MK, and tested the effect of expression on polyploidization. Western blot and immunofluorescent analyses were used to assess expression of cell cycle proteins in cultured MK. Populations of polyploidizing MK were separated on the basis of DNA content by flow cytometry. The gene encoding mouse polo-like kinase 1 (PLK-1) was introduced into MK by retroviral transduction, and its effects measured by flow cytometry. Polyploid mouse MK expressed lower levels of two proteins, p55CDC and PLK-1, whose activity is necessary for cell cycle progression and completion of mitosis. Comparison of sorted 2N/4N and polyploid MK indicated that PLK-1 expression was absent in polyploid MK, while expression of other cell cycle proteins was similar in both populations. Forced expression of PLK-1 during MK differentiation was associated with decreased polyploidization. These experiments suggest that PLK-1 is an important regulator of polyploidization in differentiating MK.

Alleviation of hyperglycemia in diabetic rats by intraportal injection of insulin-producing cells generated from surgically resected human pancreatic tissue.

PubMed

Shyu, Jia-Fwu; Wang, Hwai-Shi; Shyr, Yi-Ming; Wang, Shin-E; Chen, Chia-Hsiang; Tan, Joo-Shin; Lin, Meng-Feng; Hsieh, Po-Shiuan; Sytwu, Huey-Kang; Chen, Tien-Hua

2011-03-01

Although islet transplantation holds promise for the treatment of diabetes, the scarcity of donor tissue remains a major drawback. The aim of this study is to generate insulin-producing cells from adult human pancreatic cells isolated from surgically resected pancreatic tissue. To isolate pancreatic endocrine precursor cells from 57 surgically resected pancreases, the cells were cultured and propagated in conditioned medium after which they were differentiated in Matrigel. The resultant cells were characterized using morphology, immunofluorescent studies, expression of differentiated pancreatic islet-specific genes using quantitative reverse transcription-PCR, and glucose-induced insulin secretion through analysis of C-peptide secretion. The relationships between propagation of insulin-producing cells and clinical variables of the donor were also analyzed. Finally, insulin-producing cell function was examined in streptozotocin-induced diabetic rats. Pancreatic endocrine precursor cells were successfully cultured; insulin-producing cells cultured from soft pancreas parenchyma had a significantly higher success rate. Morphological examination revealed islet-like cluster formation upon transfer to Matrigel. The presence of the neural stem cell marker nestin, duct cell marker cytokeratin 19, and endocrine cell markers C-peptide and pancreatic and duodenal homeobox 1, was also observed. In addition, glucose-stimulated C-peptide release was significantly increased in the insulin-producing cells. Furthermore, in diabetic rats, transplantation of insulin-producing cells reduced hyperglycemia. Isolated pancreatic endocrine precursor cells from surgically resected pancreatic tissue differentiated into insulin-producing cells and showed characteristics of functional endocrine cells. Thus, surgically resected pancreatic tissue may represent an alternative source of functional insulin-producing cells.

Differentiation of lepidoptera scale cells from epidermal stem cells followed by ecdysone-regulated DNA duplication and scale secreting.

PubMed

Yuan, Shenglei; Huang, Wuren; Geng, Lei; Beerntsen, Brenda T; Song, Hongsheng; Ling, Erjun

2017-01-01

Integuments are the first line to protect insects from physical damage and pathogenic infection. In lepidopteran insects, they undergo distinct morphology changes such as scale formation during metamorphosis. However, we know little about integument development and scale formation during this stage. Here, we use the silkworm, Bombyx mori, as a model and show that stem cells in the integument of each segment, but not intersegmental membrane, divide into two scale precursor cells during the spinning stage. In young pupae, the scale precursor cell divides again. One of the daughter cells becomes a mature scale-secreting cell that undergoes several rounds of DNA duplication and the other daughter cell undergoes apoptosis later on. This scale precursor cell division is crucial to the development and differentiation of scale-secreting cells because scale production can be blocked after treatment with the cell division inhibitor paclitaxel. Subsequently, the growth of scale-secreting cells is under the control of 20-hydroxyecdysone but not juvenile hormone since injection of 20-hydroxyecdysone inhibited scale formation. Further work demonstrated that 20-hydroxyecdysone injection inhibits DNA duplication in scale-secreting cells while the expression of scale-forming gene ASH1 was down-regulated by BR-C Z2. Therefore, this research demonstrates that the scale cells of the silkworm develops through stem cell division prior to pupation and then another wave of cell division differentiates these cells into scale secreting cells soon after entrance into the pupal stage. Additionally, DNA duplication and scale production in the scale-secreting cells were found to be under the regulation of 20-hydroxyecdysone.

13C nuclear magnetic resonance and gas chromatography-mass spectrometry studies of carbon metabolism in the actinomycin D producer Streptomyces parvulus by use of 13C-labeled precursors.

PubMed Central

Inbar, L; Lapidot, A

1991-01-01

Fructose and glutamate metabolism was monitored in cell suspensions of streptomyces parvulus by 13C nuclear magnetic resonance. The experiments were performed for cells grown with various 13C sources in a growth medium containing D-[U-13C]fructose, L-[13C]glutamate, or L-[U-13C]aspartate and with nonlabeled precursors to compare intracellular pools in S. parvulus cells at different periods of the cell life cycle. The transport of fructose into the cells was biphasic in nature; during rapid transport, mannitol, fructose, and glucose 6-phosphate were accumulated intracellularly, whereas during the passive diffusion of fructose, the intracellular carbohydrate pool comprised mainly trehalose (1,1'-alpha-alpha-D-glucose). The regulation of fructokinase activity by the intracellular intermediates may play an important role in fructose catabolism in S. parvulus. Transaldolase activity in S. parvulus was determined from the 13C nuclear magnetic resonance labeling pattern of trehalose carbons obtained from cells grown in medium containing either L-[U-13C]aspartate or L-[U-13C]glutamate. Only carbons 4, 5, and 6 of the disaccharide were labeled. Isotopomer analysis of the trehalose carbons led us to conclude that the flux through the reverse glycolytic pathway, condensation of glyceraldehyde 3-phosphate with dihydroxyacetone phosphate, makes at best a minor contribution to the 13C-labeled glucose units observed in trehalose. The pentose pathway and transaldolase activity can explain the labeling pattern of 4,5,6-13C3 of trehalose. Moreover, the transfer of the 13C label of L-[U-13C]aspartate into the different isotopomers of trehalose C4, C5, and C6 by the transaldolase activity allowed us to calculate the relative fluxes from oxaloacetate via gluconeogenesis and through the tricarboxylic acid cycle. The ratio of the two fluxes is approximately 1. However, the main carbon source for trehalose synthesis in S. parvulus is fructose and not glutamate or aspartate. The 13C enrichment and isotopomer population, measured by nuclear magnetic resonance and gas chromatography-mass spectrometry, of the actinomycin D peptide ring enabled us to specify the origins of the five amino acids of actinomycin D. Threonine and proline exhibited isotopomer populations similar to that of the extracellular L-[13C]glutamate, indicating that protein catabolism is the origin of their 13C label, whereas the isotopomer populations of sarcosine and N-methylvaline were similar to those of the new intracellular pool of S. parvulus that originated from D-[U-13C]fructose during the production of actinomycin D. PMID:1744035

Induction of CD4 T cell memory by local cellular collectivity.

PubMed

Polonsky, Michal; Rimer, Jacob; Kern-Perets, Amos; Zaretsky, Irina; Miller, Stav; Bornstein, Chamutal; David, Eyal; Kopelman, Naama Meira; Stelzer, Gil; Porat, Ziv; Chain, Benjamin; Friedman, Nir

2018-06-15

Cell differentiation is directed by signals driving progenitors into specialized cell types. This process can involve collective decision-making, when differentiating cells determine their lineage choice by interacting with each other. We used live-cell imaging in microwell arrays to study collective processes affecting differentiation of naïve CD4 + T cells into memory precursors. We found that differentiation of precursor memory T cells sharply increases above a threshold number of locally interacting cells. These homotypic interactions involve the cytokines interleukin-2 (IL-2) and IL-6, which affect memory differentiation orthogonal to their effect on proliferation and survival. Mathematical modeling suggests that the differentiation rate is continuously modulated by the instantaneous number of locally interacting cells. This cellular collectivity can prioritize allocation of immune memory to stronger responses. Copyright © 2018, American Association for the Advancement of Science.

Superenhancer reprogramming drives a B-cell–epithelial transition and high-risk leukemia

PubMed Central

Hu, Yeguang; Zhang, Zhihong; Kashiwagi, Mariko; Yoshida, Toshimi; Joshi, Ila; Jena, Nilamani; Somasundaram, Rajesh; Emmanuel, Akinola Olumide; Sigvardsson, Mikael; Fitamant, Julien; El-Bardeesy, Nabeel; Gounari, Fotini; Van Etten, Richard A.; Georgopoulos, Katia

2016-01-01

IKAROS is required for the differentiation of highly proliferative pre-B-cell precursors, and loss of IKAROS function indicates poor prognosis in precursor B-cell acute lymphoblastic leukemia (B-ALL). Here we show that IKAROS regulates this developmental stage by positive and negative regulation of superenhancers with distinct lineage affiliations. IKAROS defines superenhancers at pre-B-cell differentiation genes together with B-cell master regulators such as PAX5, EBF1, and IRF4 but is required for a highly permissive chromatin environment, a function that cannot be compensated for by the other transcription factors. IKAROS is also highly enriched at inactive enhancers of genes normally expressed in stem–epithelial cells. Upon IKAROS loss, expression of pre-B-cell differentiation genes is attenuated, while a group of extralineage transcription factors that are directly repressed by IKAROS and depend on EBF1 relocalization at their enhancers for expression is induced. LHX2, LMO2, and TEAD–YAP1, normally kept separate from native B-cell transcription regulators by IKAROS, now cooperate directly with them in a de novo superenhancer network with its own feed-forward transcriptional reinforcement. Induction of de novo superenhancers antagonizes Polycomb repression and superimposes aberrant stem–epithelial cell properties in a B-cell precursor. This dual mechanism of IKAROS regulation promotes differentiation while safeguarding against a hybrid stem–epithelial–B-cell phenotype that underlies high-risk B-ALL. PMID:27664237

Curtailed T-cell activation curbs effector differentiation and generates CD8+ T cells with a naturally-occurring memory stem cell phenotype.

PubMed

Zanon, Veronica; Pilipow, Karolina; Scamardella, Eloise; De Paoli, Federica; De Simone, Gabriele; Price, David A; Martinez Usatorre, Amaia; Romero, Pedro; Mavilio, Domenico; Roberto, Alessandra; Lugli, Enrico

2017-09-01

Human T memory stem (T SCM ) cells with superior persistence capacity and effector functions are emerging as important players in the maintenance of long-lived T-cell memory and are thus considered an attractive population to be used in adoptive transfer-based immunotherapy of cancer. However, the molecular signals regulating their generation remain poorly defined. Here we show that curtailed T-cell receptor stimulation curbs human effector CD8 + T-cell differentiation and allows the generation of CD45RO - CD45RA + CCR7 + CD27 + CD95 + -phenotype cells from highly purified naïve T-cell precursors, resembling naturally-occurring human T SCM . These cells proliferate extensively in vitro and in vivo, express low amounts of effector-associated genes and transcription factors and undergo considerable self-renewal in response to IL-15 while retaining effector differentiation potential. Such a phenotype is associated with a lower number of mitochondria compared to highly-activated effector T cells committed to terminal differentiation. These results shed light on the molecular signals that are required to generate long-lived memory T cells with potential application in adoptive cell transfer immunotherapy. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co.KGaA, Weinheim.

Transcriptional Profiling of Antigen-Dependent Murine B Cell Differentiation and Memory Formation1

PubMed Central

Bhattacharya, Deepta; Cheah, Ming T.; Franco, Christopher B.; Hosen, Naoki; Pin, Christopher L.; Sha, William C.; Weissman, Irving L.

2015-01-01

Humoral immunity is characterized by the generation of Ab-secreting plasma cells and memory B cells that can more rapidly generate specific Abs upon Ag exposure than their naive counterparts. To determine the intrinsic differences that distinguish naive and memory B cells and to identify pathways that allow germinal center B cells to differentiate into memory B cells, we compared the transcriptional profiles of highly purified populations of these three cell types along with plasma cells isolated from mice immunized with a T-dependent Ag. The transcriptional profile of memory B cells is similar to that of naive B cells, yet displays several important differences, including increased expression of activation-induced deaminase and several antiapoptotic genes, chemotactic receptors, and costimulatory molecules. Retroviral expression of either Klf2 or Ski, two transcriptional regulators specifically enriched in memory B cells relative to their germinal center precursors, imparted a competitive advantage to Ag receptor and CD40-engaged B cells in vitro. These data suggest that humoral recall responses are more rapid than primary responses due to the expression of a unique transcriptional program by memory B cells that allows them to both be maintained at high frequencies and to detect and rapidly respond to antigenic re-exposure. PMID:17982071

Formation and specification of a Drosophila dopaminergic precursor cell.

PubMed

Watson, Joseph D; Crews, Stephen T

2012-09-01

Dopaminergic neurons play important roles in animal behavior, including motivation, reward and locomotion. The Drosophila dopaminergic H-cell interneuron is an attractive system for studying the genetics of neural development because analysis is focused on a single neuronal cell type. Here we provide a mechanistic understanding of how MP3, the precursor to the H-cell, forms and acquires its identity. We show that the gooseberry/gooseberry-neuro (gsb/gsb-n) transcription factor genes act to specify MP3 cell fate. It is proposed that single-minded commits neuroectodermal cells to a midline fate, followed by a series of signaling events that result in the formation of a single gsb(+)/gsb-n(+) MP3 cell per segment. The wingless signaling pathway establishes a midline anterior domain by activating expression of the forkhead transcription factors sloppy paired 1 and sloppy paired 2. This is followed by hedgehog signaling that activates gsb/gsb-n expression in a subgroup of anterior cells. Finally, Notch signaling results in the selection of a single MP3, with the remaining cells becoming midline glia. In MP3, gsb/gsb-n direct H-cell development, in large part by activating expression of the lethal of scute and tailup H-cell regulatory genes. Thus, a series of signaling and transcriptional events result in the specification of a unique dopaminergic precursor cell. Additional genetic experiments indicate that the molecular mechanisms that govern MP3/H-cell development might also direct the development of non-midline dopaminergic neurons.

Formation and specification of a Drosophila dopaminergic precursor cell

PubMed Central

Watson, Joseph D.; Crews, Stephen T.

2012-01-01

Dopaminergic neurons play important roles in animal behavior, including motivation, reward and locomotion. The Drosophila dopaminergic H-cell interneuron is an attractive system for studying the genetics of neural development because analysis is focused on a single neuronal cell type. Here we provide a mechanistic understanding of how MP3, the precursor to the H-cell, forms and acquires its identity. We show that the gooseberry/gooseberry-neuro (gsb/gsb-n) transcription factor genes act to specify MP3 cell fate. It is proposed that single-minded commits neuroectodermal cells to a midline fate, followed by a series of signaling events that result in the formation of a single gsb+/gsb-n+ MP3 cell per segment. The wingless signaling pathway establishes a midline anterior domain by activating expression of the forkhead transcription factors sloppy paired 1 and sloppy paired 2. This is followed by hedgehog signaling that activates gsb/gsb-n expression in a subgroup of anterior cells. Finally, Notch signaling results in the selection of a single MP3, with the remaining cells becoming midline glia. In MP3, gsb/gsb-n direct H-cell development, in large part by activating expression of the lethal of scute and tailup H-cell regulatory genes. Thus, a series of signaling and transcriptional events result in the specification of a unique dopaminergic precursor cell. Additional genetic experiments indicate that the molecular mechanisms that govern MP3/H-cell development might also direct the development of non-midline dopaminergic neurons. PMID:22874915

Autocrine CSF-1R signaling drives mesothelioma chemoresistance via AKT activation

PubMed Central

Cioce, M; Canino, C; Goparaju, C; Yang, H; Carbone, M; Pass, H I

2014-01-01

Clinical management of malignant pleural mesothelioma (MPM) is very challenging because of the uncommon resistance of this tumor to chemotherapy. We report here increased expression of macrophage colony-stimulating-factor-1-receptor (M-CSF/CSF-1R) mRNA in mesothelioma versus normal tissue specimens and demonstrate that CSF-1R expression identifies chemoresistant cells of mesothelial nature in both primary cultures and mesothelioma cell lines. By using RNAi or ligand trapping, we demonstrate that the chemoresistance properties of those cells depend on autocrine CSF-1R signaling. At the single-cell level, the isolated CSF-1Rpos cells exhibit a complex repertoire of pluripotency, epithelial–mesenchymal transition and detoxifying factors, which define a clonogenic, chemoresistant, precursor-like cell sub-population. The simple activation of CSF-1R in untransformed mesothelial cells is sufficient to confer clonogenicity and resistance to pemetrexed, hallmarks of mesothelioma. In addition, this induced a gene expression profile highly mimicking that observed in the MPM cells endogenously expressing the receptor and the ligands, suggesting that CSF-1R expression is mainly responsible for the phenotype of the identified cell sub-populations. The survival of CSF1Rpos cells requires active AKT (v-akt murine thymoma viral oncogene homolog 1) signaling, which contributed to increased levels of nuclear, transcriptionally competent β-catenin. Inhibition of AKT reduced the transcriptional activity of β-catenin-dependent reporters and sensitized the cells to senescence-induced clonogenic death after pemetrexed treatment. This work expands what is known on the non-macrophage functions of CSF-1R and its role in solid tumors, and suggests that CSF-1R signaling may have a critical pathogenic role in a prototypical, inflammation-related cancer such as MPM and therefore may represent a promising target for therapeutic intervention. PMID:24722292

IsomiR expression profiles in human lymphoblastoid cell lines exhibit population and gender dependencies

PubMed Central

Loher, Phillipe; Londin, Eric R.; Rigoutsos, Isidore

2014-01-01

For many years it was believed that each mature microRNA (miRNA) existed as a single entity with fixed endpoints and a ‘static’ and unchangeable primary sequence. However, recent evidence suggests that mature miRNAs are more ‘dynamic’ and that each miRNA precursor arm gives rise to multiple isoforms, the isomiRs. Here we report on our identification of numerous and abundant isomiRs in the lymphoblastoid cell lines (LCLs) of 452 men and women from five different population groups. Unexpectedly, we find that these isomiRs exhibit an expression profile that is population-dependent and gender-dependent. This is important as it indicates that the LCLs of each gender/population combination have their own unique collection of mature miRNA transcripts. Moreover, each identified isomiR has its own characteristic abundance that remains consistent across biological replicates indicating that these are not degradation products. The primary sequences of identified isomiRs differ from the known miRBase miRNA either at their 5´-endpoint (leads to a different ‘seed’ sequence and suggests a different targetome), their 3´-endpoint, or both simultaneously. Our analysis of Argonaute PAR-CLIP data from LCLs supports the association of many of these newly identified isomiRs with the Argonaute silencing complex and thus their functional roles through participation in the RNA interference pathway. PMID:25229428

IsomiR expression profiles in human lymphoblastoid cell lines exhibit population and gender dependencies.

PubMed

Loher, Phillipe; Londin, Eric R; Rigoutsos, Isidore

2014-09-30

For many years it was believed that each mature microRNA (miRNA) existed as a single entity with fixed endpoints and a 'static' and unchangeable primary sequence. However, recent evidence suggests that mature miRNAs are more 'dynamic' and that each miRNA precursor arm gives rise to multiple isoforms, the isomiRs. Here we report on our identification of numerous and abundant isomiRs in the lymphoblastoid cell lines (LCLs) of 452 men and women from five different population groups. Unexpectedly, we find that these isomiRs exhibit an expression profile that is population-dependent and gender-dependent. This is important as it indicates that the LCLs of each gender/population combination have their own unique collection of mature miRNA transcripts. Moreover, each identified isomiR has its own characteristic abundance that remains consistent across biological replicates indicating that these are not degradation products. The primary sequences of identified isomiRs differ from the known miRBase miRNA either at their 5´-endpoint (leads to a different 'seed' sequence and suggests a different targetome), their 3´-endpoint, or both simultaneously. Our analysis of Argonaute PAR-CLIP data from LCLs supports the association of many of these newly identified isomiRs with the Argonaute silencing complex and thus their functional roles through participation in the RNA interference pathway.

The role of lipids in host microbe interactions.

PubMed

Lang, Roland; Mattner, Jochen

2017-06-01

Lipids are one of the major subcellular constituents and serve as signal molecules, energy sources, metabolic precursors and structural membrane components in various organisms. The function of lipids can be modified by multiple biochemical processes such as (de-)phosphorylation or (de-)glycosylation, and the organization of fatty acids into distinct cellular pools and subcellular compartments plays a pivotal role for the morphology and function of various cell populations. Thus, lipids regulate, for example, phagosome formation and maturation within host cells and thus, are critical for the elimination of microbial pathogens. Vice versa, microbial pathogens can manipulate the lipid composition of phagosomal membranes in host cells, and thus avoid their delivery to phagolysosomes. Lipids of microbial origin belong also to the strongest and most versatile inducers of mammalian immune responses upon engagement of distinct receptors on myeloid and lymphoid cells. Furthermore, microbial lipid toxins can induce membrane injuries and cell death. Thus, we will review here selected examples for mutual host-microbe interactions within the broad and divergent universe of lipids in microbial defense, tissue injury and immune evasion.

Intracellular Protein Shuttling: A Mechanism Relevant for Myelin Repair in Multiple Sclerosis?

PubMed Central

Göttle, Peter; Küry, Patrick

2015-01-01

A prominent feature of demyelinating diseases such as multiple sclerosis (MS) is the degeneration and loss of previously established functional myelin sheaths, which results in impaired signal propagation and axonal damage. However, at least in early disease stages, partial replacement of lost oligodendrocytes and thus remyelination occur as a result of resident oligodendroglial precursor cell (OPC) activation. These cells represent a widespread cell population within the adult central nervous system (CNS) that can differentiate into functional myelinating glial cells to restore axonal functions. Nevertheless, the spontaneous remyelination capacity in the adult CNS is inefficient because OPCs often fail to generate new oligodendrocytes due to the lack of stimulatory cues and the presence of inhibitory factors. Recent studies have provided evidence that regulated intracellular protein shuttling is functionally involved in oligodendroglial differentiation and remyelination activities. In this review we shed light on the role of the subcellular localization of differentiation-associated factors within oligodendroglial cells and show that regulation of intracellular localization of regulatory factors represents a crucial process to modulate oligodendroglial maturation and myelin repair in the CNS. PMID:26151843

Investigation of relationship between precursor of miRNA-944 and its mature form in lung squamous-cell carcinoma - the diagnostic value.

PubMed

Powrózek, Tomasz; Mlak, Radosław; Dziedzic, Marcin; Małecka-Massalska, Teresa; Sagan, Dariusz

2018-03-01

MicroRNA (miRNA) are attractive markers of lung cancer, due to their regulatory role in cell cycle. However, we know more about function of miRNA in cancer development, there is still little known about role of their precursors (primary miRNA; pri-miRNA) in tumorgenesis. In present study we investigated potential role of miRNA-944 and its precursor pri-miRNA-944 in development of squamous-cell lung cancer (SCC) and explored interdependence between miRNA precursor and its mature form. This is a first available literature report analyzing pri-miRNA as a cancer diagnostic marker. Expression of miRNA-944 and its precursor was analyzed in 58 fresh-frozen tissues of non-small cell lung cancer and corresponding adjacent non-cancerous tissues using qRT-PCR. Expression of pri-miRNA-944 was correlated with TP63 and miRNA-944. Using ROC analysis diagnostic accuracy of studied markers was evaluated. miRNA-944 and its precursor were significantly overexspressed in SCC compared to adenocarcinoma (AC) and non-cancerous tissue. pri-miRNA-944 strongly and positively correlated with TP63 (r = 0.739, p < 0.001) and with mature miRNA-944 expression (r = 0.691, p < 0.001). Also, TP63 expression significantly correlated with mature miRNA (r = 0.785, p < 0.001). Combined analysis of pri-miRNA-944 and mature miRNA-944 allowed to distinguish SCC tissue form AC with sensitivity of 93.3% and specificity of 100% (AUC = 0.978), and SCC from non-cancerous tissue with 92.9% sensitivity and 100% specificity (AUC = 0.992). We assumed that pri-miRNA-944 and miRNA-944 may be involved in early squamous-type differentiation of lung tumors. Moreover, analysis of both markers provided high diagnostic accuracy for SCC detection. Copyright © 2018 Elsevier GmbH. All rights reserved.

Temporal Expression of Bim Limits the Development of Agonist-Selected Thymocytes and Skews Their TCRβ Repertoire

PubMed Central

Li, Kun-Po; Fahnrich, Anke; Roy, Eron; Cuda, Carla M.; Grimes, H. Leighton; Perlman, Harris R.; Kalies, Kathrin; Hildeman, David A.

2017-01-01

CD8αα TCRαβ+ intestinal intraepithelial lymphocytes play a critical role in promoting intestinal homeostasis, although mechanisms controlling their development and peripheral homeostasis remain unclear. In this study, we examined the spatiotemporal role of Bim in the thymic selection of CD8αα precursors and the fate of these cells in the periphery. We found that T cell–specific expression of Bim during early/cortical, but not late/medullary, thymic development controls the agonist selection of CD8αα precursors and limits their private TCRβ repertoire. During this process, agonist-selected double-positive cells lose CD4/8 coreceptor expression and masquerade as double-negative (DN) TCRαβhi thymocytes. Although these DN thymocytes fail to re-express coreceptors after OP9-DL1 culture, they eventually mature and accumulate in the spleen where TCR and IL-15/STAT5 signaling promotes their conversion to CD8αα cells and their expression of gut-homing receptors. Adoptive transfer of splenic DN cells gives rise to CD8αα cells in the gut, establishing their precursor relationship in vivo. Interestingly, Bim does not restrict the IL-15–driven maturation of CD8αα cells that is critical for intestinal homeostasis. Thus, we found a temporal and tissue-specific role for Bim in limiting thymic agonist selection of CD8αα precursors and their TCRβ repertoire, but not in the maintenance of CD8αα intraepithelial lymphocytes in the intestine. PMID:27852740

Developmental changes in electrophysiological characteristics of human induced Pluripotent Stem Cell-derived cardiomyocytes

PubMed Central

Ben-Ari, Meital; Naor, Shulamit; Zeevi-Levin, Naama; Schick, Revital; Ben Jehuda, Ronen; Reiter, Irina; Raveh, Amit; Grijnevitch, Inna; Barak, Omri; Rosen, Michael R.; Weissman, Amir; Binah, Ofer

2016-01-01

Background Previous studies proposed that throughout differentiation of human induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) only 3 types of action potentials (AP) exist: nodal, atrial and ventricular-like. Objective To investigate whether there are precisely 3 phenotypes or a continuum exists among them, we tested 2 hypotheses: (1) during culture development a cardiac precursor cell is present that - depending on age - can evolve into the 3 phenotypes. (2) The predominant pattern is early prevalence of nodal phenotype, transient appearance of atrial phenotype, evolution to ventricular phenotype, and persistence of transitional phenotypes. Methods To test these hypotheses we: (1) performed FACS analysis of nodal, atrial and ventricular markers; (2) recorded AP from 280 7-to-95 day old iPSC-CMs; (3) analyzed AP characteristics. Results The major findings were: (1) FACS analysis of 30 and 60-day old cultures showed that an iPSC-CMs population shifts from nodal into atrial/ventricular phenotype, while including significant transitional populations.(2) The AP population did not consist of 3 distinct phenotypes; (3) Culture aging was associated with a shift from nodal to ventricular dominance, with a transient (57–70 days) appearance of atrial phenotype; (4) Beat Rate Variability was more prominent in nodal than ventricular cardiomyocytes while If density increased in older cultures. Conclusions From the onset of development the iPSC-CMs population includes nodal, atrial and ventricular AP and a broad spectrum of transitional phenotypes. The most readily distinguishable phenotype is atrial which appears only transiently, yet dominates at 57–70 days of evolution. PMID:27639456

Maintenance of “stem cell” features of cartilage cell sub-populations during in vitro propagation

PubMed Central

2013-01-01

Background The discovery of mesenchymal stem cells (MSCs) or MSC-like cells in cartilage tissue does not tie in well with the established view that MSCs derive from a perivascular niche. The presence of MSCs may raise concerns about specificity and application safety, particularly in terms of the regulatory site. The aim of the present study was to investigate the benefits or possible risks of the MSC-like properties of cells isolated from cartilage in the context of autologous chondrocyte implantation. Methods Chondrocytic cells were isolated from cartilage or intervertebral disc tissue. Flow cytometry was used to analyze the expression of cell surface antigens. MSC-like cells were either enriched or depleted by means of magnetic cell sorting (MACS) involving the monoclonal antibodies W5C5/SUSD2 and W8B2/MSCA-1. We addressed the issues of prolonged expansion of such cells as well as the influence of culture medium as a trigger for selecting a single cell type. Established protocols were used to study in vitro differentiation. In addition to histological and biochemical assessment, the acquired phenotypes were also evaluated on the mRNA transcript level. Results In the studied cells, we found strongly analogous expression of antigens typically expressed on MSCs, including CD49e, CD73, CD90, CD105, CD140b and CD166. The expression of W5C5 and W8B2 antigens in cartilage cell sub-populations did not correlate with multi-potency. We demonstrated that a chondroid precursor, but not a bona fide multipotent mesenchymal, cell type can be obtained under established in vitro culture conditions. The culture media used for expansion influenced the cell phenotype. Conclusions The risk of adverse adipose or osseous differentiation is not posed by expanded chondrocyte cultures, even after enrichment of putative MSC-like cell populations by MACS. It is possible that this limited “stemness” in chondrocytes, expanded for use in ACI, may instead be beneficial as it allows re-differentiation under appropriate conditions despite prolonged times in culture. PMID:23363653

Reduced subventricular zone proliferation and white matter damage in juvenile ferrets with kaolin-induced hydrocephalus.

PubMed

Di Curzio, Domenico L; Buist, Richard J; Del Bigio, Marc R

2013-10-01

Hydrocephalus is a neurological condition characterized by altered cerebrospinal fluid (CSF) flow with enlargement of ventricular cavities in the brain. A reliable model of hydrocephalus in gyrencephalic mammals is necessary to test preclinical hypotheses. Our objective was to characterize the behavioral, structural, and histological changes in juvenile ferrets following induction of hydrocephalus. Fourteen-day old ferrets were given an injection of kaolin (aluminum silicate) into the cisterna magna. Two days later and repeated weekly until 56 days of age, magnetic resonance (MR) imaging was used to assess ventricle size. Behavior was examined thrice weekly. Compared to age-matched saline-injected controls, severely hydrocephalic ferrets weighed significantly less, their postures were impaired, and they were hyperactive prior to extreme debilitation. They developed significant ventriculomegaly and displayed white matter destruction. Reactive astroglia and microglia detected by glial fibrillary acidic protein (GFAP) and Iba-1 immunostaining were apparent in white matter, cortex, and hippocampus. There was a hydrocephalus-related increase in activated caspase 3 labeling of apoptotic cells (7.0 vs. 15.5%) and a reduction in Ki67 labeling of proliferating cells (23.3 vs. 5.9%) in the subventricular zone (SVZ). Reduced Olig2 immunolabeling suggests a depletion of glial precursors. GFAP content was elevated. Myelin basic protein (MBP) quantitation and myelin biochemical enzyme activity showed early maturational increases. Where white matter was not destroyed, the remaining axons developed myelin similar to the controls. In conclusion, the hydrocephalus-induced periventricular disturbances may involve developmental impairments in cell proliferation and glial precursor cell populations. The ferret should prove useful for testing hypotheses about white matter damage and protection in the immature hydrocephalic brain. Copyright © 2013 Elsevier Inc. All rights reserved.

Wnt/β-catenin signaling in dermal condensates is required for hair follicle formation

PubMed Central

Tsai, Su-Yi; Sennett, Rachel; Rezza, Amélie; Clavel, Carlos; Grisanti, Laura; Zemla, Roland; Najam, Sara; Rendl, Michael

2014-01-01

Broad dermal Wnt signaling is required for patterned induction of hair follicle placodes and subsequent Wnt signaling in placode stem cells is essential for induction of dermal condensates, cell clusters of precursors for the hair follicle dermal papilla (DP). Progression of hair follicle formation then requires coordinated signal exchange between dermal condensates and placode stem cells. However, it remains unknown whether continued Wnt signaling in DP precursor cells plays a role in this process, largely due to the long-standing inability to specifically target dermal condensates for gene ablation. Here we use the Tbx18Cre knockin mouse line to ablate the Wnt-responsive transcription factor β-catenin specifically in these cells at E14.5 during the first wave of guard hair follicle formation. In the absence of β-catenin, canonical Wnt signaling is effectively abolished in these cells. Sox2+ dermal condensates initiate normally, however by E16.5 guard hair follicle numbers are strongly reduced and by E18.5 most whiskers and guard hair follicles are absent, suggesting that active Wnt signaling in dermal condensates is important for hair follicle formation to proceed after induction. To explore the molecular mechanisms by which Wnt signaling in dermal condensates regulates hair follicle formation, we analyze genome-wide the gene expression changes in embryonic β-catenin null DP precursor cells. We find altered expression of several signaling pathway genes, including Fgfs and Activin, both previously implicated in hair follicle formation. In summary, these data reveal a functional role of Wnt signaling in DP precursors for embryonic hair follicle formation and identify Fgf and Activin signaling as potential effectors of Wnt signaling-regulated events. PMID:24309208

ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3[S

PubMed Central

Yu, Man; Benham, Aaron; Logan, Sreemathi; Brush, R. Steven; Mandal, Md Nawajes A.; Anderson, Robert E.; Agbaga, Martin-Paul

2012-01-01

We hypothesized that reduction/loss of very long chain PUFAs (VLC-PUFAs) due to mutations in the ELOngase of very long chain fatty acid-4 (ELOVL4) protein contributes to retinal degeneration in autosomal dominant Stargardt-like macular dystrophy (STGD3) and age-related macular degeneration; hence, increasing VLC-PUFA in the retina of these patients could provide some therapeutic benefits. Thus, we tested the efficiency of elongation of C20-C22 PUFA by the ELOVL4 protein to determine which substrates are the best precursors for biosynthesis of VLC-PUFA. The ELOVL4 protein was expressed in pheochromocytoma cells, while green fluorescent protein-expressing and nontransduced cells served as controls. The cells were treated with 20:5n3, 22:6n3, and 20:4n6, either individually or in equal combinations. Both transduced and control cells internalized and elongated the supplemented FAs to C22-C26 precursors. Only ELOVL4-expressing cells synthesized C28-C38 VLC-PUFA from these precursors. In general, 20:5n3 was more efficiently elongated to VLC-PUFA in the ELOVL4-expressing cells, regardless of whether it was in combination with 22:6n3 or with 20:4n6. In each FA treatment group, C34 and C36 VLC-PUFAs were the predominant VLC-PUFAs in the ELOVL4-expressing cells. In summary, 20:5n3, followed by 20:4n6, seems to be the best precursor for boosting the synthesis of VLC-PUFA by ELOVL4 protein. PMID:22158834

Early embryonic sensitivity to cyclophosphamide in cardiac differentiation from human embryonic stem cells.

PubMed

Zhu, Ming-Xia; Zhao, Jin-Yuan; Chen, Gui-An; Guan, Li

2011-09-01

hESCs (human embryonic stem cells) can differentiate into tissue derivatives of all three germ layers in vitro and mimic the development of the embryo in vivo. In this study, we have investigated the potential of an hESC-based assay for the detection of toxicity to cardiac differentiation in embryonic development. First of all, we developed the protocol of cardiac induction from hESCs according to our previous work and distinguished cardiac precursor cells and late mature cardiomyocytes from differentiated cells, demonstrated by the Q-PCR (quantitative real-time PCR), immunocytochemistry and flow cytometry analysis. In order to test whether CPA (cyclophosphamide) induces developmental and cellular toxicity in the human embryo, we exposed the differentiating cells from hESCs to CPA (a well-known proteratogen) at different stages. We have found that a high concentration of CPA could inhibit cardiac differentiation of hESCs. Two separate exposure intervals were used to determine the effects of CPA on cardiac precursor cells and late mature cardiomyocytes respectively. The cardiac precursor cells were sensitive to CPA in non-cytotoxic concentrations for the expression of the cardiac-specific mRNA markers Nkx2.5 (NK2 transcription factor related, locus 5), GATA-4 (GATA binding protein 4 transcription factor) and TNNT2 (troponin T type 2). Non-cytotoxic CPA concentrations did not affect the mRNA markers' expression in late mature cardiomyocytes, indicating that cardiac precursors were more sensitive to CPA than late cardiomyocytes in cardiogenesis. We set up the in vitro developmental toxicity test model so as to reduce the number of test animals and expenses without compromising the safety of consumers and patients. Furthermore, such in vitro methods may be possibly suited to test a large number of chemicals than the classical employed in vivo tests.

Free and Conjugated Benzoic Acid in Tobacco Plants and Cell Cultures. Induced Accumulation upon Elicitation of Defense Responses and Role as Salicylic Acid Precursors1

PubMed Central

Chong, Julie; Pierrel, Marie-Agnès; Atanassova, Rossitza; Werck-Reichhart, Danièle; Fritig, Bernard; Saindrenan, Patrick

2001-01-01

Salicylic acid (SA) is a key endogenous component of local and systemic disease resistance in plants. In this study, we investigated the role of benzoic acid (BA) as precursor of SA biosynthesis in tobacco (Nicotiana tabacum cv Samsun NN) plants undergoing a hypersensitive response following infection with tobacco mosaic virus or in tobacco cell suspensions elicited with β-megaspermin, an elicitor from Phytophthora megasperma. We found a small pool of conjugated BA in healthy leaves and untreated cell suspensions of tobacco, whereas free BA levels were barely detectable. Infection of plants with tobacco mosaic virus or elicitation of cells led to a rapid de novo synthesis and accumulation of conjugated BA, whereas free BA was weakly induced. In presence of diphenylene iodonium, an inhibitor of superoxide anion formation, SA accumulation was abolished in elicited cells and much higher BA levels were concomitantly induced, mainly as a conjugated form. Furthermore, piperonylic acid, an inhibitor of cinnamate-4-hydroxylase was used as a powerful tool to redirect the metabolic flow from the main phenylpropanoid pathway into the SA biosynthetic branch. Under these conditions, in vivo labeling and radioisotope dilution experiments with [14C]trans-cinnamic acid as precursor clearly indicated that the free form of BA produced in elicited tobacco cells is not the major precursor of SA biosynthesis. The main conjugated form of BA accumulating after elicitation of tobacco cells was identified for the first time as benzoyl-glucose. Our data point to the likely role of conjugated forms of BA in SA biosynthesis. PMID:11154339

Specific Contributions of CSF-1 and GM-CSF to the Dynamics of the Mononuclear Phagocyte System.

PubMed

Louis, Cynthia; Cook, Andrew D; Lacey, Derek; Fleetwood, Andrew J; Vlahos, Ross; Anderson, Gary P; Hamilton, John A

2015-07-01

M-CSF (or CSF-1) and GM-CSF can regulate the development and function of the mononuclear phagocyte system (MPS). To address some of the outstanding and sometimes conflicting issues surrounding this biology, we undertook a comparative analysis of the effects of neutralizing mAbs to these CSFs on murine MPS populations in the steady-state and during acute inflammatory reactions. CSF-1 neutralization, but not of GM-CSF, in normal mice rapidly reduced the numbers of more mature Ly6C(-) monocytes in blood and bone marrow, without any effect on proliferating precursors, and also the numbers of the resident peritoneal macrophages, observations consistent with CSF-1 signaling being essential only at a relatively late state in steady-state MPS development; in contrast, GM-CSF neutralization had no effect on the numbers of these particular populations. In Ag-induced peritonitis (AIP), thioglycolate-induced peritonitis, and LPS-induced lung inflammation, CSF-1 neutralization lowered inflammatory macrophage number; in the AIP model, this reduced number was not due to suppressed proliferation. More detailed studies with the convenient AIP model indicated that CSF-1 neutralization led to a relatively uniform reduction in all inflammatory cell populations; GM-CSF neutralization, in contrast, was more selective, resulting in the preferential loss among the MPS populations of a cycling, monocyte-derived inflammatory dendritic cell population. Some mechanistic options for the specific CSF-dependent biologies enumerated are discussed. Copyright © 2015 by The American Association of Immunologists, Inc.

Two roles for CD4 cells in the control of the generation of cytotoxic T lymphocytes.

PubMed

Cassell, D; Forman, J

1991-01-01

The generation of CTL against Qa-1 Ag in C57BL/6 (B6) (Qa-1b) and B6.Tlaa (Qa-1a) congenic strains requires in vivo priming with the Qa-1 alloantigen together with a helper Ag, such as H-Y. The primed precursors obtained from these female mice generate Qa-1-specific CTL activity upon culture in vitro. Although the presence of the H-Y helper Ag is not required for the in vitro sensitization, no response occurs in the absence of CD4 cells. Addition of unprimed B6.Tlaa CD4 cells from Qa-1 incompatible radiation bone marrow chimeras (B6.Tlaa----B6), that are presumably tolerant to Qa-1b, provide helper activity for Qa-1b-specific CTL. This indicates that although CD4 cells are obligatory for the Qa-1 response, they need not be specific for alloantigens on the APC to generate helper activity in in vitro cultures. Addition of unirradiated B6 CD8-depleted spleen cells to CD4-depleted B6.Tlaa anti-B6 cultures in the presence of either B6.Tlaa CD4 cells or rIL-2 prevents the generation of Qa-1 specific CTL. This inhibition is not due to an anti-idiotypic Ts cell since B6.Tlaa----B6 chimeric cells do not suppress an anti-Qa-1b response. Rather, this finding is consistent with that of a veto cell mechanism. To determine whether CD4 cells themselves exhibit veto activity, highly purified CD4 populations were tested for their ability to inhibit the generation of Qa-1-specific CTL. CD4 cells precultured for 2 to 3 days with Con A and rIL-2 specifically inhibit CTL activity whereas resting cells do not, similar to that noted for CD8 veto cells. The relative efficiency of activated CD4 cells is greater than that of resting NK cells but is less than that of activated CD8 or NK cells. Thus, CD4 cells not only provide helper activity for CTL precursors, but also act as veto cells to prevent the generation of CTL activity.

Stimulation of Mucosal Mast Cell Growth in Normal and Nude Rat Bone Marrow Cultures

NASA Astrophysics Data System (ADS)

Haig, David M.; McMenamin, Christine; Gunneberg, Christian; Woodbury, Richard; Jarrett, Ellen E. E.

1983-07-01

Mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) appear and proliferate to form the predominant cell type in rat bone marrow cultures stimulated with factors from antigen- or mitogen-activated lymphocytes. Conditioned media causing a selective proliferation of MMC were derived from mesenteric lymph node cells of Nippostrongylus brasiliensis-infected rats restimulated in vitro with specific antigen or from normal or infected rat mesenteric lymph node cells stimulated with concanavalin A. MMC growth factor is not produced by T-cell-depleted mesenteric lymph node cells or by the mesenteric lymph node cells of athymic rats. By contrast, MMC precursors are present in the bone marrow of athymic rats and are normally receptive to the growth factor produced by the lymphocytes of thymus-intact rats. The thymus dependence of MMC hyperplasia is thus based on the requirement of a thymus-independent precursor for a T-cell-derived growth promoter.

Prx1 and 3.2 kb Col1a1 promoters target distinct bone cell populations in transgenic mice

PubMed Central

Ouyang, Zhufeng; Chen, Zhijun; Ishikawa, Masakazu; Yue, Xiuzhen; Kawanami, Aya; Leahy, Patrick; Greenfield, Edward M.; Murakami, Shunichi

2014-01-01

Bones consist of a number of cell types including osteoblasts and their precursor cells at various stages of differentiation. To analyze cellular organization within the bone, we generated Col1a1CreER-DsRed transgenic mice that express, in osteoblasts, CreER and DsRed under the control of a mouse 3.2 kb Col1a1 promoter. We further crossed Col1a1CreER-DsRed mice with Prx1CreER-GFP mice that express CreER and GFP in osteochondro progenitor cells under the control of a 2.4 kb Prx1 promoter. Since the 3.2 kb Col1a1 promoter becomes active in osteoblasts at early stages of differentiation, and Prx1CreER-GFP-expressing periosteal cells show endogenous Col1a1 expression, we expected to find a cell population in which both the 2.4 kb Prx1 promoter and the 3.2 kb Col1a1 promoter are active. However, our histological and flow cytometric analyses demonstrated that these transgenes are expressed in distinct cell populations. In the periosteum of long bones, Col1a1CreER-DsRed is expressed in the innermost layer directly lining the bone surface, while Prx1CreER-GFP-expressing cells are localized immediately outside of the Col1a1CreER-DsRed-expressing osteoblasts. In the calvaria, Prx1CreER-GFP-expressing cells are also localized in the cranial suture mesenchyme. Our experiments further showed that Col1a1CreER-DsRed-expressing cells lack chondrogenic potential, while the Prx1CreER-GFP-expressing cells show both chondrogenic and osteogenic potential. Our results indicate that Col1a1CreER-DsRed-expressing cells are committed osteoblasts, while Prx1CreER-GFP-expressing cells are osteochondro progenitor cells. The Prx1CreER-GFP and Col1a1CreER-DsRed transgenes will offer novel approaches for analyzing lineage commitment and early stages of osteoblast differentiation under physiologic and pathologic conditions. PMID:24513582

Histochemical in situ identification of bovine embryonic blood cells reveals differences to the adult haematopoietic system and suggests a close relationship between haematopoietic stem cells and primordial germ cells.

PubMed

Kritzenberger, Michaela; Wrobel, Karl-Heinz

2004-04-01

Cryostat sections of bovine embryos of exactly known age (obtained from artificial insemination), ranging from 32 to 60 days post-insemination, were treated with a wide range of antibodies directed against cell surface antigens or lineage-specific factors in order to demonstrate different types of fetal blood cells and their precursors. An antibody specific to bovine c-kit (bk-1) stained not only presumptive haematopoietic stem cells in the dorsal aorta and the embryonic liver, but also a subpopulation of putative primordial germ cells in the gonadal anlage, the latter being further characterised by a positive labelling with the lectins STA, WFA and WGA and a histochemical reaction for alkaline phosphatase. The antibody against CD 45, commonly regarded as a pan-leukocyte marker, reacted in the bovine embryo with different types of blood cells, as well as with presumptive vasculogenetic cells and a subpopulation of putative primordial germ cells. CD 61 immunoreaction proved to be a useful tool for demonstrating megakaryocytopoiesis in the embryonic liver, in addition to the lumen of blood vessels and the mesonephros. Staining with BM-2 was restricted to a single population of medium-sized, round to oval cells, forming small groups within the parenchymal strands of the liver. Characterised furthermore by a U-shaped nucleus, this BM-2-positive cell type apparently represents a developmental stage in the granulopoietic lineage. B-lymphocytopoiesis in the bovine liver was detected with antibodies directed against WC-4 and IgM, but not until day 58 post-insemination. Using antibodies to CD 14, no positive results could be obtained in embryonic tissues, although anti-CD 14-positive macrophages were easily recognised in lymph nodes of adult bovines. The antibody against CD 68, however, identified two populations of primitive macrophages in our samples. One population was located in parenchymal strands of the embryonic liver, probably acting as nursing cells for haematopoietic foci, and the other was observed intravasally in the sinusoids of the liver, most probably representing primitive Kupffer cells.

Pathogenic mechanisms of allergic inflammation: atopic asthma as a paradigm.

PubMed

Holt, Patrick G; Strickland, Deborah H; Bosco, Anthony; Jahnsen, Frode L

2009-01-01

Prospective studies tracking birth cohorts over periods of years indicate that the seeds for atopic asthma in adulthood are sewn during early life. The key events involve programming of functional phenotypes within the immune and respiratory systems which determine long-term responsiveness to ubiquitous environmental stimuli, particularly respiratory viruses and aeroallergens. A crucial component of asthma pathogenesis is early sensitization to aeroallergens stemming from a failure of mucosal tolerance mechanisms during the preschool years, which is associated with delayed postnatal maturation of a range of adaptive and innate immune functions. These maturational defects also increase risk for severe respiratory infections, and the combination of sensitization and infections maximizes risk for early development of the persistent asthma phenotype. Interactions between immunoinflammatory pathways stimulated by these agents also sustain the disease in later life as major triggers of asthma exacerbations. Recent studies on the nature of these interactions suggest the operation of an infection-associated lung:bone marrow axis involving upregulation of FcERlalpha on myeloid precursor populations prior to their migration to the airways, thus amplifying local inflammation via IgE-mediated recruitment of bystander atopic effector mechanisms. The key participants in the disease process are airway mucosal dendritic cells and adjacent epithelial cells, and transiting CD4(+) effector and regulatory T-cell populations, and increasingly detailed characterization of their roles at different stages of pathogenesis is opening up novel possibilities for therapeutic control of asthma. Of particular interest is the application of genomics-based approaches to drug target identification in cell populations of interest, exemplified by recent findings discussed below relating to the gene network(s) triggered by activation of Th2-memory cells from atopics. 2009 Elsevier Inc. All rights reserved.

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

PubMed Central

Agley, Chibeza C.; Rowlerson, Anthea M.; Velloso, Cristiana P.; Lazarus, Norman L.; Harridge, Stephen D. R.

2015-01-01

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56– and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (>95% myogenic cells) and good yield (~2.8 x 106 ± 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56– cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package. PMID:25650991

Vaticaffinol, a resveratrol tetramer, exerts more preferable immunosuppressive activity than its precursor in vitro and in vivo through multiple aspects against activated T lymphocytes

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

Feng, Li-Li; Wu, Xue-Feng; Liu, Hai-Liang

2013-03-01

In the present study, we aimed to investigate the immunosuppressive activity of vaticaffinol, a resveratrol tetramer isolated from Vatica mangachapoi, on T lymphocytes both in vitro and in vivo, and further explored its potential molecular mechanism. Resveratrol had a wide spectrum of healthy beneficial effects with multiple targets. Interestingly, its tetramer, vaticaffinol, exerted more intensive immunosuppressive activity than resveratrol. Vaticaffinol significantly inhibited T cells proliferation activated by concanavalin A (Con A) or anti-CD3 plus anti-CD28 in a dose- and time-dependent manner. It also induced Con A-activated T cells undergoing apoptosis through mitochondrial pathway. Moreover, this compound prevented cells from enteringmore » S phase and G2/M phase during T cells activation. In addition, vaticaffinol inhibited ERK and AKT signaling pathways in Con A-activated T cells. Furthermore, vaticaffinol significantly ameliorated ear swelling in a mouse model of picryl chloride-induced ear contact dermatitis in vivo. In most of the aforementioned experiments, however, resveratrol had only slight effects on the inhibition of T lymphocytes compared with vaticaffinol. Taken together, our findings suggest that vaticaffinol exerts more preferable immunosuppressive activity than its precursor resveratrol both in vitro and in vivo by affecting multiple targets against activated T cells. - Graphical abstract: Vaticaffinol, a resveratrol tetramer isolated from Vatica mangachapoi, exerts more intensive immunosuppressive activity than its precursor resveratrol does in vitro and in vivo. Its mechanism may involve multiple effects against activated T cells: regulation of signalings involved in cell proliferation, G0/G1 arrest of T cells, as well as an apoptosis induction in activated effector T cells. Highlights: ► Vaticaffinol, a resveratrol tetramer, exerts more potent activity than its precursor. ► It inhibited T cells proliferation and prevented them from entering cell cycles. ► It led to apoptosis of activated T cells through mitochondrial pathway. ► It down-regulated ERK and AKT signaling pathways in Con A-activated T cells. ► It significantly ameliorated picryl chloride-induced ear swelling.« less

Differential sensitivity to cadmium of immunomarkers measured in hemocyte subpopulations of zebra mussel Dreissena polymorpha.

PubMed

Evariste, Lauris; Rioult, Damien; Brousseau, Pauline; Geffard, Alain; David, Elise; Auffret, Michel; Fournier, Michel; Betoulle, Stéphane

2017-03-01

Increasing discharge of industrial wastes into the environment results in pollution transfer towards hydrosystems. These activities release heavy metals such as cadmium, known as persistent pollutant that is accumulated by molluscs and exercise immunotoxicological effects. Among molluscs, the zebra mussel, Dreissena polymorpha constitutes a suitable support for freshwater ecotoxicological studies. In molluscs, homeostasis maintain is ensured in part by hemocytes that are composed of several cell populations involved in multiple physiological processes such as cell-mediated immune response or metal metabolism. Thus, hemocytes constitute a target of concern to study adverse effects of heavy metals. The objectives of this work were to determine whether immune-related endpoints assessed were of different sensitivity to cadmium and whether hemocyte functionalities were differentially affected depending on hemocyte subpopulation considered. Hemocytes were exposed ex vivo to concentrations of cadmium ranging from 10 -6 M to 10 -3 M for 21h prior flow cytometric analysis of cellular markers. Measured parameters (viability, phagocytosis, oxidative activity, lysosomal content) decreased in a dose-dependent manner with sensitivity differences depending on endpoint and cell type considered. Our results indicated that phagocytosis related endpoints were the most sensitive studied mechanisms to cadmium compared to other markers with EC 50 of 3.71±0.53×10 -4 M for phagocytic activity and 2.79±0.19×10 -4 M considering mean number of beads per phagocytic cell. Lysosomal content of granulocytes was less affected compared to other cell types, indicating lower sensitivity to cadmium. This suggests that granulocyte population is greatly involved in metal metabolism. Mitochondrial activity was reduced only in blast-like hemocytes that are considered to be cell precursors. Impairment of these cell functionalities may potentially compromise functions ensured by differentiated cells. We concluded that analysis of hemocyte activities should be performed at sub-population scale for more accurate results in ecotoxicological studies. Copyright © 2016 Elsevier Inc. All rights reserved.

Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages

PubMed Central

Laugwitz, Karl-Ludwig; Moretti, Alessandra; Lam, Jason; Gruber, Peter; Chen, Yinhong; Woodard, Sarah; Lin, Li-Zhu; Cai, Chen-Leng; Lu, Min Min; Reth, Michael; Platoshyn, Oleksandr; Yuan, Jason X.-J.; Evans, Sylvia; Chien, Kenneth R.

2017-01-01

The purification, renewal and differentiation of native cardiac progenitors would form a mechanistic underpinning for unravelling steps for cardiac cell lineage formation, and their links to forms of congenital and adult cardiac diseases1–3. Until now there has been little evidence for native cardiac precursor cells in the postnatal heart4. Herein, we report the identification of isl1+ cardiac progenitors in postnatal rat, mouse and human myocardium. A cardiac mesenchymal feeder layer allows renewal of the isolated progenitor cells with maintenance of their capability to adopt a fully differentiated cardiomyocyte phenotype. Tamoxifen-inducible Cre/lox technology enables selective marking of this progenitor cell population including its progeny, at a defined time, and purification to relative homogeneity. Co-culture studies with neonatal myocytes indicate that isl1+ cells represent authentic, endogenous cardiac progenitors (cardioblasts) that display highly efficient conversion to a mature cardiac phenotype with stable expression of myocytic markers (25%) in the absence of cell fusion, intact Ca2+-cycling, and the generation of action potentials. The discovery of native cardioblasts represents a genetically based system to identify steps in cardiac cell lineage formation and maturation in development and disease. PMID:15703750

Osteoclast size heterogeneity in rat long bones is associated with differences in adhesive ligand specificity.

PubMed

Hu, Yingwei; Ek-Rylander, Barbro; Karlström, Erik; Wendel, Mikael; Andersson, Göran

2008-02-01

Prothrombin (PT) is an RGD-containing bone-residing precursor to the serine protease thrombin (TH), which acts as an agonist for a variety of cellular responses in osteoblasts and osteoclasts. We show here that PT, TH, osteopontin (OPN) and fibronectin (FN) promoted adhesion of isolated neonatal rat long bone osteoclasts. However, the cells that adhered to PT and TH were smaller in size, rounded and contained 3-4 nuclei, in comparison to the cells adhering to OPN and FN, which were larger with extended cytoplasmic processes and 6-7 nuclei. Attachment of the larger osteoclasts to OPN and FN was inhibited by antibodies towards beta 3 and beta 1 integrin subunits, respectively. Whereas an RGD-containing peptide inhibited adhesion of the smaller osteoclasts to PT and TH, this was not seen with the beta 3 or beta 1 antibodies. In contrast, the beta 1 antibody augmented osteoclast adhesion to PT and TH in an RGD-dependent manner. Small osteoclasts were less efficient in resorbing mineralized bovine bone slices, as well as expressed lower mRNA levels of MMP-9 and the cathepsins K and L compared to large osteoclasts. The small osteoclast adhering to PT and TH may represent either an immature, less functional precursor to the large osteoclast or alternatively constitute a distinct osteoclast population with a specific role in bone.

Precursor to the TRICARE Next Generation Program

DTIC Science & Technology

2004-06-01

Precursor to T-NEX 6 Appendix A - Maps of Interest 84 Appendix B - International Classification of Diseases Codes 86...utilization management, and disease management (Lupo, 2003). Health management will be demonstrated by measurable quality improvements, population...National Association of County and City Health Officials and the Center for Disease Control and Prevention that helps key officials review community

40 CFR Appendix D to Part 58 - Network Design Criteria for Ambient Air Quality Monitoring

Code of Federal Regulations, 2013 CFR

2013-07-01

... determine the extent of regional pollutant transport among populated areas; and in support of secondary... sources within the area, transport of O3 and its precursors, and the photochemical processes related to O3... precursor concentrations entering the area and will identify those areas which are subjected to transport...

40 CFR Appendix D to Part 58 - Network Design Criteria for Ambient Air Quality Monitoring

Code of Federal Regulations, 2014 CFR

2014-07-01

... determine the extent of regional pollutant transport among populated areas; and in support of secondary... sources within the area, transport of O3 and its precursors, and the photochemical processes related to O3... precursor concentrations entering the area and will identify those areas which are subjected to transport...

Circulating CXCR5+CXCR3+PD-1lo Tfh-like cells in HIV-1 controllers with neutralizing antibody breadth

PubMed Central

Martin-Gayo, Enrique; Cronin, Jacqueline; Hickman, Taylor; Ouyang, Zhengyu; Lindqvist, Madelene; Kolb, Kellie E.; Schulze zur Wiesch, Julian; Cubas, Rafael; Porichis, Filippos; Shalek, Alex K.; van Lunzen, Jan; Haddad, Elias K.; Walker, Bruce D.; Kaufmann, Daniel E.; Lichterfeld, Mathias; Yu, Xu G.

2017-01-01

HIV-1–specific broadly neutralizing antibodies (bnAbs) typically develop in individuals with continuous high-level viral replication and increased immune activation, conditions that cannot be reproduced during prophylactic immunization. Understanding mechanisms supporting bnAb development in the absence of high-level viremia may be important for designing bnAb-inducing immunogens. Here, we show that the breadth of neutralizing antibody responses in HIV-1 controllers was associated with a relative enrichment of circulating CXCR5+CXCR3+PD-1lo CD4+ T cells. These CXCR3+PD-1lo Tfh-like cells were preferentially induced in vitro by functionally superior dendritic cells from controller neutralizers, and able to secrete IL-21 and support B cells. In addition, these CXCR3+PD-1lo Tfh-like cells contained higher proportions of stem cell–like memory T cells, and upon antigenic stimulation differentiated into PD-1hi Tfh-like cells in a Notch-dependent manner. Together, these data suggest that CXCR5+CXCR3+PD-1lo cells represent a dendritic cell–primed precursor cell population for PD-1hi Tfh-like cells that may contribute to the generation of bnAbs in the absence of high-level viremia. PMID:28138558

Nuclear localization of foamy virus Gag precursor protein.

PubMed Central

Schliephake, A W; Rethwilm, A

1994-01-01

All foamy viruses give rise to a strong nuclear staining when infected cells are reacted with sera from infected hosts. This nuclear fluorescence distinguishes foamy viruses from all other retroviruses. The experiments reported here indicate that the foamy virus Gag precursor protein is transiently located in the nuclei of infected cells and this is the likely reason for the typical foamy virus nuclear fluorescence. By using the vaccinia virus expression system, a conserved basic sequence motif in the nucleocapsid domain of foamy virus Gag proteins was identified to be responsible for the nuclear transport of the gag precursor molecule. This motif was also found to be able to direct a heterologous protein, the Gag protein of human immunodeficiency virus, into the nucleus. Images PMID:8035493

High volume method of making low-cost, lightweight solar materials

DOEpatents

Blue, Craig A.; Clemens, Art; Duty, Chad E.; Harper, David C.; Ott, Ronald D.; Rivard, John D.; Murray, Christopher S.; Murray, Susan L.; Klein, Andre R.

2014-07-15

A thin film solar cell and a method fabricating thin film solar cells on flexible substrates. The method includes including providing a flexible polymeric substrate, depositing a photovoltaic precursor on a surface of the substrate, such as CdTe, ZrTe, CdZnTe, CdSe or Cu(In,Ga)Se.sub.2, and exposing the photovoltaic precursor to at least one 0.5 microsecond to 10 second pulse of predominately infrared light emitted from a light source having a power output of about 20,000 W/cm.sup.2 or less to thermally convert the precursor into a crystalline photovoltaic material having a photovoltaic efficiency of greater than one percent, the conversion being carried out without substantial damage to the substrate.

Estimating human exposure to PFOS isomers and PFCA homologues: the relative importance of direct and indirect (precursor) exposure.

PubMed

Gebbink, Wouter A; Berger, Urs; Cousins, Ian T

2015-01-01

Contributions of direct and indirect (via precursors) pathways of human exposure to perfluorooctane sulfonic acid (PFOS) isomers and perfluoroalkyl carboxylic acids (PFCAs) are estimated using a Scenario-Based Risk Assessment (SceBRA) modelling approach. Monitoring data published since 2008 (including samples from 2007) are used. The estimated daily exposures (resulting from both direct and precursor intake) for the general adult population are highest for PFOS and perfluorooctanoic acid (PFOA), followed by perfluorohexanoic acid (PFHxA) and perfluorodecanoic acid (PFDA), while lower daily exposures are estimated for perfluorobutanoic acid (PFBA) and perfluorododecanoic acid (PFDoDA). The precursor contributions to the individual perfluoroalkyl acid (PFAA) daily exposures are estimated to be 11-33% for PFOS, 0.1-2.5% for PFBA, 3.7-34% for PFHxA, 13-64% for PFOA, 5.2-66% for PFDA, and 0.7-25% for PFDoDA (ranges represent estimated precursor contributions in a low- and high-exposure scenario). For PFOS, direct intake via diet is the major exposure pathway regardless of exposure scenario. For PFCAs, the dominant exposure pathway is dependent on perfluoroalkyl chain length and exposure scenario. Modelled PFOS and PFOA concentrations in human serum using the estimated intakes from an intermediate-exposure scenario are in agreement with measured concentrations in different populations. The isomer pattern of PFOS resulting from total intakes (direct and via precursors) is estimated to be enriched with linear PFOS (84%) relative to technical PFOS (70% linear). This finding appears to be contradictory to the observed enrichment of branched PFOS isomers in recent human serum monitoring studies and suggests that either external exposure is not fully understood (e.g. there are unknown precursors, missing or poorly quantified exposure pathways) and/or that there is an incomplete understanding of the isomer-specific human pharmacokinetic processes of PFOS, its precursors and intermediates. Copyright © 2014. Published by Elsevier Ltd.

Platinum-free catalysts for low temperature fuel cells

NASA Astrophysics Data System (ADS)

Lastovina, Tatiana; Pimonova, Julia; Budnyk, Andriy

2017-04-01

In this work, we have successfully prepared Zn/Co-N/C and Zn/Co-Fe/N/C composites, both derived from single zeolitic imidazolate framework (ZIF) precursor Zn/Co-ZIF containing equivalent quantities of Zn and Co metal sites. The composites were formed by pyrolysis of the precursor at 700 °C in inert gas atmosphere as such and after mixing it with Fe(II) salt and 1,10-phenontraline in ethanol. Catalytic tests for oxygen reduction reaction (ORR) in electrochemical cell demonstrated promising results allowing us to consider these composites as potential Pt-free catalysts for low temperature fuel cells.

From the outside, from within: Biological and therapeutic relevance of signal transduction in T-cell acute lymphoblastic leukemia.

PubMed

Oliveira, Mariana L; Akkapeddi, Padma; Alcobia, Isabel; Almeida, Afonso R; Cardoso, Bruno A; Fragoso, Rita; Serafim, Teresa L; Barata, João T

2017-10-01

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer that arises from clonal expansion of transformed T-cell precursors. In this review we summarize the current knowledge on the external stimuli and cell-intrinsic lesions that drive aberrant activation of pivotal, pro-tumoral intracellular signaling pathways in T-cell precursors, driving transformation, leukemia expansion, spread or resistance to therapy. In addition to their pathophysiological relevance, receptors and kinases involved in signal transduction are often attractive candidates for targeted drug development. As such, we discuss also the potential of T-ALL signaling players as targets for therapeutic intervention. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The effect of magnetic nanoparticles on neuronal differentiation of induced pluripotent stem cell-derived neural precursors

PubMed Central

Jiráková, Klára; Šeneklová, Monika; Jirák, Daniel; Turnovcová, Karolína; Vosmanská, Magda; Babič, Michal; Horák, Daniel; Veverka, Pavel; Jendelová, Pavla

2016-01-01

Introduction Magnetic resonance (MR) imaging is suitable for noninvasive long-term tracking. We labeled human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) with two types of iron-based nanoparticles, silica-coated cobalt zinc ferrite nanoparticles (CZF) and poly-l-lysine-coated iron oxide superparamagnetic nanoparticles (PLL-coated γ-Fe2O3) and studied their effect on proliferation and neuronal differentiation. Materials and methods We investigated the effect of these two contrast agents on neural precursor cell proliferation and differentiation capability. We further defined the intracellular localization and labeling efficiency and analyzed labeled cells by MR. Results Cell proliferation was not affected by PLL-coated γ-Fe2O3 but was slowed down in cells labeled with CZF. Labeling efficiency, iron content and relaxation rates measured by MR were lower in cells labeled with CZF when compared to PLL-coated γ-Fe2O3. Cytoplasmic localization of both types of nanoparticles was confirmed by transmission electron microscopy. Flow cytometry and immunocytochemical analysis of specific markers expressed during neuronal differentiation did not show any significant differences between unlabeled cells or cells labeled with both magnetic nanoparticles. Conclusion Our results show that cells labeled with PLL-coated γ-Fe2O3 are suitable for MR detection, did not affect the differentiation potential of iPSC-NPs and are suitable for in vivo cell therapies in experimental models of central nervous system disorders. PMID:27920532

Lack of effect of a granulocyte proliferation inhibitor or their committed precursor cells.

PubMed

Lord, B I; Testa, N G; Wright, E G; Banerjee, R K

1977-05-01

Using the agar culture technique, we have measured the effect of granulocyte extracts GCE (and of erythrocyte-RCE and lymph node extracts-LNE) on the growth and proliferation of the committed granulocytic precursor cells, CFU-C. In addition we have determined their effects on the proliferation of the developing colony cells and on the ultimate cell production in the colonies. The results show that GCE has no effect on the growth or proliferative activity on the CFU-C. It does, however, reduce both the autoradiographic labelling indices of the developing colony cells and the net colony cellularities, acting as a cell cycle modulator. These are effects specific to the GCE since at the dose levels used, neither RCE nor LNE affected these measurements.

Bone Morphogenetic Protein Regulation of Enteric Neuronal Phenotypic Diversity: Relationship to Timing of Cell Cycle Exit

PubMed Central

Chalazonitis, Alcmène; Pham, Tuan.D.; Li, Zhishan; Roman, Daniel; Guha, Udayan; Gomes, William; Kan, Lixin; Kessler, John A.; Gershon, Michael D.

2008-01-01

The effects of bone morphogenetic protein (BMP) signaling on enteric neuron development were examined in transgenic mice over expressing either the BMP inhibitor, noggin, or BMP4 under control of the neuron specific enolase (NSE) promoter. Noggin antagonism of BMP signaling increased total numbers of enteric neurons and those of subpopulations derived from precursors that exit the cell cycle early in neurogenesis (serotonin, calretinin, calbindin). In contrast, noggin overexpression decreased numbers of neurons derived from precursors that exit the cell cycle late (γ-aminobutyric acid, tyrosine hydroxylase [TH], dopamine transporter, calcitonin gene related peptide, TrkC). Numbers of TH- and TrkC-expressing neurons were increased by overexpression of BMP4. These observations are consistent with the idea that phenotypic expression in the enteric nervous system (ENS) is determined, in part, by the number of proliferative divisions neuronal precursors undergo before their terminal mitosis. BMP signaling may thus regulate enteric neuronal phenotypic diversity by promoting the exit of precursors from the cell cycle. BMP2 increased the numbers of TH- and TrkC-expressing neurons developing in vitro from immunoselected enteric crest-derived precursors; BMP signaling may thus also specify or promote the development of dopaminergic TrkC/NT-3-dependent neurons. The developmental defects in the ENS of noggin overexpressing mice caused a relatively mild disturbance of motility (irregular rapid transit and increased stool frequency, weight, and water content). Although the function of the gut thus displays a remarkable tolerance for ENS defects, subtle functional abnormalities in motility or secretion may arise when ENS defects short of aganglionosis occur during development. PMID:18537141

The Amyloid Precursor Protein (APP) Triplicated Gene Impairs Neuronal Precursor Differentiation and Neurite Development through Two Different Domains in the Ts65Dn Mouse Model for Down Syndrome*

PubMed Central

Trazzi, Stefania; Fuchs, Claudia; Valli, Emanuele; Perini, Giovanni; Bartesaghi, Renata; Ciani, Elisabetta

2013-01-01

Intellectual disability in Down syndrome (DS) appears to be related to severe proliferation impairment during brain development. Recent evidence shows that it is not only cellular proliferation that is heavily compromised in DS, but also cell fate specification and dendritic maturation. The amyloid precursor protein (APP), a gene that is triplicated in DS, plays a key role in normal brain development by influencing neural precursor cell proliferation, cell fate specification, and neuronal maturation. APP influences these processes via two separate domains, the APP intracellular domain (AICD) and the soluble secreted APP. We recently found that the proliferation impairment of neuronal precursors (NPCs) from the Ts65Dn mouse model for DS was caused by derangement of the Shh pathway due to overexpression of patched1(Ptch1), its inhibitory regulator. Ptch1 overexpression was related to increased levels within the APP/AICD system. The overall goal of this study was to determine whether APP contributes to neurogenesis impairment in DS by influencing in addition to proliferation, cell fate specification, and neurite development. We found that normalization of APP expression restored the reduced neuronogenesis, the increased astrogliogenesis, and the reduced neurite length of trisomic NPCs, indicating that APP overexpression underpins all aspects of neurogenesis impairment. Moreover, we found that two different domains of APP impair neuronal differentiation and maturation in trisomic NPCs. The APP/AICD system regulates neuronogenesis and neurite length through the Shh pathway, whereas the APP/secreted AP system promotes astrogliogenesis through an IL-6-associated signaling cascade. These results provide novel insight into the mechanisms underlying brain development alterations in DS. PMID:23740250

MASTL is essential for anaphase entry of proliferating primordial germ cells and establishment of female germ cells in mice

PubMed Central

Risal, Sanjiv; Zhang, Jingjing; Adhikari, Deepak; Liu, Xiaoman; Shao, Jingchen; Hu, Mengwen; Busayavalasa, Kiran; Tu, Zhaowei; Chen, Zijiang; Kaldis, Philipp; Liu, Kui

2017-01-01

In mammals, primordial germ cells (PGCs) are the embryonic cell population that serve as germ cell precursors in both females and males. During mouse embryonic development, the majority of PGCs are arrested at the G2 phase when they migrate into the hindgut at 7.75–8.75 dpc (days post coitum). It is after 9.5 dpc that the PGCs undergo proliferation with a doubling time of 12.6 h. The molecular mechanisms underlying PGC proliferation are however not well studied. In this work. Here we studied how MASTL (microtubule-associated serine/threonine kinase-like)/Greatwall kinase regulates the rapid proliferation of PGCs. We generated a mouse model where we specifically deleted Mastl in PGCs and found a significant loss of PGCs before the onset of meiosis in female PGCs. We further revealed that the deletion of Mastl in PGCs did not prevent mitotic entry, but led to a failure of the cells to proceed beyond metaphase-like stage, indicating that MASTL-mediated molecular events are indispensable for anaphase entry in PGCs. These mitotic defects further led to the death of Mastl-null PGCs by 12.5 dpc. Moreover, the defect in mitotic progression observed in the Mastl-null PGCs was rescued by simultaneous deletion of Ppp2r1a (α subunit of PP2A). Thus, our results demonstrate that MASTL, PP2A, and therefore regulated phosphatase activity have a fundamental role in establishing female germ cell population in gonads by controlling PGC proliferation during embryogenesis. PMID:28224044

Immunological and biochemical characterization of processing products from the neurotensin/neuromedin N precursor in the rat medullary thyroid carcinoma 6-23 cell line.

PubMed Central

Bidard, J N; de Nadai, F; Rovere, C; Moinier, D; Laur, J; Martinez, J; Cuber, J C; Kitabgi, P

1993-01-01

Neurotensin (NT) and neuromedin N (NN) are two related biologically active peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide starting with an N-terminal signal peptide and containing in its C-terminal region one copy each of NT and NN. NN precedes NT and is separated from it by a Lys-Arg sequence. Two other Lys-Arg sequences flank the N-terminus of NN and the C-terminus of NT. A fourth Lys-Arg sequence occurs near the middle of the precursor and is followed by an NN-like sequence. Finally, an Arg-Arg pair is present within the NT moiety. The four Lys-Arg doublets represent putative processing sites in the precursor molecule. The present study was designed to investigate the post-translational processing of the NT/NN precursor in the rat medullary thyroid carcinoma (rMTC) 6-23 cell line, which synthesizes large amounts of NT upon dexamethasone treatment. Five region-specific antisera recognizing the free N- or C-termini of sequences adjacent to the basic doublets were produced, characterized and used for immunoblotting and radioimmunoassay studies in combination with gel filtration, reverse-phase h.p.l.c. and trypsin digestion of rMTC 6-23 cell extracts. Because two of the antigenic sequences, i.e. NN and the NN-like sequence, start with a lysine residue that is essential for recognition by their respective antisera, a micromethod by which trypsin specifically cleaves at arginine residues was developed. The results show that dexamethasone-treated rMTC 6-23 cells produced comparable amounts of NT, NN and a peptide corresponding to a large N-terminal precursor fragment lacking the NN and NT moieties. This large fragment was purified. N-Terminal sequencing revealed that it started at residue Ser23 of the prepro-NT/NN sequence, and thus established the Cys22-Ser23 bond as the cleavage site of the signal peptide. Two other large N-terminal fragments bearing respectively the NN and NT sequences at their C-termini were present in lower amounts. The NN-like sequence was internal to all the large fragments. There was no evidence for the presence of peptides with the NN-like sequence at their N-termini. This shows that, in rMTC 6-23 cells, the precursor is readily processed at the three Lys-Arg doublets that flank and separate the NT and NN sequences. In contrast, the Lys-Arg doublet that precedes the NN-like sequence is not processed in this system.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 3 PMID:8471039

Meninges: from protective membrane to stem cell niche.

PubMed

Decimo, Ilaria; Fumagalli, Guido; Berton, Valeria; Krampera, Mauro; Bifari, Francesco

2012-01-01

Meninges are a three tissue membrane primarily known as coverings of the brain. More in depth studies on meningeal function and ultrastructure have recently changed the view of meninges as a merely protective membrane. Accurate evaluation of the anatomical distribution in the CNS reveals that meninges largely penetrate inside the neural tissue. Meninges enter the CNS by projecting between structures, in the stroma of choroid plexus and form the perivascular space (Virchow-Robin) of every parenchymal vessel. Thus, meninges may modulate most of the physiological and pathological events of the CNS throughout the life. Meninges are present since the very early embryonic stages of cortical development and appear to be necessary for normal corticogenesis and brain structures formation. In adulthood meninges contribute to neural tissue homeostasis by secreting several trophic factors including FGF2 and SDF-1. Recently, for the first time, we have identified the presence of a stem cell population with neural differentiation potential in meninges. In addition, we and other groups have further described the presence in meninges of injury responsive neural precursors. In this review we will give a comprehensive view of meninges and their multiple roles in the context of a functional network with the neural tissue. We will highlight the current literature on the developmental feature of meninges and their role in cortical development. Moreover, we will elucidate the anatomical distribution of the meninges and their trophic properties in adult CNS. Finally, we will emphasize recent evidences suggesting the potential role of meninges as stem cell niche harbouring endogenous precursors that can be activated by injury and are able to contribute to CNS parenchymal reaction.

Design and crystal structure of a native-like HIV-1 envelope trimer that engages multiple broadly neutralizing antibody precursors in vivo

DOE PAGES

Medina-Ramírez, Max; Garces, Fernando; Escolano, Amelia; ...

2017-08-28

Induction of broadly neutralizing antibodies (bNAbs) by HIV-1 envelope glycoprotein immunogens would be a major advance toward an effective vaccine. A critical step in this process is the activation of naive B cells expressing germline (gl) antibody precursors that have the potential to evolve into bNAbs. Here, we reengineered the BG505 SOSIP.664 glycoprotein to engage gl precursors of bNAbs that target either the trimer apex or the CD4-binding site. The resulting BG505 SOSIP.v4.1-GT1 trimer binds multiple bNAb gl precursors in vitro. Immunization experiments in knock-in mice expressing gl-VRC01 or gl-PGT121 show that this trimer activates B cells in vivo, resultingmore » in the secretion of specific antibodies into the sera. A crystal structure of the gl-targeting trimer at 3.2-Å resolution in complex with neutralizing antibodies 35O22 and 9H+109L reveals a native-like conformation and the successful incorporation of design features associated with binding of multiple gl-bNAb precursors.« less

Design and crystal structure of a native-like HIV-1 envelope trimer that engages multiple broadly neutralizing antibody precursors in vivo

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

Medina-Ramírez, Max; Garces, Fernando; Escolano, Amelia

Induction of broadly neutralizing antibodies (bNAbs) by HIV-1 envelope glycoprotein immunogens would be a major advance toward an effective vaccine. A critical step in this process is the activation of naive B cells expressing germline (gl) antibody precursors that have the potential to evolve into bNAbs. Here, we reengineered the BG505 SOSIP.664 glycoprotein to engage gl precursors of bNAbs that target either the trimer apex or the CD4-binding site. The resulting BG505 SOSIP.v4.1-GT1 trimer binds multiple bNAb gl precursors in vitro. Immunization experiments in knock-in mice expressing gl-VRC01 or gl-PGT121 show that this trimer activates B cells in vivo, resultingmore » in the secretion of specific antibodies into the sera. A crystal structure of the gl-targeting trimer at 3.2-Å resolution in complex with neutralizing antibodies 35O22 and 9H+109L reveals a native-like conformation and the successful incorporation of design features associated with binding of multiple gl-bNAb precursors.« less

Induction of Skin-Derived Precursor Cells from Human Induced Pluripotent Stem Cells.

PubMed

Sugiyama-Nakagiri, Yoriko; Fujimura, Tsutomu; Moriwaki, Shigeru

2016-01-01

The generation of full thickness human skin from dissociated cells is an attractive approach not only for treating skin diseases, but also for treating many systemic disorders. However, it is currently not possible to obtain an unlimited number of skin dermal cells. The goal of this study was to develop a procedure to produce skin dermal stem cells from induced pluripotent stem cells (iPSCs). Skin-derived precursor cells (SKPs) were isolated as adult dermal precursors that could differentiate into both neural and mesodermal progenies and could reconstitute the dermis. Thus, we attempted to generate SKPs from iPSCs that could reconstitute the skin dermis. Human iPSCs were initially cultured with recombinant noggin and SB431542, an inhibitor of activin/nodal and TGFβ signaling, to induce neural crest progenitor cells. Those cells were then treated with SKP medium that included CHIR99021, a WNT signal activator. The induction efficacy from neural crest progenitor cells to SKPs was more than 97%. No other modifiers tested were able to induce those cells. Those human iPSC-derived SKPs (hiPSC-SKPs) showed a similar gene expression signature to SKPs isolated from human skin dermis. Human iPSC-SKPs differentiated into neural and mesodermal progenies, including adipocytes, skeletogenic cell types and Schwann cells. Moreover, they could be induced to follicular type keratinization when co-cultured with human epidermal keratinocytes. We here provide a new efficient protocol to create human skin dermal stem cells from hiPSCs that could contribute to the treatment of various skin disorders.

Dual role for Drosophila lethal of scute in CNS midline precursor formation and dopaminergic neuron and motoneuron cell fate

PubMed Central

Stagg, Stephanie B.; Guardiola, Amaris R.; Crews, Stephen T.

2011-01-01

Dopaminergic neurons play important behavioral roles in locomotion, reward and aggression. The Drosophila H-cell is a dopaminergic neuron that resides at the midline of the ventral nerve cord. Both the H-cell and the glutamatergic H-cell sib are the asymmetric progeny of the MP3 midline precursor cell. H-cell sib cell fate is dependent on Notch signaling, whereas H-cell fate is Notch independent. Genetic analysis of genes that could potentially regulate H-cell fate revealed that the lethal of scute [l(1)sc], tailup and SoxNeuro transcription factor genes act together to control H-cell gene expression. The l(1)sc bHLH gene is required for all H-cell-specific gene transcription, whereas tailup acts in parallel to l(1)sc and controls genes involved in dopamine metabolism. SoxNeuro functions downstream of l(1)sc and controls expression of a peptide neurotransmitter receptor gene. The role of l(1)sc may be more widespread, as a l(1)sc mutant shows reductions in gene expression in non-midline dopaminergic neurons. In addition, l(1)sc mutant embryos possess defects in the formation of MP4-6 midline precursor and the median neuroblast stem cell, revealing a proneural role for l(1)sc in midline cells. The Notch-dependent progeny of MP4-6 are the mVUM motoneurons, and these cells also require l(1)sc for mVUM-specific gene expression. Thus, l(1)sc plays an important regulatory role in both neurogenesis and specifying dopaminergic neuron and motoneuron identities. PMID:21558367

Early postnatal GFAP-expressing cells produce multilineage progeny in cerebrum and astrocytes in cerebellum of adult mice.

PubMed

Guo, Zhibao; Wang, Xijuan; Xiao, Jun; Wang, Yihui; Lu, Hong; Teng, Junfang; Wang, Wei

2013-09-26

Early postnatal GFAP-expressing cells are thought to be immature astrocytes. However, it is not clear if they possess multilineage capacity and if they can generate different lineages (astrocytes, neurons and oligodendrocytes) in the brain of adult mice. In order to identify the fate of astroglial cells in the postnatal brain, hGFAP-Cre-ER(T2) transgenic mice were crossed with the R26R Cre reporter mouse strains which exhibit constitutive expression of β-galactosidase (β-gal). Mice carrying the hGFAP-Cre-ER(T2)/R26R transgene were treated with Tamoxifen to induce Cre recombination in astroglial cells at postnatal (P) day 6 and Cre recombinase-expressing cells were identified by X-gal staining. Immunohistochemical staining was used to identify the type(s) of these reporter-tagged cells. Sixty days after recombination, X-gal-positive cells in different cerebral regions of the adult mice expressed the astroglial markers Blbp and GFAP, the neuronal marker NeuN, the oligodendrocyte precursor cell marker NG2 and the mature oligodendrocyte marker CC1. X-gal-positive cells in the cerebellum coexpressed the astroglial marker Blbp, but not the granule cell marker NeuN, Purkinje cell marker Calbindin or oligodendrocyte precursor cell marker NG2. Our genetic fate mapping data demonstrated that early postnatal GFAP-positive cells possessed multilineage potential and eventually differentiated into neurons, astrocytes, and oligodendrocyte precursor cells in the cerebrum and into astrocytes (including Bergmann glia) in the cerebellum of adult mice. © 2013 Elsevier B.V. All rights reserved.

Dedifferentiated Schwann Cell Precursors Secreting Paracrine Factors Are Required for Regeneration of the Mammalian Digit Tip.

PubMed

Johnston, Adam P W; Yuzwa, Scott A; Carr, Matthew J; Mahmud, Neemat; Storer, Mekayla A; Krause, Matthew P; Jones, Karen; Paul, Smitha; Kaplan, David R; Miller, Freda D

2016-10-06

Adult mammals have lost multi-tissue regenerative capacity, except for the distal digit, which is able to regenerate via mechanisms that remain largely unknown. Here, we show that, after adult mouse distal digit removal, nerve-associated Schwann cell precursors (SCPs) dedifferentiate and secrete growth factors that promote expansion of the blastema and digit regeneration. When SCPs were dysregulated or ablated, mesenchymal precursor proliferation in the blastema was decreased and nail and bone regeneration were impaired. Transplantation of exogenous SCPs rescued these regeneration defects. We found that SCPs secrete factors that promote self-renewal of mesenchymal precursors, and we used transcriptomic and proteomic analysis to define candidate factors. Two of these, oncostatin M (OSM) and platelet-derived growth factor AA (PDGF-AA), are made by SCPs in the regenerating digit and rescued the deficits in regeneration caused by loss of SCPs. As all peripheral tissues contain nerves, these results could have broad implications for mammalian tissue repair and regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

Leukemia Inhibitory Factor Enhances Endogenous Cardiomyocyte Regeneration after Myocardial Infarction

PubMed Central

Kanda, Masato; Nagai, Toshio; Takahashi, Toshinao; Liu, Mei Lan; Kondou, Naomichi; Naito, Atsuhiko T.; Akazawa, Hiroshi; Sashida, Goro; Iwama, Atsushi; Komuro, Issei; Kobayashi, Yoshio

2016-01-01

Cardiac stem cells or precursor cells regenerate cardiomyocytes; however, the mechanism underlying this effect remains unclear. We generated CreLacZ mice in which more than 99.9% of the cardiomyocytes in the left ventricular field were positive for 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) staining immediately after tamoxifen injection. Three months after myocardial infarction (MI), the MI mice had more X-gal-negative (newly generated) cells than the control mice (3.04 ± 0.38/mm2, MI; 0.47 ± 0.16/mm2, sham; p < 0.05). The cardiac side population (CSP) cell fraction contained label-retaining cells, which differentiated into X-gal-negative cardiomyocytes after MI. We injected a leukemia inhibitory factor (LIF)-expression construct at the time of MI and identified a significant functional improvement in the LIF-treated group. At 1 month after MI, in the MI border and scar area, the LIF-injected mice had 31.41 ± 5.83 X-gal-negative cardiomyocytes/mm2, whereas the control mice had 12.34 ± 2.56 X-gal-negative cardiomyocytes/mm2 (p < 0.05). Using 5-ethynyl-2'-deoxyurinide (EdU) administration after MI, the percentages of EdU-positive CSP cells in the LIF-treated and control mice were 29.4 ± 2.7% and 10.6 ± 3.7%, respectively, which suggests that LIF influenced CSP proliferation. Moreover, LIF activated the Janus kinase (JAK)signal transducer and activator of transcription (STAT), mitogen-activated protein kinase/extracellular signal-regulated (MEK)extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)–AKT pathways in CSPs in vivo and in vitro. The enhanced green fluorescent protein (EGFP)-bone marrow-chimeric CreLacZ mouse results indicated that LIF did not stimulate cardiogenesis via circulating bone marrow-derived cells during the 4 weeks following MI. Thus, LIF stimulates, in part, stem cell-derived cardiomyocyte regeneration by activating cardiac stem or precursor cells. This approach may represent a novel therapeutic strategy for cardiogenesis. PMID:27227407

Role of Bruton’s tyrosine kinase in myeloma cell migration and induction of bone disease

PubMed Central

Bam, Rakesh; Ling, Wen; Khan, Sharmin; Pennisi, Angela; Venkateshaiah, Sathisha Upparahalli; Li, Xin; van Rhee, Frits; Usmani, Saad; Barlogie, Bart; Shaughnessy, John; Epstein, Joshua; Yaccoby, Shmuel

2014-01-01

Myeloma cells typically grow in bone, recruit osteoclast precursors and induce their differentiation and activity in areas adjacent to tumor foci. Bruton’s tyrosine kinase (BTK), of the TEC family, is expressed in hematopoietic cells and is particularly involved in B-lymphocyte function and osteoclastogenesis. We demonstrated BTK expression in clinical myeloma plasma cells, interleukin (IL) –6– or stroma–dependent cell lines and osteoclasts. SDF-1 induced BTK activation in myeloma cells and BTK inhibition by small hairpin RNA or the small molecule inhibitor, LFM-A13, reduced their migration toward stromal cell-derived factor-1 (SDF-1). Pretreatment with LFM-A13 also reduced in vivo homing of myeloma cells to bone using bioluminescence imaging in the SCID-rab model. Enforced expression of BTK in myeloma cell line enhanced cell migration toward SDF-1 but had no effect on short-term growth. BTK expression was correlated with cell-surface CXCR4 expression in myeloma cells (n = 33, r = 0.81, P < 0.0001), and BTK gene and protein expression was more profound in cell-surface CXCR4-expressing myeloma cells. BTK was not upregulated by IL-6 while its inhibition had no effect on IL-6 signaling in myeloma cells. Human osteoclast precursors also expressed BTK and cell-surface CXCR4 and migrated toward SDF-1. LFM-A13 suppressed migration and differentiation of osteoclast precursors as well as bone-resorbing activity of mature osteoclasts. In primary myeloma-bearing SCID-rab mice, LFM-A13 inhibited osteoclast activity, prevented myeloma-induced bone resorption and moderately suppressed myeloma growth. These data demonstrate BTK and cell-surface CXCR4 association in myeloma cells and that BTK plays a role in myeloma cell homing to bone and myeloma-induced bone disease. PMID:23456977

ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes

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

Miao, Y.C.; Liu, C.

2010-12-28

Lignin is a complex biopolymer derived primarily from the condensation of three monomeric precursors, the monolignols. The synthesis of monolignols occurs in the cytoplasm. To reach the cell wall where they are oxidized and polymerized, they must be transported across the cell membrane. However, the molecular mechanisms underlying the transport process are unclear. There are conflicting views about whether the transport of these precursors occurs by passive diffusion or is an energized active process; further, we know little about what chemical forms are required. Using isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitorsmore » in the assays, we examined the uptake of monolignols and their derivatives by these native membrane vesicles. We demonstrate that the transport of lignin precursors across plasmalemma and their sequestration into vacuoles are ATP-dependent primary-transport processes, involving ATP-binding cassette-like transporters. Moreover, we show that both plasma and vacuolar membrane vesicles selectively transport different forms of lignin precursors. In the presence of ATP, the inverted plasma membrane vesicles preferentially take up monolignol aglycones, whereas the vacuolar vesicles are more specific for glucoconjugates, suggesting that the different ATP-binding cassette-like transporters recognize different chemical forms in conveying them to distinct sites, and that glucosylation of monolignols is necessary for their vacuolar storage but not required for direct transport into the cell wall in Arabidopsis.« less

Differentiation potentials of perivascular cells in the bone tissue remodeling zones under microgravity

NASA Astrophysics Data System (ADS)

Rodionova, Natalia; Katkova, Olena

Adaptive remodeling processes in the skeleton bones occur in the close topographical interconnection with blood capillaries followed by perivascular cells. Radioautographic studies with 3H- thymidine (Kimmel D.B., Fee W.S., 1980; Rodionova N.V., 1989, 2006) has shown that in osteogenesis zones there is sequential differentiation process of the perivascular cells into osteogenic ones. Using electron microscopy and cytochemistry we studied perivsacular cells in metaphysis of the rats femoral bones under conditions of modeling microgravity (28 days duration) and in femoral bones metaphyses of rats flown on board of the space laboratory (Spacelab - 2) It was revealed that population of the perivascular cells is not homogeneous in adaptive zones of the remodeling in both control and test groups (lowering support loading). This population comprises adjacent to endothelium little differentiated forms and isolated cells with differentiation features (specific volume of rough endoplasmic reticulum in cytoplasm is increased). Majority of the perivascular cells in the control group reveals reaction to alkaline phosphatase (marker of the osteogenic differentiation). In little differentiated cells this reaction is registered in nucleolus, nucleous and cytoplasm. In differentiating cells activity of the alkaline phosphatase is also detected on the outer surface of the cellular membrane. Unlike the control group in the bones of animals under microgravitaty reaction to the alkaline phosphatase is registered not for all cells of perivascular population. Part of the differentiating perivascular cells does not contain a product of the reaction. There is also visible trend of individual alkaline phosphatase containing perivascular cells amounts decrease (i.e. osteogenic cells-precursors). Under microgravity some little differentiated perivascular cells reveal destruction signs. Found decrease trend of the alkaline phosphatase containing cells (i.e. osteogenic cells) number in perivascular cells population. It is one of the mechanisms of the osteogenic process intensity decrease in bones due to lowering support loading on the bone skeleton. In particular this is confirmed by the fact that in the zones of adaptive remodeling we found fibroblasts and fibrosis zones - areas filled with non mineralized collagen fibrils on the bones surfaces. Hence it should be considered that lowering (removal) support loading slows down (or blocks) osteogenic differentiation of the perivascular cells part and stimulates differentiation of the fibroblast cells. Obtained data considered as one of the cellular mechanisms of the adaptive reactions development in spongy bone under microgravity which could lead to the bone mass loss.

Cultured networks of excitatory projection neurons and inhibitory interneurons for studying human cortical neurotoxicity

PubMed Central

Xu, Jin-Chong; Fan, Jing; Wang, Xueqing; Eacker, Stephen M.; Kam, Tae-In; Chen, Li; Yin, Xiling; Zhu, Juehua; Chi, Zhikai; Jiang, Haisong; Chen, Rong; Dawson, Ted M.; Dawson, Valina L.

2017-01-01

Translating neuroprotective treatments from discovery in cell and animal models to the clinic has proven challenging. To reduce the gap between basic studies of neurotoxicity and neuroprotection and clinically relevant therapies, we developed a human cortical neuron culture system from human embryonic stem cells (ESCs) or inducible pluripotent stem cells (iPSCs) that generated both excitatory and inhibitory neuronal networks resembling the composition of the human cortex. This methodology used timed administration of retinoic acid (RA) to FOXG1 neural precursor cells leading to differentiation of neuronal populations representative of the six cortical layers with both excitatory and inhibitory neuronal networks that were functional and homeostatically stable. In human cortical neuron cultures, excitotoxicity or ischemia due to oxygen and glucose deprivation led to cell death that was dependent on N-methyl-D-aspartate (NMDA) receptors, nitric oxide (NO), and the poly (ADP-ribose) polymerase (PARP)-dependent cell death, a cell death pathway designated parthanatos to separate it from apoptosis, necroptosis and other forms of cell death. Neuronal cell death was attenuated by PARP inhibitors that are currently in clinical trials for cancer treatment. This culture system provides a new platform for the study of human cortical neurotoxicity and suggests that PARP inhibitors may be useful for ameliorating excitotoxic and ischemic cell death in human neurons. PMID:27053772

CAR-pNK Cell Immunotherapy in CD7 Positive Leukemia and Lymphoma

ClinicalTrials.gov

2016-12-04

Acute Myeloid Leukemia; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; T-cell Prolymphocytic Leukemia; T-cell Large Granular Lymphocytic Leukemia; Peripheral T-cell Lymphoma, NOS; Angioimmunoblastic T-cell Lymphoma; Extranodal NK/T-cell Lymphoma, Nasal Type; Enteropathy-type Intestinal T-cell Lymphoma; Hepatosplenic T-cell Lymphoma

Neuroprotective Properties of Endocannabinoids N-Arachidonoyl Dopamine and N-Docosahexaenoyl Dopamine Examined in Neuronal Precursors Derived from Human Pluripotent Stem Cells.

PubMed

Novosadova, E V; Arsenyeva, E L; Manuilova, E S; Khaspekov, L G; Bobrov, M Yu; Bezuglov, V V; Illarioshkin, S N; Grivennikov, I A

2017-11-01

Neuroprotective properties of endocannabinoids N-arachidonoyl dopamine (NADA) and N-docosahexaenoyl dopamine (DHDA) were examined in neuronal precursor cells differentiated from human induced pluripotent stem cells and subjected to oxidative stress. Both compounds exerted neuroprotective activity, which was enhanced by elevating the concentration of the endocannabinoids within the 0.1-10 µM range. However, both agents at 10 µM concentration showed a marked toxic effect resulting in death of ~30% of the cells. Finally, antagonists of cannabinoid receptors as well as the receptor of the TRPV1 endovanilloid system did not hamper the neuroprotective effects of these endocannabinoids.

Method for producing textured substrates for thin-film photovoltaic cells

DOEpatents

Lauf, R.J.

1996-04-02

The invention pertains to the production of ceramic substrates used in the manufacture of thin-film photovoltaic cells used for directly converting solar energy to electrical energy. Elongated ribbon-like sheets of substrate precursor containing a mixture of ceramic particulates, a binder, and a plasticizer are formed and then while green provided with a mechanically textured surface region used for supporting the thin film semiconductor of the photovoltaic cell when the sheets of the substrate precursor are subsequently cut into substrate-sized shapes and then sintered. The textured surface pattern on the substrate provides enhanced light trapping and collection for substantially increasing the, solar energy conversion efficiency of thin-film photovoltaic cells. 4 figs.

Method for producing textured substrates for thin-film photovoltaic cells

DOEpatents

Lauf, R.J.

1994-04-26

The invention pertains to the production of ceramic substrates used in the manufacture of thin-film photovoltaic cells used for directly converting solar energy to electrical energy. Elongated ribbon-like sheets of substrate precursor containing a mixture of ceramic particulates, a binder, and a plasticizer are formed and then while green provided with a mechanically textured surface region used for supporting the thin film semiconductor of the photovoltaic cell when the sheets of the substrate precursor are subsequently cut into substrate-sized shapes and then sintered. The textured surface pattern on the substrate provides enhanced light trapping and collection for substantially increasing the solar energy conversion efficiency of thin-film photovoltaic cells. 4 figures.

Method for producing textured substrates for thin-film photovoltaic cells

DOEpatents

Lauf, Robert J.

1994-01-01

The invention pertains to the production of ceramic substrates used in the manufacture of thin-film photovoltaic cells used for directly converting solar energy to electrical energy. Elongated ribbon-like sheets of substrate precursor containing a mixture of ceramic particulates, a binder, and a plasticizer are formed and then while green provided with a mechanically textured surface region used for supporting the thin film semiconductor of the photovoltaic cell when the sheets of the substrate precursor are subsequently cut into substrate-sized shapes and then sintered. The textured surface pattern on the substrate provides enhanced light trapping and collection for substantially increasing the solar energy conversion efficiency of thin-film photovoltaic cells.

Method for producing textured substrates for thin-film photovoltaic cells

DOEpatents

Lauf, Robert J.

1996-01-01

The invention pertains to the production of ceramic substrates used in the manufacture of thin-film photovoltaic cells used for directly converting solar energy to electrical energy. Elongated ribbon-like sheets of substrate precursor containing a mixture of ceramic particulates, a binder, and a plasticizer are formed and then while green provided with a mechanically textured surface region used for supporting the thin film semiconductor of the photovoltaic cell when the sheets of the substrate precursor are subsequently cut into substrate-sized shapes and then sintered. The textured surface pattern on the substrate provides enhanced light trapping and collection for substantially increasing the, solar energy conversion efficiency of thin-film photovoltaic cells.

In vivo visualisation of different modes of action of biological DMARDs inhibiting osteoclastic bone resorption.

PubMed

Matsuura, Yoshinobu; Kikuta, Junichi; Kishi, Yuika; Hasegawa, Tetsuo; Okuzaki, Daisuke; Hirano, Toru; Minoshima, Masafumi; Kikuchi, Kazuya; Kumanogoh, Atsushi; Ishii, Masaru

2018-04-28

Osteoclasts play critical roles in inflammatory bone destruction. Precursor cell migration, cell differentiation, and functional cell activation are all in play. Biological disease-modifying antirheumatic drugs (DMARDs) have been shown to significantly inhibit both bone erosion as well as synovitis, although how such agents reduce osteoclastic bone destruction in vivo has not been fully explained. Here, we used an intravital time-lapse imaging technique to directly visualise mature osteoclasts and their precursors, and explored how different biological DMARDs acted in vivo . Lipopolysaccharide (LPS) was injected into the calvarial periosteum of fluorescent reporter mice to induce inflammatory bone destruction. Time-lapse imaging was performed via intravital multiphoton microscopy 5 days after LPS injection. Biological DMARDs, including monoclonal antibodies (mAbs) against the interleukin (IL) 6 receptor (IL-6R) and tumour necrosis factor α (TNFα), or cytotoxic T-lymphocyte-associated protein 4 (CTLA4)-Ig, were intraperitoneally administered at the time of LPS injection. We determined CD80/86 expression levels in mature osteoclasts and their precursors by flow cytometry, quantitative PCR and immunohistochemistry. Of the biologicals tested, anti-IL-6R and anti-TNFα mAbs affected mature osteoclasts and switched bone-resorbing osteoclasts to non-resorbing cells. CTLA4-Ig had no action on mature osteoclasts but mobilised osteoclast precursors, eliminating their firm attachment to bone surfaces. In agreement with these results, CD80/86 (the target molecules of CTLA4-Ig) were prominently expressed only in osteoclast precursor cells, being suppressed during osteoclast maturation. Intravital imaging revealed that various biological DMARDs acted at specific therapeutic time points during osteoclastic bone destruction, with different efficacies. These results enable us to grasp the real modes of action of drugs, optimising the usage of drug regimens. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Xin, an actin binding protein, is expressed within muscle satellite cells and newly regenerated skeletal muscle fibers.

PubMed

Hawke, Thomas J; Atkinson, Daniel J; Kanatous, Shane B; Van der Ven, Peter F M; Goetsch, Sean C; Garry, Daniel J

2007-11-01

Xin is a muscle-specific actin binding protein of which its role and regulation within skeletal muscle is not well understood. Here we demonstrate that Xin mRNA is robustly upregulated (>16-fold) within 12 h of skeletal muscle injury and is localized to the muscle satellite cell population. RT-PCR confirmed the expression pattern of Xin during regeneration, as well as within primary muscle myoblast cultures, but not other known stem cell populations. Immunohistochemical staining of single myofibers demonstrate Xin expression colocalized with the satellite cell marker Syndecan-4 further supporting the mRNA expression of Xin in satellite cells. In situ hybridization of regenerating muscle 5-7 days postinjury illustrates Xin expression within newly regenerated myofibers. Promoter-reporter assays demonstrate that known myogenic transcription factors [myocyte enhancer factor-2 (MEF2), myogenic differentiation-1 (MyoD), and myogenic factor-5 (Myf-5)] transactivate Xin promoter constructs supporting the muscle-specific expression of Xin. To determine the role of Xin within muscle precursor cells, proliferation, migration, and differentiation analysis using Xin, short hairpin RNA (shRNA) were undertaken in C2C12 myoblasts. Reducing endogenous Xin expression resulted in a 26% increase (P < 0.05) in cell proliferation and a 20% increase (P < 0.05) in myoblast migratory capacity. Skeletal muscle myosin heavy chain protein levels were increased (P < 0.05) with Xin shRNA administration; however, this was not accompanied by changes in myoglobin protein (another marker of differentiation) nor overt morphological differences relative to differentiating control cells. Taken together, the present findings support the hypothesis that Xin is expressed within muscle satellite cells during skeletal muscle regeneration and is involved in the regulation of myoblast function.

Vinpocetine inhibits oligodendroglial precursor cell differentiation.

PubMed

Torres, Klintsy Julieta; Göttle, Peter; Kremer, David; Rivera, Jose Flores; Aguirre-Cruz, Lucinda; Corona, Teresa; Hartung, Hans-Peter; Küry, Patrick

2012-01-01

In multiple sclerosis during periods of remission a limited degree of myelin repair can be observed mediated by oligodendroglial precursor cells. Phosphodiesterase inhibitors act as anti-inflammatory agents and might hold promise for future multiple sclerosis treatment. To investigate whether phosphodiesterase inhibitors could also influence myelin repair. We stimulated primary oligodendroglial precursor cells with cilostazol, rolipram and vinpocetine and assessed their effects on repair related cellular processes. We found that vinpocetine exerted a strong negative effect on myelin expression while cilostazol and rolipram did not show such effects. In addition, vinpocetine decreased morphological complexities suggesting an overall negative impact on oligodendroglial cell maturation. We provide evidence that this is not mediated via a blockade of phosphodiesterase-1 but rather by inhibition of IĸB kinase. These findings suggest that vinpocetine via IĸB inhibition exerts a strong negative impact on oligodendroglial cell maturation and may therefore provide the rationale to restrict its application during periods of remission in multiple sclerosis patients. This is of particular interest since vinpocetine is widely used as a health supplement thought to act as a cognitive and memory enhancer for healthy people and patients with neurological or muscle diseases. Copyright © 2012 S. Karger AG, Basel.

Sol-Gel Deposited Double Layer TiO₂ and Al₂O₃ Anti-Reflection Coating for Silicon Solar Cell.

PubMed

Jung, Jinsu; Jannat, Azmira; Akhtar, M Shaheer; Yang, O-Bong

2018-02-01

In this work, the deposition of double layer ARC on p-type Si solar cells was carried out by simple spin coating using sol-gel derived Al2O3 and TiO2 precursors for the fabrication of crystalline Si solar cells. The first ARC layer was created by freshly prepared sol-gel derived Al2O3 precursor using spin coating technique and then second ARC layer of TiO2 was deposited with sol-gel derived TiO2 precursor, which was finally annealed at 400 °C. The double layer Al2O3/TiO2 ARC on Si wafer exhibited the low average reflectance of 4.74% in the wavelength range of 400 and 1000 nm. The fabricated solar cells based on double TiO2/Al2O3 ARC attained the conversion efficiency of ~13.95% with short circuit current (JSC) of 35.27 mA/cm2, open circuit voltage (VOC) of 593.35 mV and fill factor (FF) of 66.67%. Moreover, the fabricated solar cells presented relatively low series resistance (Rs) as compared to single layer ARCs, resulting in the high VOC and FF.

GATA-3 function in innate and adaptive immunity.

PubMed

Tindemans, Irma; Serafini, Nicolas; Di Santo, James P; Hendriks, Rudi W

2014-08-21

The zinc-finger transcription factor GATA-3 has received much attention as a master regulator of T helper 2 (Th2) cell differentiation, during which it controls interleukin-4 (IL-4), IL-5, and IL-13 expression. More recently, GATA-3 was shown to contribute to type 2 immunity through regulation of group 2 innate lymphoid cell (ILC2) development and function. Furthermore, during thymopoiesis, GATA-3 represses B cell potential in early T cell precursors, activates TCR signaling in pre-T cells, and promotes the CD4(+) T cell lineage after positive selection. GATA-3 also functions outside the thymus in hematopoietic stem cells, regulatory T cells, CD8(+) T cells, thymic natural killer cells, and ILC precursors. Here we discuss the varied functions of GATA-3 in innate and adaptive immune cells, with emphasis on its activity in T cells and ILCs, and examine the mechanistic basis for the dose-dependent, developmental-stage- and cell-lineage-specific activity of this transcription factor. Copyright © 2014 Elsevier Inc. All rights reserved.

Development of Mouse Embryonic Stem (ES) Cells: IV Differentiation to Mature T and B Lymphocytes after Implantation of Embryoid Bodies Into Nude Mice

PubMed Central

Mok, Hoyan

1995-01-01

Mouse embryonic stem (ES) cells in culture can differentiate into late stages of many lineage-committed precursor cells. Under appropriate organ-culture conditions, ES cels differentiate into lymphoidlike cells at a stage equivalent to lymphoid cells found in fetal liver. These hematopoietic precursors are located in cup-shaped structures found in some embryoid bodies; we called such embryoid bodies “ES fetuses.” In this study, we have followed the maturation of hematopoietic cells after implantation of ES fetuses into nude mice for 3 weeks. ES-cell-derived lymphoid cells-pre-B cells, mature B cells, and mature T cells were found in all lymphoid organs. Interestingly, there was also an increase of T cells of host origin. Because native nude mouse lack thymus, these T cells might be educated by thymuslike epithelium generated from ES fetuses. Practical applications of this combined in vitro and in vivo system are discussed. PMID:9700357

Unusual neutral oligosaccharides in mature Sindbis virus glycoproteins are synthesized from truncated precursor oligosaccharides in Chinese hamster ovary cells.

PubMed

Davidson, S K; Hunt, L A

1983-03-01

We have previously demonstrated the presence of unusual small asparaginyl-oligosaccharides [(Man)3GlcNAc2-ASN] in the mature glycoproteins of Sindbis virus released from both wild-type and lectin-resistant Chinese hamster ovary cells, but the mechanism of synthesis of these structures was not determined. Gel filtration and endo-beta-N-acetylglucosaminidase analyses of Pronase-digested glycopeptides from [3H]mannose-labelled Sindbis virus released at different times after infection of a phytohaemagglutinin-resistant line of Chinese hamster ovary cells demonstrated that these small asparaginyl-oligosaccharides were present in similar relative amounts in virus released throughout the virus infection, rather than arising primarily at late times when cytopathic effects were maximal. Similar analyses of pulse-labelled, cell-associated viral glycopeptides suggested that these small oligosaccharides on mature virus glycoprotein resulted from the normal alpha 1,2-mannosidase processing of truncated precursor oligosaccharides (containing five rather than nine mannoses), rather than from aberrant processing or degradation of the full-size precursor oligosaccharides or normal intermediates.