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Results for “stem cell differentiation”

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PubMed2006

Reorganization of actin filaments enhances chondrogenic differentiation of cells derived from murine embryonic stem cells.

Differentiation of embryonic stem cells is of great interest to developmental biology and regenerative medicine. This study investigated the effects of cytochalasin D (CD) on the distribution of actin filaments in mouse embryoid body (EB)-derived cells. Furthermore, CD was applied to chondrogenic medium to examine its chondrogenic effect. CD at a concentration of 1 microg/ml disrupted stress fibers in EB-derived cells. Actin filaments in treated cells reorganized into a peripheral pattern, and type II collagen was detected by immunocytochemistry. The expression of type II collagen, Sox9, and at a later time point, aggrecan was up-regulated after CD treatment. In the CD-treated cells, Oct4 and Sox2, representing undifferentiation, were down-regulated as well as Sox1, AFP, and CTN-1, representing ectoderm, endoderm, and cardiogenesis, respectively. In conclusion, CD treatment enhances chondrogenesis of EB-derived cells. Moreover, it promotes a more complete stem cell differentiation toward chondrogenesis, when cultured in chondrogenic medium.

Actin Cytoskeleton↗

PubMed2006

Differential expression of glucose transporter isoforms during embryonic stem cell differentiation.

In mouse blastocysts six facilitative glucose transporter isoforms (GLUT)1-4, 8 and 9 are expressed. We have used the mouse embryonic stem (ES) cell line D3 and spontaneously differentiating embryoid bodies (EB) to investigate GLUT expression and the influence of glucose during differentiation of early embryonic cells. Both ES cells and EBs (2d-20d) expressed GLUT1, 3, and 8, whereas the isoforms 2 and 4 were detectable exclusively in EBs. Differentiation-associated expression of GLUT was analyzed by double staining with stage-specific embryonic antigen (SSEA-1), cytokeratins (CK18, 19), nestin, and desmin. Similar to trophoblast cells in mouse blastocysts the outer cell layer of endoderm-like cells showed a high GLUT3 expression in early EBs. In 20-day-old EBs no GLUT3 protein and only minor GLUT3 mRNA amounts could be detected. A minimal glucose concentration of 5 mM applied during 2 and 8 days of EB culture resulted in up-regulated GLUT4, Oct-4 and SSEA-1 levels and a delay in EB differentiation. We conclude that GLUT expression depends on cellular differentiation and that the expression is modulated by glucose concentration. The developmental and glucose-dependent regulation of GLUT strongly suggests a functional role of glucose and glucose transporters in ES cell differentiation and embryonic development.

Animals↗

PubMed2004

Regulation of human skeletal stem cells differentiation by Dlk1/Pref-1.

UNLABELLED: Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells. INTRODUCTION: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs. MATERIALS AND METHODS: As a model for hMSCs, we have stably transduced telomerase-immortalized hMSC (hMSC-TERT) with the full length of human Dlk1/Pref-1 cDNA and tested its effect on hMSC growth and differentiation into osteoblasts or adipocytes as assessed by cytochemical staining, FACS analysis, and real time PCR. Ex vivo calvaria organ cultures assay was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation. RESULTS: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high levels of Dlk1/Pref-1 protein. Overexpression of Dlk1/Pref-1 did not affect the proliferation rate of hMSC, but the ability to form mature adipocytes, mineralized matrix in vitro, and new bone formation in neonatal murine calvariae organ cultures was reduced. These effects were associated with inhibition of gene expression markers of late stages of adipocyte (adipocyte fatty acid-binding protein [aP2], peroxisome proliferator-activated receptor-gamma2 [PPARgamma2], and adiponectin [APM1]) and osteoblast differentiation (alkaline phosphatase [ALP], collagen type I [Col1], and osteocalcin [OC]). Lineage commitment markers for adipocytes (adipocyte determination and differentiation factor -1 [ADD1]) and osteoblasts (core binding factor/runt-related binding factor 2 [Cbfa1/Runx2]) were not affected. CONCLUSION: During hMSC differentiation, Dlk1/Pref-1 maintains the size of the bipotential progenitor cell pool by inhibiting the formation of mature osteoblasts and adipocytes.

Adipocytes↗

PubMed2005

Correction of factor IX deficiency in mice by embryonic stem cells differentiated in vitro.

Murine embryonic stem (ES) cells are pluripotent, but significant functional engraftment does not occur when they are introduced into the liver. However, here we demonstrate that functional liver engraftment does occur if the ES cells (from strain 129 mice) are first differentiated in vitro for 7 days in the presence of FGF. Strikingly, when these differentiated cells, termed putative endodermal precursors (PEPs), were injected into their livers, two of six C57BL/6 and four of eight BALB/c factor IX (F-IX)-deficient mice survived for >7 days, even though the recipients were of a different strain and, in the case of the BALB/c recipients, had a complete MHC mismatch. F-IX was detected in all six of the PEP-injected survivors. Two mice subsequently died of causes unrelated to F-IX; the others survived until death at 38 or 115 days after the transplantation. No uninjected control F-IX-deficient mice survived for >7 days. Large confluent regions of sinusoidal PEP engraftment were demonstrated by immunofluorescence in the long-term BALB/c survivors. The PEP engraftment was not associated with detectable cell fusion, and the transplantation was accompanied with only a low incidence of teratoma formation.

Animals↗

PubMed2005

[Potential of human adipose tissue derived adult stem cells differentiate into endothelial cells].

OBJECTIVE: To investigate whether human adipose derived adult stem (hADAS) cells can differentiate into endothelial cells. METHODS: Stem cells were isolated and expanded from adipose tissue and then induced to differentiate into cells of osteogenic, adipogenic and neurogenic lineages in vitro. hADAS cells were induced with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) to endothelial cells differentiation. hADAS cells were intravenously injected into mouse hindlimb ischemic models to test their ability to differentiate endothelial cells in vivo. RESULTS: hADAS cells were easily isolated and expanded in vitro. They had the ability to differentiate into osteogenic, adipogenic and neurogenic lineages. The cells expressed vascular endothelial growth factor receptor-2 (VEGFR-2, Flk1), and expressed endothelial markers when cultured with VEGF and bFGF. In response to local cues, hADAS cells in vivo differentiate into endothelial cells that contributed to neoangiogenesis in hindlimb ischemia models. CONCLUSIONS: Flk1+ hADAS cells have multipotential not only similar to bone marrow mesenchymal stem cells, but also exhibiting characteristics of endothelial progenitor cells. They may be a potential source of endothelial cells for cellular pro-angiogenic therapies.

Adipose Tissue↗

PubMed1985

Inhibitors of cholera toxin-induced adenosine diphosphate ribosylation of membrane-associated proteins block stem cell differentiation.

Two potent inhibitors of mono-adenosine diphosphate (ADP) ribosylation have recently been described and characterized, named p-methoxylbenzylaminodecamethylene guanidine sulfate (MBAMG) and benzylaminododecylguanine hydrochloride (BADGH). We have used these agents to investigate the role of ADP ribosylation in hematopoiesis using long-term marrow cultures. The addition of MBAMG (10(-6) mol/L) or BADGH (5 X 10(-4) mol/L) led to both an inhibition of mature cell production and the development of colony-stimulating factor (CSF-1)-responsive GM-CFC, but had no effect upon spleen colony-forming units (CFU-S) or on progenitor cells which respond to the multilineage stimulating factor present in WEHI-3B cell-conditioned medium. These data indicate that these inhibitors of mono-ADP ribosylation can block the commitment and/or differentiation of stem cells and infers that ADP ribosylation may be of some importance in the hematopoietic process.

Adenosine Diphosphate Ribose↗

PubMed2005

Cultured human epithelium: human umbilical cord blood stem cells differentiate into keratinocytes under in vitro conditions.

BACKGROUND: Stem cells have the capacity to renew or to give rise to a specialized cell types. Human umbilical cord blood (HUCB) has been explored as an alternative source of stem cells. However, its potential to differentiate into cells of other tissues is still under discussion. The aim of our study was to evaluate if HUCB stem cells could differentiate into epithelial cells under in vitro conditions. METHODS: Human keratinocytes derived from adult female skin donors, were isolated and cultured on fibrin glue/fibroblast gels-control group. In the umbilical cord blood cell group, male umbilical cord blood cells were added at a 1:10 ratio to keratinocytes and co-cultured on the fibrin glue/fibroblasts gel. After 15 days of culture, the sheets were analyzed by use of histochemistry and FISH. DNA was extracted and evaluated by use of polymerase chain reaction (PCR) for detection of Y-chromosome-specific sequences. RESULTS: In both groups a regular epithelial sheet consisting of three to four layers of cells was formed. Using PCR and FISH, in the umbilical cord blood cell group the presence of Y-chromosome-specific sequences in the cultured keratinocytes could be detected. In the control group, no Y-chromosome-specific sequences could be detected. CONCLUSION: Our findings indicate that umbilical cord blood stem cells differentiate into epithelial cells under in vitro conditions and thereby, might serve as a starting material for isolation and expansion of cells for transplantation in patients with large skin defects.

Adult↗

PubMedDate not supplied

[The need of B-helpers for the T-lymphocyte regulation of the process of hematopoietic stem cell differentiation].

We have detected and characterized a subpopulation of immunoregulatory cells, i.e., B-helpers capable to enhance the activity of Td-lymphocytes and controlling differentiation of syngeneic hemopoietic stem cells in mouse spleen and bone marrow. B-helpers found in the spleen and lymphatic nodes are resistant to radiation (at a dose of 6 Gr) but are impaired when irradiated at 9 Gr. Manifestation of the helper activity does not require either DNA or RNA synthesis but depends on protein synthesis and is mediated by soluble transmitter substances. Initial activation of B-helpers by lipopolysaccharide or alloantigens does not affect their helper functions. In the absence of T-lymphocytes B-cells do not affect differentiation of hemopoietic stem cells; interaction of B-helpers with differentiating Td-lymphocytes is not genetically restricted. Using preparative electrophoresis, we could isolate fractions of Td-lymphocytes which require or do not require B-helper cells in order to induce change in differentiation of hemopoietic stem cells from mainly erythroid to preferentially granulocyte pathway.

Animals↗

PubMed2003

[Embryonic stem cell differentiation: the role of growth factors].

In vitro differentiation of embryonic stem cells is expected as a useful means to prepare target cells for transplantation therapy. If normal and functional cells can be induced to differentiate only by adding a specific growth factor, it should be a safer and simpler method. Some of the examples showed, however, that it is necessary to follow an actual differentiation process taking place in embryogenesis to induce a specific cell lineage, so that it is difficult to selectively obtain target cells by using a single growth factor. Yet, it is also suggested that cell specification may be controlled by a specific growth factor or its combination in a simplified situation such as a single cell culture in a serum-free medium.

Animals↗

PubMed1999

GERp95, a membrane-associated protein that belongs to a family of proteins involved in stem cell differentiation.

A panel of mAbs was elicited against intracellular membrane fractions from rat pancreas. One of the antibodies reacted with a 95-kDa protein that localizes primarily to the Golgi complex or the endoplasmic reticulum (ER), depending on cell type. The corresponding cDNA was cloned and sequenced and found to encode a protein of 97.6 kDa that we call GERp95 (Golgi ER protein 95 kDa). The protein copurifies with intracellular membranes but does not contain hydrophobic regions that could function as signal peptides or transmembrane domains. Biochemical analysis suggests that GERp95 is a cytoplasmically exposed peripheral membrane protein that exists in a protease-resistant complex. GERp95 belongs to a family of highly conserved proteins in metazoans and Schizosaccharomyces pombe. It has recently been determined that plant and Drosophila homologues of GERp95 are important for controlling the differentiation of stem cells (Bohmert et al., 1998; Cox et al., 1998; Moussian et al., 1998). In Caenorhabditis elegans, there are at least 20 members of this protein family. To this end, we have used RNA interference to show that the GERp95 orthologue in C. elegans is important for maturation of germ-line stem cells in the gonad. GERp95 and related proteins are an emerging new family of proteins that have important roles in metazoan development. The present study suggests that these proteins may exert their effects on cell differentiation from the level of intracellular membranes.

Amino Acid Sequence↗

PubMed1997

Overexpression of human stem cell factor impairs melanocyte, mast cell, and thymocyte development: a role for receptor tyrosine kinase-mediated mitogen activated protein kinase activation in cell differentiation.

Stem cell factor (SCF) is synthesized as both soluble (S) and membrane-associated (MA) proteins. Indirect insight into the function of MA and S isoforms of SCF has come from studies performed in Steel (Sl) mutant mice. However, the physiologic role(s) of these two isoforms remain unknown. In an attempt to better understand the in vivo role of c-kit/SCF interactions on various cell lineages, transgenic mice were generated that overexpress MA isoform of human SCF (hSCF). In murine cells, hSCF behaves as an antagonist to normal SCF function, due to interference with the interaction between endogenous murine SCF and its receptor, c-kit, encoded by the dominant white spotting (W) gene. Mice expressing the hSCF transgene display a variety of phenotypic abnormalities, which are accentuated when combined with W alleles. Here we show that mice homozygous for the hSCF transgene demonstrate a coat color deficiency seen in some mice homozygous for mild W alleles. Specifically, homozygous hSCF transgenic mice (hSCF220) display a pronounced forehead blaze, with additional white spots over the cervical region, as well as a very large belly spot. Doubly heterozygous animals that carry both a mutated W allele and the hSCF transgene also display an unusual pigment defect and a dramatic reduction in the number of dermal mast cells. Furthermore, overexpression of MA hSCF in the thymus results in abnormal thymocyte differentiation and proliferation, which is associated with reduced mitogen activated protein (MAP) kinase activation. Thus, MAP kinase activation by a receptor tyrosine kinase, such as c-kit, may be critical for the differentiation of thymocytes in vivo.

Animals↗

PubMed2006

Hairless and Wnt signaling: allies in epithelial stem cell differentiation.

Nuclear receptors and Wnt signaling are both important regulators of developmental and physiological processes. Recent work linking these pathways in epithelial stem cell differentiation has come from studies analyzing the in vivo function of the nuclear receptor corepressor, Hairless (HR). The HR protein has long been suspected to regulate a stem cell-mediated process, hair cycling, as mutations in the Hr gene cause hair loss in both mice and men. The discovery that the HR protein is a nuclear receptor corepressor indicated that HR function in hair cycling is by regulating gene expression. A recent study revealed that HR represses expression of Wise, an inhibitor of Wnt signaling, leading to a model in which HR controls the timing of Wnt signaling required for hair cycling. Here we review these data, and provide new data showing that HR corepressor activity is essential for its in vivo function, and identify an additional putative Wnt inhibitor regulated by HR. This work complements previous studies demonstrating the role of Wnt signaling in epithelial stem cell differentiation.

Animals↗

PubMed2004

Adult skeletal muscle stem cells differentiate into endothelial lineage and ameliorate renal dysfunction after acute ischemia.

We previously demonstrated that endothelial cells are severely damaged during renal ischemia-reperfusion and that transplantation of adult human endothelial cells into athymic nude rats subjected to renal ischemia resulted in a dramatic protection of the kidney against injury and dysfunction. Morphological studies demonstrated the engraftment of transplanted cells into renal microvasculature. The goal of the present study was to determine the potential efficacy of in vitro expanded skeletal muscle-derived stem cells (MDSC) differentiated along the endothelial lineage in ameliorating acute renal injury. MDSC obtained from the Tie-2-green fluorescent protein (GFP) mice were used as donors of differentiated and nondifferentiated stem cells. FVB mice, used as recipients, were subjected to renal ischemia and transplanted with the above MDSC. The differentiation of MDSC along the endothelial lineage was monitored by the appearance of Tie-2 promotor-driven expression of GFP. These mouse endothelial cell antigen-, endothelial nitric oxide synthase (eNOS)-, Flk-1-, Flt-1-, and CD31-positive cells engrafted into renal microvasculature and significantly protected short-term renal function after ischemia. Transplantation of nondifferentiated MDSC characterized by the expression of Sca-1 (low levels of CD34, Flk-1, and cKit, and negative for GFP, eNOS, and CD31) did not improve short-term renal dysfunction. In conclusion, the data 1) provide a rich source of MDSC, 2) delineate protocols for their in vitro expansion and differentiation along the endothelial lineage, and 3) demonstrate their efficacy in preserving renal function immediately after ischemic insult.

Acute Disease↗

PubMed2004

[Mesenchymal stem cells. Differentiation and alternative source of neural tissue].

Embryonic stem cells are a population of cells located in the blastocyst, committed to specific differentiation according to spatial and temporal factors such as age and place of final location. Despite the final fate of hematic cells, hemopoietic cells retain a relative degree of plasticity dependent on environmental factors. Mesenchymal cells are a well differentiated population of bone marrow derived non hemopoietic cells with totipotential properties. The medical interest of such totipotentiality rests in the potential of such cells to repair damaged tissues. Particularly neuronal differentiation from progenitors obtained from mesenchymae non hemopoietic cells offers a new possibility in the field of neural transplantation and tissue engineering to repair functional entities in the nervous system.

Bone Marrow Transplantation↗

PubMedDate not supplied

A theory on the differentiation of bone-marrow stem cells.

Differentiation of pluripotent stem cells is viewed in parallel with maturation of lymphoid cells. Both processes share many features each occurring within a reticular framework with its component RE cells. RE cells are seen as providing control over stem cell differentiation in marrow thus minimizing stem cell competition by concentrating differentiation stimuli of a particular kind. The differentiation molecules are considered to be histocompatibility and tissue specific antigen components derived from aging or activated mature blood cells. They are probably low molecular weight glycoproteins. Evidence that histocompatibility antigens are of importance in cellular differentiation is presented. The hypothesis presupposes positive feedback mechanisms to bone-marrow with threshold responses being determined by RE cells.

Cell Differentiation↗

PubMed2003

Stem cell differentiation and the effects of deficiency.

Stem cells have several unique attributes, the key features being their potency and plasticity. They have the ability to give rise to multiple cell lineages and to transdifferentiate into totally different cell type(s) when relocated to a novel stem cell niche. Most self-renewing tissues are served by stem cells. At the ocular surface, the corneo-scleral limbus is believed to provide the niche for corneal epithelial stem cells. A large body of circumstantial evidence, both clinical and basic, supports this view. However, specific identification of limbal stem cells has proved elusive. Cytokeratin markers, vimentin, epidermal growth factor receptors, p63, and others have been used to identify epithelial cell populations at the limbus, which could harbour putative stem cells. In contrast, none of the known haematopoietic stem cell markers namely, CD34 and CD133, stain any specific subset of corneal or limbal epithelial cells. Singly or collectively, none of these markers point to any unique cell(s) that could be regarded as stem cells, supporting the notion that the corneal epithelium is served by 'committed progenitors' rather than by stem cells. Disease or destruction of the corneo-scleral limbus is associated with consequential events that eventually lead to visual impairment or blindness. Conjunctivalisation and vascularisation of the corneal surface and persistent or recurring epithelial defects are hallmarks of limbal deficiency.

Cell Differentiation↗

PubMed1990

Characteristics of embryonic stem cell differentiation: a comparison with two embryonal carcinoma cell lines.

Embryonic stem (ES) cells isolated from late blastocysts can now be maintained in culture in an undifferentiated state provided they are grown in the presence of a specific differentiation inhibitor, known variously as leukaemia inhibiting factor (LIF) or differentiation inhibiting activity (DIA), found at high concentrations in medium conditioned by Buffalo rat liver (BRL) cells. ES cells acquired a differentiated phenotype in monolayer, either when in the absence of LIF/DIA or in the presence of retinoic acid (RA). We have now characterized this bipotential differentiation of ES cells in terms of a series of extracellular matrix and cell surface proteins as well as cytokeratin expression, and compared it with the changes observed during the differentiation of two embryonal carcinoma (EC) cell lines, P19 and F9. ES cells exposed to RA in the presence of LIF/DIA largely resembled F9 EC + RA after 5 days, while ES cells deprived of LIF/DIA formed a culture with mixed phenotype resembling P19 EC + RA. This study therefore establishes the predominantly parietal endoderm-like phenotype of cells derived from ES by RA induction, and suggests that a mixed population of endoderm- and ill-defined mesoderm-like cells are formed after removal of specific inhibitor(s) of differentiation.

Animals↗

PubMed1989

Isolation of mutants showing temperature-sensitive cell growth from embryonal carcinoma cells: control of stem cell differentiation by incubation temperatures.

Embryonal carcinoma(EC) cells, the undifferentiated stem cells of teratocarcinomas, have many properties in common with pluripotent embryonic cells, and thus provide an excellent system for studying the early events involved in embryonic development and stem cell differentiation. We have isolated three novel mutants with temperature-sensitive(ts) cell growth that were able to differentiate at a non-permissive temperature for cell growth. These mutations affect the progression of the cell cycle, leading to the transient accumulation of cells in a specific phase, the S phase, of the cell cycle, which is likely to be the primary cause of stem cell differentiation of EC cells at non-permissive temperature. Isolation of these mutants strongly supports the notion that there is a close association between the inhibition of DNA synthesis and EC cell differentiation.

Animals↗