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Results for “stem cell differentiation”

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PubMed2002

Matrix assembly, regulation, and survival functions of laminin and its receptors in embryonic stem cell differentiation.

Laminin-1 is essential for early embryonic basement membrane assembly and differentiation. Several steps can be distinguished, i.e., the expression of laminin and companion matrix components, their accumulation on the cell surface and assembly into basement membrane between endoderm and inner cell mass, and the ensuing differentiation of epiblast. In this study, we used differentiating embryoid bodies derived from mouse embryonic stem cells null for gamma1-laminin, beta1-integrin and alpha/beta-dystroglycan to dissect the contributions of laminin domains and interacting receptors to this process. We found that (a) laminin enables beta1-integrin-null embryoid bodies to assemble basement membrane and achieve epiblast with beta1-integrin enabling expression of the laminin alpha1 subunit; (b) basement membrane assembly and differentiation require laminin polymerization in conjunction with cell anchorage, the latter critically dependent upon a heparin-binding locus within LG module-4; (c) dystroglycan is not uniquely required for basement membrane assembly or initial differentiation; (d) dystroglycan and integrin cooperate to sustain survival of the epiblast and regulate laminin expression; and (e) laminin, acting via beta1-integrin through LG1-3 and requiring polymerization, can regulate dystroglycan expression.

Animals↗
PubMed2004

Neural stem cell differentiation in a cell-collagen-bioreactor culture system.

Neural stem cells and neural progenitors (NSCs/NPs) are capable of self-renewal and can give rise to both neurons and glia. Such cells have been isolated from the embryonic brain and immobilized in three dimensional collagen gels. The collagen-entrapped NSCs/NPs recapitulate CNS stem cell development and form functional synapses and neuronal circuits. However, the cell-collagen constructs from static conditions contain hypoxic, necrotic cores and the cells are short-lived. In the present study, NSCs/NPs isolated from embryonic day 13 rat cortical neuroepithelium are immobilized in type I collagen gels and cultured in NASA-designed rotating wall vessel (RWV) bioreactors for up to 9 weeks. Initially, during the first 2 weeks of culture, a lag phase of cellular growth and differentiation is observed in the RWV bioreactors. Accelerated growth and differentiation, with the cells beginning to form large aggregates (approximately 1 mm in diameter) without death cores, begins during the third week. The collagen-entrapped NSCs/NPs cultured in RWV show active neuronal generation followed by astrocyte production. After 6 weeks in rotary culture, the cell-collagen constructs contain over 10 fold greater nestin+ and GFAP+ cells and two-fold more TuJ1 gene expression than those found in static cultures. In addition, TuJ1+ neurons in RWV culture give rise to extensive neurite outgrowth and considerably more synapsin I+ pre-synaptic puncta surrounding MAP2+ cell bodies and dendrites. These results strongly suggest that the cell-collagen-bioreactor culture system supports long-term NSC/NP growth and differentiation, and RWV bioreactors can be useful in generating neural tissue like constructs, which may have the potential for cell replacement therapy.

Animals↗
PubMed1996

Kinase-negative mutant epidermal growth factor receptor (EGFR) expression during embryonal stem cell differentiation favours EGFR-independent lineages.

EGF receptors are expressed on most fetal and adult cells but their precise roles are not well known. We previously reported that, in P19 embryonal carcinoma cells, the expression of kinase-negative EGFR inhibits retinoic acid (RA)-induced differentiation to nervous tissue, suggesting that EGFR plays a role in differentiation (J.-X. Wu and E. D. Adamson (1993) Dev. Biol. 159, 208-222). Embryo stem (ES) cells differentiate into a wide range of tissue types after the removal of the cytokine LIF from the culture medium. We demonstrate here that the induction of some early markers of differentiation, tissue-type plasminogen activator (tPA), AFP and keratins 8 and 19 is inhibited, whilst brachyury and myosin are increased, in clones containing kinase-negative mutant EGFR. After an extended period of differentiation, the cell types present in mutant and control cultures differed. Mutant clones produced frequent cardiac and skeletal muscle as the predominant differentiated cell types in vitro; other cells types were sparse or absent. Teratocarcinomas formed by EGFR-deltakinase-expressing ES cells contained frequent skeletal and cardiac muscle as well as apoptotic nuclei, while normal ES cells produced no detectable muscle and less apoptoses. Since mutant differentiated cultures had slower growth rates and increased levels of cell death, we concluded that: (1) inactive EGFR does not allow some cell types to survive and/or proliferate; (2) tissues that do not require EGFR for their survival, development or function predominate in long-term mutant cultures; (3) EGFR activity is not necessary for cardiac and skeletal muscle or endoderm formation and (4) Impaired survival of EGF-dependent lineages leads to preferential selection of muscle in differentiating ES cells.

Animals↗
PubMed2005

Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice.

We report that prospectively isolated, human CNS stem cells grown as neurospheres (hCNS-SCns) survive, migrate, and express differentiation markers for neurons and oligodendrocytes after long-term engraftment in spinal cord-injured NOD-scid mice. hCNS-SCns engraftment was associated with locomotor recovery, an observation that was abolished by selective ablation of engrafted cells by diphtheria toxin. Remyelination by hCNS-SCns was found in both the spinal cord injury NOD-scid model and myelin-deficient shiverer mice. Moreover, electron microscopic evidence consistent with synapse formation between hCNS-SCns and mouse host neurons was observed. Glial fibrillary acidic protein-positive astrocytic differentiation was rare, and hCNS-SCns did not appear to contribute to the scar. These data suggest that hCNS-SCns may possess therapeutic potential for CNS injury and disease.

Animals↗
PubMed2005

Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells differentiation into nerve-like cells.

BACKGROUND: The two most basic properties of mesenchymal stem cells (MSCs) are the capacities to self-renew indefinitely and differentiate into multiple cells and tissue types. The cells from human umbilical cord Wharton's Jelly have properties of MSCs and represent a rich source of primitive cells. This study was conducted to explore the possibility of inducing human umbilical cord Wharton's Jelly-derived MSCs to differentiate into nerve-like cells. METHODS: MSCs were cultured from the Wharton's Jelly taken from human umbilical cord of babies delivered after full-term normal labor. Salvia miltiorrhiza and beta-mercaptoethanol were used to induce the human umbilical cord-derived MSCs to differentiate. The expression of neural protein markers was shown by immunocytochemistry. The induction process was monitored by phase contrast microscopy, electron microscopy (EM), and laser scanning confocal microscopy (LSCM). The pleiotrophin and nestin genes were measured by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: MSCs in the Wharton's Jelly were easily attainable and could be maintained and expanded in culture. They were positive for markers of MSCs, but negative for markers of hematopoietic cells and graft-versus-host disease (GVHD)-related cells. Treatment with Salvia miltiorrhiza caused Wharton's Jelly cells to undergo profound morphological changes. The induced MSCs developed rounded cell bodies with multiple neurite-like extensions. Eventually they developed processes that formed networks reminiscent of primary cultures of neurons. Salvia miltiorrhiza and beta-mercaptoethanol also induced MSCs to express nestin, beta-tubulinIII, neurofilament (NF) and glial fibrillary acidic protein (GFAP). It was confirmed by RT-PCR that MSCs could express pleiotrophin both before and after induction by Salvia miltiorrhiza. The expression was markedly enhanced after induction and the nestin gene was also expressed. CONCLUSIONS: MSCs could be isolated from human umbilical cord Wharton's Jelly. They were capable of differentiating into nerve-like cells using Salvia miltiorrhiza or beta-mercaptoethanol. The induced MSCs not only underwent morphologic changes, but also expressed the neuron-related genes and neuronal cell markers. They may represent an alternative source of stem cells for central nervous system cell transplantation.

Cell Differentiation↗
PubMed2001

Diethylstilbestrol regulates trophoblast stem cell differentiation as a ligand of orphan nuclear receptor ERR beta.

The orphan nuclear receptor ERR beta is expressed in undifferentiated trophoblast stem cell lines and extraembryonic ectoderm, and genetic ablation of ERR beta results in abnormal trophoblast proliferation and precocious differentiation toward the giant cell lineage. Here, we show that the synthetic estrogen diethylstilbestrol (DES) promotes coactivator release from ERR beta and inhibits its transcriptional activity. Strikingly, treatment of trophoblast stem cells with DES led to their differentiation toward the polyploid giant cell lineage. In addition, DES-treated pregnant mice exhibited abnormal early placenta development associated with an overabundance of trophoblast giant cells and an absence of diploid trophoblast. These results define a novel pathway for DES action and provide evidence for steroidlike control of trophoblast development.

Animals↗
PubMed2000

Herpesvirus saimiri-based gene delivery vectors maintain heterologous expression throughout mouse embryonic stem cell differentiation in vitro.

In order to achieve a high efficiency of gene delivery into rare cell types like stem cells the use of viral vectors is presently without alternative. An ideal stem cell gene therapy vector would be able to infect primitive progenitor cells and sustain or activate gene expression in differentiated progeny. However, many viral vectors are inactivated when introduced in developing systems where cell differentiation occurs. To this end, we have developed a mouse in vitro model for testing herpesvirus saimiri (HVS)-based gene therapy vectors. We demonstrate here for the first time that HVS is able to infect totipotent mouse embryonic stem (ES) cells with high efficiency. We have transduced ES cells with a recombinant virus carrying the enhanced green fluorescent protein (EGFP) gene and the neomycin resistance gene (NeoR) driven by a CMV promoter and the SV40 promoter, respectively. ES cells maintain the viral episomal genome and can be terminally differentiated into mature haematopoietic cells. Moreover, heterologous gene expression is maintained throughout in vitro differentiation. Besides its obvious use in gene therapy, this unique expression system has wide ranging applications in studies aimed at understanding gene function and expression in cell differentiation and development.

Animals↗
PubMed2005

High dose chemotherapy and transplantation of hematopoietic progenitors from murine D3 embryonic stem cells.

Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors, useful for both basic research and clinical applications. To date, characteristics of specific factors capable of influencing hematopoietic cell fate from ES cells remains elusive. We report that mMSC Feeder Layer and the combination of VEGF, SCF and TPO strongly promote hematopoietic differentiation. The results showed that the cells induced from ES-D3 expressed hematopoietic progenitor antigens (CD34 and CD117), myelocyte cell antigen (CD11b), erythrocyte cell antigen (Ter119), and transcription factors (Flk-1, GATA-2, SCL, beta-H1 and beta-major). Furthermore, those induced differentiated cells were injected into female C57BL/6 mice which were treated with high dose topotecan chemotherapy to restore part of their blood system function. We observed rapid white blood cell recovery, which suggested that the infusion of differentiated cells has a positive impact on hematopoiesis. The Sry gene in peripheral blood, bone marrow and spleen of transplanted female mice was confirmed by PCR analysis, which affirmed the existence of the chimera.

Animals↗
PubMed2005

Intervertebral disc cell-mediated mesenchymal stem cell differentiation.

Low back pain is one of the largest health problems in the Western world today, and intervertebral disc degeneration has been identified as a main cause. Currently, treatments are symptomatic, but cell-based tissue engineering methods are realistic alternatives for tissue regeneration. However, the major problem for these strategies is the generation of a suitable population of cells. Adult bone marrow-derived mesenchymal stem cells (MSCs) are undifferentiated, multipotent cells that have the ability to differentiate into a number of cell types, including the chondrocyte-like cells found within the nucleus pulposus (NP) of the intervertebral disc; however, no method exists to differentiate these cells in an accessible monolayer environment. We have conducted coculture experiments to determine whether cells from the human NP can initiate the differentiation of human MSCs with or without cell-cell contact. Fluorescent labeling of the stem cell population and high-speed cell sorting after coculture with cell-cell contact allowed examination of individual cell populations. Real-time quantitative polymerase chain reaction showed significant increases in NP marker genes in stem cells when cells were cocultured with contact for 7 days, and this change was regulated by cell ratio. No significant change in NP marker gene expression in either NP cells or stem cells was observed when cells were cultured without contact, regardless of cell ratio. Thus, we have shown that human NP and MSC coculture with contact is a viable method for generating a large population of differentiated cells that could be used in cell-based tissue engineering therapies for regeneration of the degenerate intervertebral disc.

Adolescent↗
PubMed2002

Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart.

BACKGROUND: Cellular cardiomyoplasty has been proposed as an alternative strategy for augmenting the function of diseased myocardium. We investigated the potential of human mesenchymal stem cells (hMSCs) from adult bone marrow to undergo myogenic differentiation once transplanted into the adult murine myocardium. METHODS AND RESULTS: A small bone marrow aspirate was taken from the iliac crest of healthy human volunteers, and hMSCs were isolated as previously described. The stem cells, labeled with lacZ, were injected into the left ventricle of CB17 SCID/beige adult mice. At 4 days after injection, none of the engrafted hMSCs expressed myogenic markers. A limited number of cells survived past 1 week and over time morphologically resembled the surrounding host cardiomyocytes. Immunohistochemistry revealed de novo expression of desmin, beta-myosin heavy chain, alpha-actinin, cardiac troponin T, and phospholamban at levels comparable to those of the host cardiomyocytes; sarcomeric organization of the contractile proteins was observed. In comparison, neither cardiac troponin T nor phospholamban was detected in the myotubes formed in vitro by MyoD-transduced hMSCs. CONCLUSIONS: The purified hMSCs from adult bone marrow engrafted in the myocardium appeared to differentiate into cardiomyocytes. The persistence of the engrafted hMSCs and their in situ differentiation in the heart may represent the basis for using these adult stem cells for cellular cardiomyoplasty.

Adenoviridae↗
PubMed2005

The effects of solvents on embryonic stem cell differentiation.

Dimethyl sulfoxide (DMSO) and ethanol are common organic solvents used for dissolving lipophilic substances for in vitro testing. However, DMSO is known to induce differentiation in embryonic stem (ES) and embryonic teratocarcinoma (EC) cells. In order to clarify if solvents like DMSO and ethanol have an influence on in vitro developmental toxicity test systems, the presented study has evaluated their effects on differentiation by using different test systems. ES and EC cells were transfected with a construct containing the mTert promoter combined with the green fluorescent protein gene (GFP). A down-regulation of mTert, a marker for undifferentiated cells, results in a lower expression of GFP, which could be measured by flow cytometry. Taking the specific characteristics of ES and EC cells into account this effect could be a hint for the interaction of DMSO with embryonic development. Additionally, the effects of the solvents ethanol and DMSO on Oct-4 expression, another marker for undifferentiated cells, were measured in wild-type ES cells. Both selected molecular markers demonstrated an induction of differentiation after exposure to DMSO; in wild-type ES cells at a concentration of 0.125% and in transgenic EC cells at a concentration of 0.25% DMSO. All other differences from controls, including those which attained a level of statistical significance, were minor or not dosage related in degree, or were not consistent over time and are, therefore, considered to be of little toxicological importance. In addition, a cytotoxicity test demonstrated that the solvents affected the employed molecular markers in non-cytotoxic concentrations. The ES cells were the most sensitive towards the cytotoxic effects of the solvent DMSO while the EC cells were more sensitive when treated with the solvent ethanol.

Animals↗
PubMed2006

The role of the adapter protein SHB in embryonic stem cell differentiation into the pancreatic beta-cell and endothelial lineages.

Embryonic stem (ES) cells represent an attractive tool not only for the study of the development of various cell types but also as a potential source of cells for transplantation. Previous studies suggested a role of the signal transduction protein SRC homology 2(SH2) protein of Beta-cells (SHB) for the development of both pancreatic 3-cells and blood vessels. SHB is an SH2 domain-containing adapter protein involved in the generation of signaling complexes in response to activation of a variety of receptors, several of which have been implicated in developmental processes. Moreover, microarray analysis of ES cells expressing mutant SHB has revealed decreased expression of several genes of developmental importance. Here, we present protocols that may be used for transfection of mouse ES cells and to study the differentiation of ES cell-derived embryoid bodies (EBs) into the pancreatic Beta-cell lineage as well as into vascular structures with special reference to the effect of SHB. Moreover, we also provide a protocol that may be used for enrichment by fluorescence-activated cell sorting of specific cell lineages in EBs.

Animals↗
PubMed2006

Synergistic effects of CoCl(2) and ROCK inhibition on mesenchymal stem cell differentiation into neuron-like cells.

Bone-marrow-derived mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after neurodegenerative diseases. Recently, several studies suggested that oxygen-dependent gene expression is of crucial importance in governing the essential steps of neurogenesis such as cell proliferation, survival and differentiation. In this context, we analysed the effect of the HIF-1 (hypoxia inducible factor-1) activation-mimicking agent CoCl(2) on MSCs. CoCl(2) treatment increased the expression of the anti-proliferative gene BTG2/PC3 and decreased cyclin D1 expression. Expression of HIF-1alpha and its target genes EPO, VEGF and p21 was also upregulated. These changes were followed by inhibition of cell proliferation and morphological changes resulting in neuron-like cells, which had increased neuronal marker expression and responded to neurotransmitters. Echinomycin, a molecule inhibiting HIF-1 DNA-binding activity, blocked the CoCl(2) effect on MSCs. Additionally, by using Y-27632, we demonstrated that Rho kinase (ROCK) inhibition potentiated CoCl(2)-induced MSC differentiation in particular into dopaminergic neuron-like cells as attested by its effect on tyrosine hydroxylase expression. Altogether, these results support the ability of MSCs to differentiate into neuron-like cells in response to CoCl(2), an effect that might act, in part, through HIF-1 activation and cell-cycle arrest, and which is potentiated by inhibition of ROCK.

Amides↗