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Results for “stem cell differentiation”

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At least 217 records · Page 12Linked to original sources
PubMed2005

Expression of regulatory genes for pancreas development during murine embryonic stem cell differentiation.

Insulin-producing cells derived from embryonic stem cells could be surrogates for beta cells in diabetes therapy. However, their derivation remains hard to achieve with current protocols which rely on initial embryoid body formation. We assume that factors known to inhibit pancreas development contribute to this limitation in vitro. To evaluate this hypothesis, embryoid bodies were examined after different culture periods by real time RT-PCR to profile the expression of genes known to regulate embryonic pancreas development. Our data indicate that transcripts for pancreas markers (insulin, glucagon and amylase ) were expressed during differentiation, but the highest levels achieved were at least 10(5) times lower than in the adult mouse pancreas. Notch signalling was activated as suggested by Delta, Jagged, Ngn3 and NeuroD1 profiles. However, Sonic hedgehog, a known inhibitor of pancreas induction in vivo drastically increased in day 6 embryoid bodies, while Inhibin betaA and betaB were down-regulated and follistatin up-regulated. Members of the Fibroblast- and Transforming Growth Factor families which pattern the endoderm were expressed at low levels, while those that inhibit pancreas development were highly transcribed. The profile of pancreas regulators expressed in embryoid bodies is therefore not compatible with differentiation of pancreatic and insulin-producing cells. These findings provide an explanation for the limited derivation of such cells to date, in addition to basic information for establishing novel differentiation protocols.

Animals↗
PubMed2004

Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4.

Embryonic stem (ES) cells homozygous for a Shp-2 mutation (Shp-2(Delta46-110)) demonstrate leukemia inhibitory factor (LIF) hypersensitivity and increased LIF-stimulated phosphorylation of signal transducer and activator of transcription (STAT3). We hypothesized that LIF-responsive genes in Shp-2(Delta46-110) cells would represent potential candidates for molecules vital for ES cell self-renewal. Using microarray analysis, we detected 41 genes whose expression was modified by LIF in Shp-2(Delta46-110) ES cells. Induction of 2 significantly up-regulated genes, suppressor of cytokine signaling-3 (SOCS-3) and Kruppel-like factor 4 (Klf4), was verified using Northern blotting. ES cells overexpressing SOCS-3 had an increased capacity to differentiate to hematopoietic progenitors, rather than to self-renew. In contrast, ES cells overexpressing Klf4 had a greater capacity to self-renew based on secondary embryoid body (EB) formation. Klf4-transduced d6 EBs expressed higher levels of Oct-4, consistent with the notion that Klf4 promotes ES cell self-renewal. These findings verify the negative role of SOCS-3 on LIF signaling and provide a novel role for Klf4 in ES cell function.

Animals↗
PubMed2005

Lamin A/C expression is a marker of mouse and human embryonic stem cell differentiation.

Nuclear lamins comprise the nuclear lamina, a scaffold-like structure that lines the inner nuclear membrane. B-type lamins are present in almost all cell types, but A-type lamins are expressed predominantly in differentiated cells, suggesting a role in maintenance of the differentiated state. Previous studies have shown that lamin A/C is not expressed during mouse development before day 9, nor in undifferentiated mouse embryonic carcinoma cells. To further investigate the role of lamins in cell phenotype maintenance and differentiation, we examined lamin expression in undifferentiated mouse and human embryonic stem (ES) cells. Wide-field and confocal immunofluorescence microscopy and semiquantitative reverse transcription-polymerase chain reaction analysis revealed that undifferentiated mouse and human ES cells express lamins B1 and B2 but not lamin A/C. Mouse ES cells display high levels of lamins B1 and B2 localized both at the nuclear periphery and throughout the nucleoplasm, but in human ES cells, B1 and B2 expression is dimmer and localized primarily at the nuclear periphery. Lamin A/C expression is activated during human ES cell differentiation before downregulation of the pluripotency marker Oct-3/4 but not before the downregulation of the pluripotency markers Tra-1-60, Tra-1-81, and SSEA-4. Our results identify the absence of A-type lamin expression as a novel marker for undifferentiated ES cells and further support a role for nuclear lamins in cell maintenance and differentiation.

Animals↗
PubMed2004

A role for nucleoprotein Zap3 in the reduction of telomerase activity during embryonic stem cell differentiation.

Telomerase, the enzyme which maintains the ends of linear chromosomes in eukaryotic cells is found in murine embryonic stem cells; however, its activity is downregulated during in vitro differentiation. Previous work has indicated that this is due to the transcriptional downregulation of murine reverse transcriptase unit (mTert) of telomerase. To investigate the factors that cause the transcriptional repression of mTert we defined a 300 bp region which is essential for its transcription and performed site directed mutagenesis and electrophoretic mobility shift assays. This analysis indicated that Sp1, Sp3 and c-Myc bind to the GC-boxes and E-boxes, respectively, within the promoter and help activate the transcription of mTert gene. We also identified a novel binding sequence, found repeated within the mTert core region, which when mutated caused increased mTert expression. Yeast one hybrid screening combined with electrophoretic mobility shift assays indicated that the nuclear protein Zap3 binds to this site and its overexpression leads to the downregulation of mTert during differentiation. This suggests that regulation of mTert transcription is a complex process which depends on a quantitative balance between transcription factors that cause activation or repression of this gene. Overexpression of Zap3 in murine embryonic stem cells results in reduction in telomerase activity and telomere length as well as reduced proliferative capacity and limited ability to contribute to the development of haematopoietic cells upon differentiation.

Animals↗
PubMed2006

The guidance of human mesenchymal stem cell differentiation in vitro by controlled modifications to the cell substrate.

Material-driven control of bone-marrow-derived mesenchymal stem cell (MSC) behaviour and differentiation is a very exciting possibility. The aim of this study was to use silane-modified surfaces to control MSC adhesion and differentiation in vitro and evaluate the use of such techniques to control MSC behaviour both in basal and stimulated conditions. A range of characterised clean glass silane-modified surfaces, methyl (-CH(3)), amino (-NH(2)), silane (-SH), hydroxyl (-OH) and carboxyl (-COOH), were produced and cultured in contact with human MSC, in conjunction with a clean glass (TAAB) control, for time periods up to 28 days in basal, chondrogenic and osteogenic stimulated media. The samples were analysed for levels of viable cell adhesion, morphology and the production of various differentiation and transcription markers using both fluorescent immunohistochemistry (collagen I, II, osteocalcin, CBFA1) and real-time polymerase chain reaction (PCR) (collagen I, II, osteocalcin, osteopontin, osteonectin, CBFA1 and Sox 9). Analysis of the results demonstrated that the range of materials could be broken down into three distinct categories. Firstly, the -TAAB control and -CH(3) surfaces maintained the MSC phenotype; secondly, the -NH(2) and -SH-modified surfaces promoted and maintained osteogenesis both in the presence and absence of biological stimuli. These surfaces did not support long-term chondrogenesis under any test conditions. Finally, the -OH and -COOH-modified surfaces promoted and maintained chondrogenesis under both basal and chondrogenic stimulated conditions, but did not support osteogenesis. These results demonstrate that intricate material properties such as surface chemistry and energy can influence MSC behaviour in vitro. These results have implications not only in promoting the efficiency of tissue-engineered constructs, but also to the wider field of MSC isolation, maintenance and expansion.

Biocompatible Materials↗
PubMed2006

TGFbeta1 induces vasculogenesis and inhibits angiogenic sprouting in an embryonic stem cell differentiation model: respective contribution of ALK1 and ALK5.

Transforming growth factor-beta1 (TGFbeta1) is a multipotent cytokine that is involved in the regulation of vasculogenesis and angiogenesis. However, the actions of TGFbeta1 on vascular cells in vitro and in vivo are extremely complex and still incompletely understood. The aim of the present study was to investigate the role of TGFbeta1 and its two type I receptors, activin receptor-like kinase-1 (ALK1) and ALK5, in an embryonic stem cell (ESC) differentiation model that recapitulates the developmental steps of vasculogenesis and sprouting angiogenesis. We show that TGFbeta1 increases endothelial cell differentiation in a vascular endothelial growth factor (VEGF)-independent manner and inhibits endothelial tube formation. Furthermore, we demonstrate that undifferentiated ESCs express ALK5 but do not express ALK1, with ALK1 being expressed only after day 5 of differentiation. Finally, we demonstrate that constitutively active forms of ALK1 and ALK5 both inhibit growth factor-induced endothelial sprouting from embryoid bodies. In conclusion, the use of this ESC differentiation model allowed us to propose the following model: at early stages of development, TGFbeta1, through the ALK5 receptor, is provasculogenic in a VEGF-independent manner. Later, in differentiated endothelial cells in which both ALK1 and ALK5 are expressed, both receptors are implicated in inhibition of sprouting angiogenesis.

Activin Receptors, Type I↗
PubMed2003

Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation.

The hematopoietic and endothelial lineages derive from mesoderm and are thought to develop through the maturation of a common progenitor, the hemangioblast. To investigate the developmental processes that regulate mesoderm induction and specification to the hemangioblast, we generated an embryonic stem cell line with the green fluorescent protein (GFP) targeted to the mesodermal gene, brachyury. After the in vitro differentiation of these embryonic stem cells to embryoid bodies, developing mesodermal progenitors could be separated from those with neuroectoderm potential based on GFP expression. Co-expression of GFP with the receptor tyrosine kinase Flk1 revealed the emergence of three distinct cell populations, GFP(-)Flk1(-), GFP(+)Flk1(-) and GFP(+)Flk1(+) cells, which represent a developmental progression ranging from pre-mesoderm to prehemangioblast mesoderm to the hemangioblast.

Animals↗
PubMed2006

Bone marrow mesenchymal stem cells differentiate into functional cardiac phenotypes by cardiac microenvironment.

Heart attacks and congestive heart failure remain among the world's most prominent health challenges despite the many breakthroughs. Bone marrow mesenchymal stem cells (BMSCs) have the potential to transdifferentiate into myocytes if an appropriate cardiac environment is provided. This study is meant to investigate the ability of BMSCs to differentiate into cardiomyocytes in a conditioned medium. BMSCs were isolated from rat femurs and tibias using Percoll gradient centrifugation method. Cells were expanded as undifferentiated cells in culture for more than 3 passages and their phenotypes were identified with flow cytometer. BMSCs were cocultured with neonatal rat ventricular myocytes in a rate of 1:10 separated by semipermeable membrane. BMSCs marker of CD29 were highly expressed (98.89+/-1.2%); however, CD34 could hardly be identified (5.61+/-0.1%). After coculturing with myocytes, some of BMSCs showed contraction which became more regular and more vigorous. As assessed by RT-PCR, SERCA2 and RyR(2) were expressed by newly formed cells from 1 to 3 weeks. Immunostaining of newly differentiated BMSCs revealed positivity for cTnT. Some of these cells were positive for sarcomeric alpha-actinin, desmin, cTnT, and cTnI. Western blotting showed that cTnI protein expression was upregulated in these cells from 1 to 3 weeks. Newly formed BMSCs exhibited ultrastructural features of sarcomere formation and inward rectifier potassium current (I(K1)). It is concluded that BMSCs possess the potential to differentiate into cardiomyocytes in the cardiac environment. BMSCs provide an excellent model for development of stem cell therapeutics, and their potential in the cardiac repair under various pathological conditions.

Animals↗
PubMed1988

An early effect of retinoic acid: cloning of an mRNA (Era-1) exhibiting rapid and protein synthesis-independent induction during teratocarcinoma stem cell differentiation.

Vitamin A and its derivatives (retinoids) exhibit profound effects on the proliferation and differentiation of many cell types. However, the molecular mechanism by which retinoids exert these effects is unknown. Cultured murine F9 teratocarcinoma stem cells, which differentiate into nontumorigenic endoderm cells in response to retinoic acid (RA), have been used to identify genes regulated by RA. A cDNA library synthesized from F9 cells treated with RA for 8 hr has been screened with a cDNA probe enriched for sequences rapidly induced by RA, and a gene that exhibits the characteristics of a primary target for RA has been identified. This gene, early retinoic acid-1 (Era-1), encodes a 2.2- to 2.4-kilobase polyadenylylated RNA; the level of Era-1 mRNA rapidly and transiently increases up to 35-fold, depending on the concentration of exogenous RA. The increase in Era-1 mRNA is dependent on the continuous presence of exogenous RA. The RA-associated increase in Era-1 mRNA is seen even in the presence of protein synthesis inhibitors, but the increase is prevented by inhibitors of RNA synthesis such as actinomycin D. This increase in the steady-state level of Era-1 mRNA in F9 cells is a very early effect of retinoic acid on gene expression in this differentiation system.

Animals↗
PubMed2005

Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo.

In this study, we isolated CD31(-), CD34(-), CD106(-) (VCAM-1(-)), and fetal liver kinase(+) (Flk1(+)) cells from adipose tissue. These cells can be induced to differentiate into cells of osteogenic and adipogenic lineages in vitro and were termed adipose derived adult stem cells (ADAS cells). We also showed that they have characteristics of endothelial progenitor cells. In vitro, ADAS cells expressed endothelial markers when cultured with VEGF. In vivo, ADAS cells can differentiate in response to local cues into endothelial cells that contributed to neoangiogenesis in hindlimb ischemia models. PI3 kinase inhibitor LY294002 blocked the differentiation of ADAS cells into endothelial cells in vitro. Because ADAS cells can be expanded in culture without obvious senescence for more than 20 population doublings, they may be a potential source of endothelial cells for cellular pro-angiogenic therapies.

Adipocytes↗
PubMed2004

Mechano-regulation of stem cell differentiation and tissue regeneration in osteochondral defects.

Cartilage defects that penetrate the subchondral bone can undergo spontaneous repair through the formation of a fibrous or cartilaginous tissue mediated primarily by mesenchymal stem cells from the bone marrow. This tissue is biomechanically inferior to normal articular cartilage, and is often observed to degrade over time. Whether or not biomechanical factors control the type and quality of the repair tissue, and its subsequent degradation, have yet to be elucidated. In this paper, we hypothesise a relationship between the mechanical environment of mesenchymal stem cells and their subsequent dispersal, proliferation, differentiation and death. The mechano-regulation stimulus is hypothesised to be a function of strain and fluid flow; these quantities are calculated using biphasic poroelastic finite element analysis. A finite element model of an osteochondral defect in the knee was created, and used to simulate the spontaneous repair process. The model predicts bone formation through both endochondral and direct intramembranous ossification in the base of the defect, cartilage formation in the centre of the defect and fibrous tissue formation superficially. Greater amounts of fibrous tissue formation are predicted as the size of the defect is increased. Large strains are predicted within the fibrous tissue at the articular surface, resulting in significant cell apoptosis. This result leads to the conclusion that repair tissue degradation is initiated in the fibrous tissue that forms at the articular surface. The success of the mechano-regulation model in predicting many of the cellular events that occur during osteochondral defect healing suggest that in the future it could be used as a tool for optimising scaffolds for tissue engineering.

Animals↗
PubMed2002

Embryonic stem cell differentiation and the analysis of mammalian development.

Molecular and cellular analysis of early mammalian development is compromised by the experimental inaccessibility of the embryo. Pluripotent embryonic stem (ES) cells are derived from and retain many properties of the pluripotent founder population of the embryo, the inner cell mass. Experimental manipulation of these cells and their environment in vitro provides an opportunity for the development of differentiation systems which can be used for analysis of the molecular and cellular basis of embryogenesis. In this review we discuss strengths and weaknesses of the available ES cell differentiation methodologies and their relationship to events in vivo. Exploitation of these systems is providing novel insight into embryonic processes as diverse as cell lineage establishment, cell progression during differentiation, patterning, morphogenesis and the molecular basis for cell properties in the early mammalian embryo.

Animals↗
PubMed1993

Identification of four murine cDNAs encoding putative protein kinases from primitive embryonic stem cells differentiated in vitro.

Protein kinases transduce signals from extracellular ligands in the hematopoietic and other systems through direct phosphorylation of tyrosine, serine, or threonine residues. Little is known about the ligands and receptors that are important in the earliest stages of development--i.e., stem cell self-renewal and lineage commitment. We have made use of the lineage differentiation potential of the murine embryonic stem cell system to clone partial cDNAs encoding four putative protein kinases. Three of the four genes contain the highly conserved residues Asp-Phe-Gly in domain VII of the protein kinase family. These genes are candidates for receptors or downstream effectors of cytokines that regulate self-renewal and lineage commitment in embryogenesis.

Amino Acid Sequence↗
PubMed2005

Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model.

BACKGROUND: Bone marrow-derived stem cells are under investigation as a treatment for ischemic heart disease. Mesenchymal stem cells (MSCs) have been used preferentially in the acute ischemia model; data in the chronic ischemia model are lacking. METHODS AND RESULTS: Twelve dogs underwent ameroid constrictor placement. Thirty days later, they received intramyocardial injections of either MSCs (100x10(6) MSCs/10 mL saline) (n=6) or saline only (10 mL) (controls) (n=6). All were euthanized at 60 days. Resting and stress 2D echocardiography was performed at 30 and 60 days after ameroid placement. White blood cell count (WBC), C-reactive protein (CRP), creatine kinase MB (CK-MB), and troponin I levels were measured. Histopathological and immunohistochemical analyses were performed. Mean left ventricular ejection fraction was similar in both groups at baseline but significantly higher in treated dogs at 60 days. WBC and CRP levels were similar over time in both groups. CK-MB and troponin I increased from baseline to 48 hours, eventually returning to baseline. There was a trend toward reduced fibrosis and greater vascular density in the treated group. MSCs colocalized with endothelial and smooth muscle cells but not with myocytes. CONCLUSIONS: In a canine chronic ischemia model, MSCs differentiated into smooth muscle cells and endothelial cells, resulting in increased vascularity and improved cardiac function.

Animals↗
PubMed1990

Thyroid hormones induce hemopoietic pluripotent stem cell differentiation toward erythropoiesis through the production of pluripoietin-like factors.

We have previously reported that E pluripoietins are produced in mice after a single 20-mg injection of cytosine arabinoside (Ara-C) and that they are able to initiate the determination of hemopoietic pluripotent stem cells (CFU-S) toward the erythrocytic lineage. However, the mechanism of E pluripoietin release is still unclear. Since the stimulating effect of thyroid hormone on erythropoiesis is well known, we postulated a link between this hormone and the E pluripoietins. In previous papers we demonstrated that L-triiodothyronine (LT3) exhibits the capacity of inducing CFU-S differentiation toward erythropoiesis in vitro. Two series of data presented here suggest that LT3 acts indirectly on CFU-S determination by promoting the release of E pluripoietin-like factors. First, the Ara-C injection which induces the production of E pluripoietins in mice also promotes an increase in the LT3 plasma level. Second, medium conditioned with bone marrow cells exposed in vitro for 90 min to LT3 (even though this medium does not contain LT3) has E pluripoietin-like effects, inducing CFU-S differentiation toward the erythrocytic lineage.

Animals↗