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Results for “Embryonic stem cells”

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PubMed2003

[Effect of bone marrow mesenchymal stem cells on hematopoietic differentiation of murine embryonic stem cells].

Mesenchymal stem cells (MSCs), precursors of diverse stromal cells, can support hematopoiesis in vitro and can promote the implantation of hematopoietic stem cells in vivo when co-transplanted with CD34(+) cells. The aim of this study was to investigate the potential effect of MSCs on the hematopoietic development of embryonic stem cells (ES cells) and the feasibility of a novel system in which ES cells will be co-cultured with MSCs. The murine bone marrow MSCs were isolated and cultured and then their phenotype and differentiation function were identified with FCM and histochemical technique. The CCE cells, murine ES cell line, were co-cultured with the isolated MSCs and the hematopoietic differentiation of CCE cells was observed with hematopoietic clonogenic assay and RT-PCR. The results showed that the morphology of MSCs became gradually homogeneous with the passage culture of cells. After passage 4, the marker of Sca-1, CD29, CD44 and CD105 were highly expressed, however, CD34 and CD45, the specific marker of hematopoietic and endothelial cells, could hardly be identified. The isolated MSCs differentiated into adipocytes and osteoblasts in specific induction culture system. After maintaining culture on mouse embryonic fibroblasts, CCE cells were plated in suspended culture system with only differentiation inductive agents and co-culture system in which MSCs were added. Compared with CCE cell suspended culture, the cells differentiated into embryoid body were obviously enhanced and there were no colony-forming cells in the co-culture system of ES cells and MSCs. In addition, transcription factor Oct-4 in co-cultured CCE cells was expressed and hematopoietic markers, Flk-1, GATA-1 and beta-H1, were negative. The ability of embryoid bodies derived from the co-culture system to produce hematopoietic colonies was markedly higher than that from the suspended culture system. It is concluded that MSCs inhibit the initial differentiation of ESC and enhance hematopoietic differentiation ability of the co-cultured ES cells.

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PubMed2004

Human placenta feeder layers support undifferentiated growth of primate embryonic stem cells.

Various undifferentiated embryonic stem (ES) cells can grow on mouse embryonic fibroblast (MEF) feeders. However, the risk of zoonosis from animal feeders to human ES cells generally excludes the clinical use of these human ES cells. We have found that human placenta is a useful source of feeder cells for the undifferentiated growth of primate ES cells. As on MEF feeders, primate ES cells cultured on human amniotic epithelial (HAE) feeder cells and human chorionic plate (HCP) cells had undifferentiated growth. The cultured primate ES cells expressed Oct-4, alkaline phosphatase, and SSEA-4. The primate ES cells on HAE feeder cells produced typical immature teratomas in vivo after injection into severe combined immunodeficient mice. Human placenta is quite novel and important because it would provide a relatively available source of feeders for the growth of human ES cells for therapeutic purposes that are also free of ethical complications.

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PubMedDate not supplied

[Isolation and long-term cultivation of rabbit embryonic stem cells].

A cell line similar to embryonic stem cells has been isolated from 5-day rabbit embryos. After the disruption of zona pellucida, embryos were mechanically separated into cel aggregates, which were then cultivated on a feeder layer of primary mouse embryonic fibroblasts. Cells similar to embryonic stem cells had high nuclear-to-cytoplasm ratio. It has been demonstrated for the first time that the inner cell mass and embryonic stem cells of rabbits expressed the activity of alkaline phosphatase. Between 10 and 30% of cells retained this expression and pluripotent characteristics up to the 23rd transfer. After 14-17 transfers the obtained embryonic cells could be induced to differentiation in vitro, which resulted in the formation of embryoid bodies. After the 23rd transfer, the line of rabbit embryonic cells was lost as a result of its differentiation.

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PubMed1997

Differentiation of embryonic stem cells into adipocytes in vitro.

Embryonic stem cells, derived from the inner cell mass of murine blastocysts, can be maintained in a totipotent state in vitro. In appropriate conditions embryonic stem cells have been shown to differentiate in vitro into various derivatives of all three primary germ layers. We describe in this paper conditions to induce differentiation of embryonic stem cells reliably and at high efficiency into adipocytes. A prerequisite is to treat early developing embryonic stem cell-derived embryoid bodies with retinoic acid for a precise period of time. Retinoic acid could not be substituted by adipogenic hormones nor by potent activators of peroxisome proliferator-activated receptors. Treatment with retinoic acid resulted in the subsequent appearance of large clusters of mature adipocytes in embryoid body outgrowths. Lipogenic and lipolytic activities as well as high level expression of adipocyte specific genes could be detected in these cultures. Analysis of expression of potential adipogenic genes, such as peroxisome proliferator-activated receptors gamma and delta and CCAAT/enhancer binding protein beta, during differentiation of retinoic acid-treated embryoid bodies has been performed. The temporal pattern of expression of genes encoding these nuclear factors resembled that found during mouse embryogenesis. The differentiation of embryonic stem cells into adipocytes will provide an invaluable model for the characterisation of the role of genes expressed during the adipocyte development programme and for the identification of new adipogenic regulatory genes.

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PubMed2006

Ciliated cells differentiated from mouse embryonic stem cells.

In the present study, we demonstrated that the mouse embryonic stem cells were differentiated into ciliated epithelial cells, with characteristics of normal ciliated cells. These cells expressed ciliary marker proteins, such as beta-tubulin IV and hepatocyte nuclear factor-3/forkhead homolog 4 (HFH-4), and processed microtubules were arranged in the 9 + 2 structure, which is the same specific alignment observed in normal ciliary microtubules. The cilia of these cells were beating at a frequency of 17-20 Hz. The differentiated embryoid bodies (EBs) containing these ciliated cells expressed respiratory marker genes such as thyroid transcription factor-1 and surfactant protein-C. For the induction of ciliated cells, culture of EBs in serum-free medium during the initial 2 days of the attachment was indispensable. When EBs were treated with bone morphogenetic proteins, the expression of HFH-4 was decreased, and the ciliated cells were scarcely differentiated. Previous methods for inducing ciliated cells in vitro from embryonic or adult tissues involved an air-liquid interface. The system used in this study more closely mimics the normal development of ciliated cells; thus, an added advantage of the system is as a tool for studying the differentiation mechanism of normal ciliated epithelial cells.

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PubMed1996

Retinoic acid promotes neural and represses mesodermal gene expression in mouse embryonic stem cells in culture.

Mouse embryonic stem cells treated with retinoic acid are induced to differentiate into neuron-like cells (Bain et al. (1995) Dev. Biol. 168, 342-357). Here we have examined the expression of a set of neural- and mesoderm-specific genes during this in vitro differentiation process. mRNAs encoding the neural genes Wnt-1, MASH1, the light and medium isoforms of neurofilaments, and the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase are all strongly upregulated by retinoic acid treatment; expression of these genes occurs in a temporal pattern resembling that in the developing brain. In contrast, retinoic acid blocks the expression of the mesodermal genes Brachyury, cardiac actin, and zeta-globin. Thus, retinoic acid exerts both pro-neuronal and anti-mesodermal activities on mouse embryonic stem cells in culture.

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PubMed1999

Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation.

The Mre11/Rad50 protein complex functions in diverse aspects of the cellular response to double-strand breaks (DSBs), including the detection of DNA damage, the activation of cell cycle checkpoints, and DSB repair. Whereas genetic analyses in Saccharomyces cerevisiae have provided insight regarding DSB repair functions of this highly conserved complex, the implication of the human complex in Nijmegen breakage syndrome reveals its role in cell cycle checkpoint functions. We established mRad50 mutant mice to examine the role of the mammalian Mre11/Rad50 protein complex in the DNA damage response. Early embryonic cells deficient in mRad50 are hypersensitive to ionizing radiation, consistent with a role for this complex in the repair of ionizing radiation-induced DSBs. However, the null mrad50 mutation is lethal in cultured embryonic stem cells and in early developing embryos, indicating that the mammalian Mre11/Rad50 protein complex mediates functions in normally growing cells that are essential for viability.

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PubMed2006

Neural differentiation of embryonic stem cells is induced by signalling from non-neural niche cells.

BACKGROUND/AIMS: Embryonic stem cell (ESC) transplantation offers new therapeutic strategies for neurodegenerative diseases and injury. However, the mechanisms underlying integration and differentiation of engrafted ESCs are poorly understood. This study elucidates the influence of exogenous signals on ESC differentiation using in vitro modelling of non-stem/stem cell interactions. METHODS: Murine ESCs were co-cultured with endothelial cells and astrocytes or conditioned medium obtained from endothelial or astrocyte cultures. After 7 days of co-culture isolated RNA was analysed using RT-PCR for the expression of pluripotency marker oct-4, neural progenitor marker nestin, and neurofilament (NFL), an early marker of neuronal lineage commitment. The presence of the glial cell surface marker A2B5 was determined in ESCs by flow cytometry. RESULTS: Neuronal differentiation was inhibited in ESCs when grown in close vicinity to cerebral endothelial or glial cells. Under these conditions, ESC differentiation was predominantly directed towards a glial fate. However, treatment of ESCs with endothelial cell- or astrocyte-conditioned medium promoted neuronal as well as glial differentiation. CONCLUSION: Our results indicate that ESC fate is determined by endothelial and glial cells that comprise the environmental niche of these stem cells in vivo. The direction of differentiation processes appears to be dependent on humoral factors secreted by adjacent cell lines.

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PubMed2006

wnt3a but not wnt11 supports self-renewal of embryonic stem cells.

wnt proteins (wnts) promote both differentiation of midbrain dopaminergic cells and self-renewal of haematopoietic stem cells. Mouse embryonic stem (ES) cells can be maintained and self-renew on mouse feeder cell layers or in media containing leukemia inhibitory factor (LIF). However, the effects of wnts on ES cells self-renewal and differentiation are not clearly understood. In the present study, we found that conditioned medium prepared from L cells expressing wnt3a can replace feeder cell layers and medium containing LIF in maintaining ES cells in the proliferation without differentiation (self-renewal) state. By contrast, conditioned medium from NIH3T3 cells expressing wnt11 did not. Alkaline phosphatase staining and compact colony formation were used as criteria of cells being in the undifferentiated state. ES cells maintained in medium conditioned by Wnt3a expressing cells underwent freezing and thawing while maintaining properties seen with LIF maintained ES cells. Purified wnt3a did not maintain self-renewal of ES cells for prolonged intervals. Thus, other factors in the medium conditioned by wnt3a expressing cells may have contributed to maintenance of ES cells in a self-renewal state. Pluripotency of ES cells was determined with the use of embryoid bodies in vitro. PD98059, a MEK specific inhibitor, promoted the growth of undifferentiated ES cells maintained in conditioned medium from wnt3a expressing cells. By contrast, the P38 MAPK inhibitor SB230580 did not, suggesting a role for the MEK pathway in self-renewal and differentiation of ES cells maintained in the wnt3a cell conditioned medium. Thus, our results show that conditioned medium from wnt3a but not wnt11 expressing cells can maintain ES cells in self-renewal and in a pluripotent state.

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PubMed2006

Ultraviolet-induced apoptosis in embryonic stem cells in vitro.

Embryonic stem (ES) cells, and the inner cell mass from which they are derived, are hypersensitive to DNA damage and appear to have specific cellular defense systems for DNA repair and the elimination of damaged cells. These mechanisms differ from somatic cells and are vital to minimize developmental defects that would potentially result from the continued proliferation and differentiation of abnormal cells into adult cell lineages. Although the DNA damage-induced signaling cascades activated in these cells are known to include p38 and c-Jun N-terminal protein kinase mitogen-activated protein kinase pathways and activation of a variety of transcription factors, including p53, nuclear factor-kappaB, and activator protein-1, the nature of the specific mechanisms unique to these cells remains to be elucidated. Here, we describe the use of homozygous knockout ES cells to investigate the role of Ets1 in the response to DNA damage in these cells. These studies demonstrate that Ets1 is required for optimal p53 function in this response and further demonstrate the potential for knockout ES cells to elucidate the role of specific genes in early embryonic cell responses.

Alkaline Phosphatase↗

PubMed1992

Establishment and characterization of lymphoid and myeloid mixed-cell populations from mouse late embryoid bodies, "embryonic-stem-cell fetuses".

Mouse embryonic stem (ES) cells have the potential to differentiate into embryoid bodies in vitro and mimic normal embryonic development. The "ES fetus" is a specific development at a late stage seen under our culture conditions. We have established several mixed populations from ES fetuses by using combinations of retroviruses carrying different oncogenes (v-abl, v-raf, c-myc), interleukins 2 and 3, and Con A. Six groups of mixed populations were characterized by immunophenotyping. For some groups, transfer of cells into sublethally irradiated mice resulted in the development of macrophages, mature T and B lymphocytes, and plasma cells of donor origin. Thus, these mixed populations may contain immortalized precursors of hematopoietic lineages. These mixed populations should be valuable for defining hematopoietic stem cells and their committed progenitors.

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PubMed2005

Multi-germ layer lineage central nervous system repair: nerve and vascular cell generation by embryonic stem cells transplanted in the injured brain.

OBJECT: To restore proper function to a damaged central nervous system (CNS) through transplantation, it is necessary to replace both neural and nonneural elements that arise from different germ layers in the embryo. Mounting evidence indicates the importance of signals related to vasculogenesis in governing neural proliferation and differentiation in early CNS development. Here, the authors examined whether embryonic stem cell (ESC)-derived progenitors can selectively generate both neural and endothelial cells after transplantation in the damaged CNS. METHODS: Injections of 20 nmol N-methyl-D-aspartate created a unilateral striatal injury in 7-day-old rats. One week postinjury, murine ESCs, neural-induced with retinoic acid, were transplanted into the injured striatum. Histological staining, laser confocal microscopy, and transmission electron microscopy of grafted ESCs were performed 1 week posttransplantation. CONCLUSIONS: Transplanted ESCs differentiated into neural cells, which segregated into multiple pools and formed neurons that conformed to host cytoarchitecture. The ESCs also generated endothelial cells, which integrated with host cells to form chimeric vasculature. The combination of ESC pluripotentiality and multiple germ layer differentiation provides a new conceptual framework for CNS repair.

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PubMed2006

Cyclic adenosine 3',5'-monophosphate induces differentiation of mouse embryonic stem cells into cardiomyocytes.

Embryonic stem (ES) cells, derived from blastocyst-stage of early mammalian embryos, have the potential to differentiate into derivatives of all three embryonic germ layers. Here we reported the first evidence that murine pluripotent ES cells could be induced to differentiate into cardiomyocytes by cyclic adenosine 3',5'-monophosphate (cAMP) in vitro. Spontaneously beating of cardiac cell clusters began to be observed within the outgrowths of embryoid bodies (EBs) as early as 2 days after the onset of differentiation. By days 5-8 after induction, a maximum level of cardiomyocyte differentiation could be achieved. Incubation of EBs with cAMP at concentrations ranging from 0.01 mg/L to 1 mg/L resulted in a significant elevation in differentiation rate, reaching a maximum value of 44.0 +/- 1.3% at 0.03 mg/L of exposure. At 0.03 mg/L concentration point, an approximately 8.1-fold increase in cardiomyocyte differentiation was observed in comparison with 5.4 +/- 0.9% of untreated controls. The differentiation rate induced by cAMP was shown to be similar to that of RA/DMSO treated controls, indicating that cAMP has the same inducing effect as RA/DMSO. However, no significant co-inducing effects between cAMP and RA/DMSO were seen. Cardiomyocytes were evident as they expressed cardiac cell specific genes and protein markers including GATA4, Nkx2.5, beta-MHC, atrial natriuretic factor (ANF) and alpha-actin when analyzed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining. The results from the present study suggested a novel role of cAMP in cardiomyocyte differentiation and provided a new research model for the study of cardiac cell biology.

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PubMed2002

Effective contribution of transplanted vascular progenitor cells derived from embryonic stem cells to adult neovascularization in proper differentiation stage.

We demonstrated that Flk-1(+) cells derived from mouse embryonic stem (ES) cells can differentiate into both endothelial cells (ECs) and mural cells (MCs) to suffice as vascular progenitor cells (VPCs). In the present study, we investigated the importance of the stage of ES cell differentiation on effective participation in adult neovascularization. We obtained Flk-1(+) LacZ-expressing undifferentiated VPCs. Additional culture of these VPCs with vascular endothelial growth factor (VEGF) resulted in a mixture of ECs and MCs (differentiated VPCs). We injected VPCs subcutaneously into tumor-bearing mice. Five days after the injection, whereas undifferentiated VPCs were often detected as nonvascular cells, differentiated VPCs were more specifically incorporated into developing vasculature mainly as ECs. VPC-derived MCs were also detected in vascular walls. Furthermore, transplantation of differentiated VPCs augmented tumor blood flow in nude mice. These results indicate that a specific vascular contribution in adult neovascularization can be achieved by selective transplantation of ES cell-derived VPCs in appropriate differentiation stages, which should be the basis for vascular regeneration schemes.

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PubMed2005

Cell cycle synchronization of embryonic stem cells: effect of serum deprivation on the differentiation of embryonic bodies in vitro.

Research on stem-cell transplantation has indicated that the success of transplantation largely depends on synchronizing donor cells into the G0/G1 phase. In this study, we investigated the profile of embryonic stem (ES) cell synchronization and its effect on the formation of embryonic bodies (EBs) using cell culture with serum deprivation. The D3 cell line of ES cells was used, and parameters such as cell proliferation and activity, EB formation, and expression of stage-specific embryonic antigen-1 and Oct-4 were investigated. Results showed that the percentage of G0/G1 stage in serum deprivation culture is significantly higher than that in culture with serum supplementation. Synchronized ES cells can reenter the normal cell cycle successfully after serum supply. EBs formed from synchronized ES cells have higher totipotency capability to differentiate into functional neuronal cells than EBs formed from unsynchronized ES cells. Our study provides a method for ES treatment before cell transplantation that possibly helps to decrease the rate of cell death after transplantation.

Cell Culture Techniques↗

PubMed1988

Regulatory factors of embryonic stem cells.

The analysis of factors which regulate cell proliferation and differentiation in early mammalian development has been facilitated by the existence of cell lines derived from pluripotential stem cells of the early embryo; embryonal carcinoma (EC) cells and embryonic stem (ES) cells. EC cells have proved to be a useful source of embryonic growth factors. A potent mitogen, ECDGF has been isolated from EC cell conditioned medium. ECDGF appears to be a novel member of the heparin binding growth factor family. A remarkable feature of heparin binding growth factors is their ability to induce mesodermal differences in Xenopus laevis animal pole explants. ES cell differentiation in vitro is controlled by exogenous factors, in particular a novel differentiation inhibitory factor DIA. Controlled bipotential differentiation of ES cells can be achieved by exposing ES cells to different combinations of regulatory signals.

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PubMed2006

Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells.

Differentiation of embryonic stem (ES) cells from a pluripotent to a committed state involves global changes in genome expression patterns. Gene activity is critically determined by chromatin structure and interactions of chromatin binding proteins. Here, we show that major architectural chromatin proteins are hyperdynamic and bind loosely to chromatin in ES cells. Upon differentiation, the hyperdynamic proteins become immobilized on chromatin. Hyperdynamic binding is a property of pluripotent cells, but not of undifferentiated cells that are already lineage committed. ES cells lacking the nucleosome assembly factor HirA exhibit elevated levels of unbound histones, and formation of embryoid bodies is accelerated. In contrast, ES cells, in which the dynamic exchange of H1 is restricted, display differentiation arrest. We suggest that hyperdynamic binding of structural chromatin proteins is a functionally important hallmark of pluripotent ES cells that contributes to the maintenance of plasticity in undifferentiated ES cells and to establishing higher-order chromatin structure.

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PubMed2005

Icariin-mediated modulation of cell cycle and p53 during cardiomyocyte differentiation in embryonic stem cells.

The aim of this study was to investigate the possible inducible effects and to clarify the modulation by icariin of cell cycle and p53 expression in the differentiation of embryonic stem cells into cardiomyocytes in vitro. Embryonic stem cells were cultivated as embryoid bodies in hanging drops and induced to differentiate into cardiomyocytes by icariin at 10(-7) M. Cardiomyocytes were characterized by the expression of sarcomeric proteins, alpha-actinin and cardiac troponin T, by immunocytochemistry. Flow cytometry revealed that 10(-7) M icariin treatment for 48 h significantly induced the accumulation of cells in G0/G1 and reduced the proportion of cells in S phase. A marked increase in apoptosis rate was observed 48 h after icariin treatment. Icariin resulted in significantly increased expressions of p53 mRNA and protein, as determined by reverse transcription-polymerase chain reaction and Western blot analysis. During day 7+0 and 7+9 cardiac developmental stage, 10(-7) M icariin increased the level of p53 mRNA, but caused a parallel decrease in the level of p53 protein. In conclusion, icariin at 10(-7) M facilitated the directional differentiation of embryonic stem cells into cardiomyocytes. Results showed p53 to be an important regulator in the differentiation in embryonic stem cells treated with 10(-7) M icariin, controlling or adjusting the balance between differentiated cells and cells undergoing apoptosis.

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