Establishment of hamster blastocyst-derived embryonic stem (ES) cells

The establishment of four ES cell lines from the Syrian "golden" hamster (Mesocricetus auratus) is described. The cells can be maintained in the undifferentiated state when grown on primary mouse embryonic fibroblast feeder layers. In suspension culture they spontaneously differentiate into embryoid bodies of increasing complexity which contain a variety of tissues including embryonic ectoderm and myocardium. All four lines--one female and three male--are karyotypically normal with 44 chromosomes. Hamster is the second species from which ES cells have been established. As in mouse, the cells should be useful for developmental and transgenic studies.     

Neural differentiation of murine embryonic stem cells ES

Pluripotent murine embryonic stem (ES) cells can differentiate into all cell types both in vivo and in vitro. Based on their capability to proliferate and differentiate, these ES cells appear as a very promising tool for cell therapy. The understanding of the molecular mechanisms underlying the neural differentiation of the ES cells is a pre-requisite for selecting adequately the cells and conditions which will be able to correctly repair damaged brain and restore altered cognitive functions. Different methods allow obtaining neural cells from ES cells. Most of the techniques differentiate ES cells by treating embryoid bodies in order to keep an embryonic organization. More recent techniques, based on conditioned media, induce a direct differentiation of ES cells into neural cells, without going through the step of embryonic bodies. Beyond the fact that these techniques allow obtaining large numbers of neural precursors and more differentiated neural cells, these approaches also provide valuable information on the process of differentiation of ES cells into neural cells. Indeed, sequential studies of this process of differentiation have revealed that globally ES cells differentiating into neural cells in vitro recapitulate the molecular events governing the in vivo differentiation of neural cells. Altogether these data suggest that murine ES cells remain a highly valuable tool to obtain large amounts of precursor and differentiated neural cells as well as to get a better understanding of the mechanisms of neural differentiation, prior to a potential move towards the use of human ES cells in therapy.     

Lithium influences differentiation and tissue-specific gene expression of mouse embryonic stem (ES) cells in vitro

The effects of lithium chloride (LiCl) on differentiation of mouse embryonic stem (ES) cells were investigated in order to evaluate the ES cell test (EST) used in a European Union validation study for screening of embryotoxic agents in vitro. We show that LiCl inhibited concentration-dependently the differentiation of ES cells into cardiac and myogenic cells. Whereas the inhibition of cardiac differentiation by high concentrations of LiCl was obvious at day 5 + 5, decreased skeletal muscle cell differentiation was observed only at day 5 + 8. Semi-quantitative RT-PCR analyses revealed significantly lower levels of mRNA encoding cardiac-specific alpha-myosin heavy chain and skeletal muscle-specific myoD. By morphological investigation, an influence of lithium on neuronal differentiation was not evident. However, mRNA levels of genes encoding synaptophysin and the 160 kDa neurofilament protein were increased by high LiCl concentrations, whereas mRNA levels of mash-1 and Engrailed-1 were decreased, suggesting a specific influence of lithium on neuronal differentiation. Furthermore, LiCl treatment resulted in a slight, but non-significant increase of beta-catenin levels in ES cell-derived embryoid bodies. Our results demonstrate that the ES cell test, EST may be suitable to detect inhibitory effects of test compounds especially on cardiac differentiation, whereas effects on neuronal cells would not be detected. Therefore, we propose that morphological analyses of cardiac differentiation alone are insufficient to detect embryotoxic effects. The assay of other cell lineages at different developmental stages, and expression analyses of tissue-specific genes should also be employed.     

CMV promotor activity during ES cell differentiation: potential insight into embryonic stem cell differentiation

The activity of the P(CMV IE) promoter was studied during the differentiation of ES cells into neurons. In order to do this, stable embryonic stem (ES) cell lines that express enhanced green fluorescent protein (EGFP) under the control of P(CMV IE) were created and these ES cells were differentiated by aggregation of cells in the presence of retinoic acid (RA). Based on our observations that the activity of P(CMV IE) was highest in undifferentiated cells, and that cell-cell interaction and addition of RA that lead to enhanced cell proliferation also increased expression from P(CMV IE), we hypothesized that the activity of P(CMV IE) was positively regulated in cycling cells. However, when analysis was done at the single cell level it was found that BrdU label and EGFP expression were not correlated. EGFP expression was found to be down-regulated in many cells that were BrdU positive and conversely there were significant numbers of BrdU negative cells that were EGFP positive. Further, P(CMV IE) activity was not observed in cells that were nestin positive or in differentiated neurons, but P(CMV IE) was active in cells with a fibroblast-like morphology. Finally, several proteins present in undifferentiated ES cells were found to bind to regulatory regions of P(CMV IE). These were absent when cells were aggregated in the presence of RA. The above results have implications for expression of transgenes in ES cells as well as providing new insight into the mechanism of lineage restriction.     

Study of hepatocyte differentiation using embryonic stem cells

The liver has many crucial functions including metabolizing dietary molecules, detoxifying compounds, and storing glycogen. The hepatocytes, comprising most of the liver organ, progressively modify their gene expression profile during the fetal development according to their roles in the different phases of development. Embryonic stem (ES) cells serve as a major tool in understanding liver development. These cells may also serve as a source of hepatic cells for cellular therapy. In this review, we aim to summarize the research that has been performed in the field of hepatocyte differentiation from mouse and human ES cells. We discuss the various methodologies for the differentiation of ES cells towards hepatic cells using either spontaneous or directed differentiation protocols. Although many protocols for differentiating ES cells to hepatic cells have been developed, the analysis of their status is not trivial and can lead to various conclusions. Hence, we discuss the issues of analyzing hepatocytes by means of the specificity of the markers for hepatocytes and the status of the cells as fetal or adult hepatocytes.     

Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture

When embryonic stem cells are cultured directly in semisolid media (methyl cellulose), they proliferate and differentiate to generate colonies known as embryoid bodies (EBs). These EBs consist of differentiated cells from a number of lineages including those of the hematopoietic system. Following 10 days of culture in the presence of 10% fetal calf serum, more than 40% of all EBs from three different ES cell lines, CCEG2, D3 and SQ1.2S8 contained visible erythropoietic cells (i.e. red with hemoglobin). Beta H1 (z globin) mRNA is detectable in EBs within 5 days of differentiation, whilst beta(maj)-globin RNA appears by day 6. In the presence of erythropoietin (Epo), the frequency of EBs with erythropoietic activity increases to greater than 60%; Epo also prolongs this erythropoietic activity. Interleukin-3 (IL-3) does not significantly increase the frequency of EBs that contain erythroid cells, but increases slightly the number of erythropoietic cells associated with them. In the presence of IL-3, in addition to cells of the erythroid lineage, macrophages, mast cells and in some instances neutrophils are found within differentiating EBs. The development of macrophages is significantly enhanced by the addition of IL-3 alone or in combination with IL-1 and M-CSF or GM-CSF. When well-differentiated EBs are allowed to attach onto tissue-culture plates and grown in the presence of IL-3, a long-term output of cells from the mast cell lineage is observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mouse cloned from embryonic stem (ES) cells synchronized in metaphase with nocodazole

Full-term development occurred when nuclei from mouse embryonic stem (ES) cells, synchronized in metaphase with nocodazole, were fused with enucleated oocytes or nuclei of reconstituted eggs and again fused with the enucleated blastomeres of fertilized two-cell embryos using inactivated Sendai virus. Two surviving male mice were derived from undifferentiated ES cell nuclei, one from single nuclear transfer and another from serial nuclear transfer. Both were noticeably small and died within 24 hr of birth for unknown reasons. These findings demonstrate that nuclear transfer of ES cells using the fusion method produces young, as does the piezoelectric-actuated nuclear transfer. J. Exp. Zool. 289:139-145, 2001.     

Enhanced differentiation of embryonic stem cells using co-cultivation with hepatocytes

We examined the effects of co-cultivated hepatocytes on the hepatospecific differentiation of murine embryonic stem (ES) cells. Utilizing an established mouse ES cell line expressing high or low levels of E-cadherin, that we have previously shown to be responsive to hepatotrophic growth factor stimulation (Dasgupta et al., 2005. Biotechnol Bioeng 92(3):257-266), we compared co-cultures of cadherin-expressing ES (CE-ES) cells with cultured rat hepatocytes, allowing for either paracrine interactions (indirect co-cultures) or both juxtacrine and paracrine interactions (direct co-cultures, random and patterned). Hepatospecific differentiation of ES cells was evaluated in terms of hepatic-like cuboidal morphology, heightened gene expression of late maturation marker, glucose-6-phosphatase in relation to early marker, alpha-fetoprotein (AFP), and the intracellular localization of albumin. Hepatocytes co-cultured with growth factor primed CE-ES cells markedly enhanced ES cell differentiation toward the hepatic lineage, an effect that was reversed through E-cadherin blockage and inhibited in control ES cells with reduced cadherin expression. Comparison of single ES cell cultures versus co-cultures show that direct contact co-cultures of hepatocytes and CE-ES cells maximally promoted ES cell commitment towards hepatodifferentiation, suggesting cooperative effects of cadherin-based juxtacrine and paracrine interactions. In contrast, E-cadherin deficient mouse ES (CD-ES) cells co-cultured with hepatocytes failed to show increased G6P expression, confirming the role of E-cadherin expression. To establish whether albumin expression in CE-ES cells was spatially regulated by co-cultured hepatocytes, we co-cultivated CE-ES cells around micropatterned, pre-differentiated rat hepatocytes. Albumin localization was enhanced "globally" within CE-ES cell colonies and was inhibited through E-cadherin antibody blockage in all but an interfacial band of ES cells. Thus, stem cell based cadherin presentation may be an effective tool to induce hepatotrophic differentiation by leveraging both distal/paracrine and contact/juxtacrine interactions with primary cells of the liver.     

Hematopoietic differentiation of embryonic stem cells

This review is focused on methods that are used to derive hematopoietic cells from embryonic stem cells (ESCs). One of the strategies that have been recently used to achieve this goal is an approach of mimicking the hematopoietic niche in vitro by using hematopoiesis-supportive feeder cells, cocktails of soluble hematopoietic growth factors and a variety of matrices. While there is clear evidence that it is possible to derive hematopoietic stem cells (HSCs) and subsequently committed hematopoietic progenitors and mature cells from ESCs, there remains the need to address multiple issues including the efficiency of HSCs derivation in vitro and their proper functionality.     

胚胎干细胞向造血系统的分化

胚胎干细胞是指从囊胚期的内细胞团中分离出来的尚未分化的胚胎细胞,可分化形成各种组织类型。在合适的条件下,胚胎干细胞可发育成造血干细胞及各类成熟血细胞,为造血干细胞移植及血细胞输注开辟了新的来源,同时也为造血发生及造血调控研究提供了有效可靠的模型。本文将综述ES细胞向造血系统分化的诱导条件、调控机制及应用前景。     

Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor (LIF)

The isolation of pluripotent murine embryonic stem (ES) cells has previously been achieved by coculturing the ES cells with fibroblast feeder cells. In this report we demonstrate that ES cell lines can be isolated from murine 129/Sv He blastocysts in the absence of feeder cells in culture medium supplemented with recombinant leukemia inhibitory factor (LIF). Three of the ES cell lines (MBL-1, MBL-2, and MBL-3) were isolated by directly explanting blastocysts, whilst two ES cell lines (MBL-4 and MBL-5) were isolated from blastocysts pretreated by immunosurgery. Three of the ES cell lines contained the Y chromosome (MBL-1, MBL-2, and MBL-5) with a high proportion of the cells displaying a normal diploid karyotype with a modal chromosome number of 40. All of the ES cell lines tested expressed the stem cell markers ECMA-7 and alkaline phosphatase, which were lost on removal of LIF when the ES cells differentiated into a variety of cell types. The full developmental potential of the ES cells was determined by injecting cells from two of the independently derived ES cell lines, MBL-1 and MBL-5, into C57BL/6J blastocysts. A high proportion of the pups born were chimeric as judged by coat pigmentation. Subsequent breeding established that the ES cells had contributed to the germ line. These results demonstrate that feeder cells are not essential for the isolation of pluripotent ES cell lines.     

Trophoblast differentiation of human embryonic stem cells

Molecular mechanisms regulating human trophoblast differentiation remain poorly understood due to difficulties in obtaining primary tissues from very early developmental stages in humans. Therefore, the use of human embryonic stem cells (hESCs) as a source for generating trophoblast tissues is of significant interest. Trophoblast-like cells have been obtained through treatment of hESCs with bone morphogenetic protein (BMP) or inhibitors of activin/nodal/transforming growth factor-β signaling, or through protocols involving formation of embryoid bodies (EBs); however, there is controversy over whether hESC-derived cells are indeed analogous to true trophoblasts found in vivo. In this review, we provide an overview of previously described efforts to obtain trophoblasts from hESCs. We also discuss the merits and limitations of hESCs as a source of trophoblast derivatives.     

Neural differentiation of human embryonic stem cells

Availability of human embryonic stem cells (hESC) has enhanced human neural differentiation research. The derivation of neural progenitor (NP) cells from hESC facilitates the interrogation of human embryonic development through the generation of neuronal subtypes and supporting glial cells. These cells will likely lead to novel drug screening and cell therapy uses. This review will discuss the current status of derivation, maintenance and further differentiation of NP cells with special emphasis on the cellular signaling involved in these processes. The derivation process affects the yield and homogeneity of the NP cells. Then when exposed to the correct environmental signaling cues, NP cells can follow a unique and robust temporal cell differentiation process forming numerous phenotypes.