小鼠胚胎干细胞的培养

ＥＳ细胞的培养应满足促进细胞增殖和抑制细胞分化而保持其高度未分化潜能。我们用胚胎成纤维细胞作为饲养层,培养基ＤＭＥＭ中加白血病抑制因子的方法来培养ＥＳ细胞。培养的ＥＳ细胞呈集落状生长,细胞排列紧密,呈未分化状态。胚胎成纤维细胞作为饲养层是分离培养ＥＳ细胞最普遍使用而且有效的方法。本文还围绕ＥＳ细胞培养中的促进增殖和抑制分化这两个方面的影响因素进行了讨论。     

At most three ES cells contribute to the somatic lineages of chimeric mice and of mice produced by ES-tetraploid complementation

Chimeric or entirely embryonic stem (ES) cell-derived mice ("ES mice") can be produced by injecting ES cells into diploid (2n) or tetraploid (4n) host blastocysts, respectively. Usually, between 10 and 15 ES cells are injected into the host blastocyst, but it is not clear how many of the injected cells contribute to the somatic lineages, thus serve as "founder cells" of the embryo proper. We have used genetically labeled ES cells to retrospectively determine the number of founder ES cells that generate the somatic lineages of chimeric and of ES mice. ES cell clones individually labeled with provirus were mixed in equal numbers and injected into 2n or 4n blastocysts to generate chimeric or ES mice. Southern analysis of DNA from the resulting animals indicated that the somatic lineages were most often derived from one or two and sometimes from up to three founder ES cells. The number of founder cells was independent of the total number of cells injected into the host blastocysts. Our results are consistent with the notion that constraints of the host embryo restrict the number of ES cells that can contribute to a chimeric or an ES mouse.     

Cdk6在维持ES细胞自我更新中的作用

细胞周期蛋白依赖性激酶6（cyclin dependent kinase 6,Cdk6）对胚胎早期发育有着重要的作用.然而,它在胚胎干（embryonic stem,ES）细胞中的生物学功能仍不清楚.在该研究中,我们运用RNA干扰技术和基因表达分析方法探索了Cdk6在小鼠胚胎干细胞中的功能及分子机制.结果表明：Cdk6的表达水平与小鼠ES细胞的自我更新密切相关.首先,维甲酸（RA）处理和白血病抑制因子（LIF）去除实验显示 ,随着ES细胞的分化,Cdk6的表达水平明显降低.更为重要的是,RNA干扰介导的Cdk6表达抑制导致ES细胞自我更新相关基因的显著下调,同时伴随细胞分化基因的表达激活,提示Cdk6对维持ES细胞自我更新至关重要.     

An in situ assessment of the routes and extents of colonisation of the mouse embryo by embryonic stem cells and their descendants

An embryonic stem (ES) cell line stably expressing lacZ under the control of an endogenous promoter has been isolated and used as a marker to follow the fate of ES cells injected into blastocysts and morulae, before midgestation. The results show a multisite pattern of blastocyst colonization by ES cells deposited into the blastocoel cavity and a low degree of mingling between ES cells and ICM cells. Furthermore, analysis of dispersal of ES cell descendants in postimplantation chimaeric embryos showed that colonization can be highly variable from one region of the embryo to another. In contrast, a high and reproducible degree of chimaerism was obtained when the ES cells were injected at the morula stage prior to ICM formation.     

Efficient generation of chimaeric mice using embryonic stem cells after long-term culture in the presence of ciliary neurotrophic factor

The aim of our study was to evaluate whether ciliary neurotrophic factor (CNTF) can substitute for leukaemia inhibitory factor (LIF) in maintaining pluripotential embryonic stem (ES) cells in culture. Two subclones of D3 ES cells were used to assess cell proliferation and differentiation in the presence of CNTF, LIF or Buffalo rat liver (BRL) cell-conditioned medium, or in the absence of exogenous differentiation inhibiting factors. ES cells maintained in medium supplemented with CNTF for up to four weeks were injected into blastocysts to investigate theirin vivo pluripotency in terms of chimaera formation. CNTF inhibited ES cell differentiation in a dose-dependent manner. The most effective concentration was 10 ng CNTF per ml of medium. The effects of CNTF on ES cell differentiation and proliferation were comparable to those of LIF at the same concentration. BRL cell-conditioned medium was less effective at preventing ES cell differentiation but induced their proliferation very markedly. Both ES cell clones efficiently formed chimaeras after long-term culture with CNTF as the only differentiation inhibiting agent. The ability of these ES cells to colonize the germ-line is the ultimate proof that CNTF can preserve the pluripotency of ES cells.     

Proliferation and differentiation of mouse embryonic stem cells in APA microcapsule: A model for studying the interaction between stem cells and their niche

Embryonic stem (ES) cells hold promise either as an in vitro model recapitulating early embryonic development or as a renewable source of therapeutically useful cells. Certain aspects of the microenvironment (or niche) play critical roles in determining the fate of ES cells. Here, we reported the feasibility of using the technique of microencapsulation to study the interaction between ES cells and their tissue niche. ES cells' growth, viability, and differentiation in vitro were evaluated when they were enclosed in solid or liquefied core APA microcapsules. In comparison with those microcapsules with solid cores, the liquefied capsules provided a more suitable culture environment for the growth of ES cells. In addition, behavior of encapsulated ES cells in vivo was observed after their being implanted into mouse peritoneal cavities. In contrast to the prolonged lag phase in vitro, ES cells encapsulated grew much faster in vivo. Typical markers for the undifferentiated ES cells, such as AP, SSEA-1, and Oct-4 gene, were also tracked by immunochemistry and RT-PCR. Results showed that expression of markers remained high over 2 weeks of culture in vitro. However, decreased expression of markers was found in those samples in vivo with time passage. These findings implied that it was the combination of the intrinsic characteristics of ES cells and their microenvironment that regulated their fate. The APA-ES cells system may provide an optimal model to study the interaction between stem cells and their tissue niches.     

An Opposite Effect of the CDK Inhibitor,p18INK4c on Embryonic Stem Cells Compared with Tumor and Adult Stem Cells

Self-renewal is a feature common to both adult and embryonic stem (ES) cells, as well as tumor stem cells (TSCs). The cyclin-dependent kinase inhibitor, p18^INK4c, is a known tumor suppressor that can inhibit self-renewal of tumor cells or adult stem cells. Here, we demonstrate an opposite effect of p18 on ES cells in comparison with teratoma cells. Our results unexpectedly showed that overexpression of p18 accelerated the growth of mouse ES cells and embryonic bodies (EB); on the contrary, inhibited the growth of late stage teratoma. Up-regulation of ES cell markers (i.e., Oct4, Nanog, Sox2, and Rex1) were detected in both ES and EB cells, while concomitant down-regulation of various differentiation markers was observed in EB cells. These results demonstrate that p18 has an opposite effect on ES cells as compared with tumor cells and adult stem cells. Mechanistically, expression of CDK4 was significantly increased with overexpression of p18 in ES cells, likely leading to a release of CDK2 from the inhibition by p21 and p27. As a result, self-renewal of ES cells was enhanced. Our current study suggests that targeting p18 in different cell types may yield different outcomes, thereby having implications for therapeutic manipulations of cell cycle machinery in stem cells.     

Tetraploid embryonic stem cells contribute to the inner cell mass of mouse blastocysts

The demonstration that mouse somatic cells can be reprogrammed following fusion with embryonic stem (ES) cells may provide an alternative to somatic cell nuclear transfer (therapeutic cloning) to generate autologous stem cells. In an attempt to produce cells with an increased pool of reprogramming factors, tetraploid ES cells were produced by polyethylene glycol mediated fusion of two ES cell lines transfected with plasmids carrying puromycin or neomycin resistance cassettes, respectively, followed by double antibiotic selection. Tetraploid ES cells retain properties characteristic of diploid ES cells, including the expression of pluripotent gene markers Oct4 and Rex1. On injection into the testis capsule of severe combined immunodeficient (SCID) mice, tetraploid ES cells are able to form teratomas containing cells representative of all three germ layers. Further, these cells demonstrated the ability to integrate into the inner cell mass of blastocysts. This study indicates that tetraploid ES cells are promising candidates as cytoplasm donors for reprogramming studies.     

Efficient production of Cre-mediated site-directed recombinants through the utilization of the puromycin resistance gene, pac: a transient gene-integration marker for ES cells.

Gene targeting in embryonic stem (ES) cells is a powerful tool for generating mice carrying specifically designed mutations in the germline. Puromycin can completely kill ES cells within 24 to 48 h whereas G418 and hygromycin cannot. We have, therefore, proposed that the puromycin N-acetyltransferase ( pac ) gene, may be utilized as a transient gene-integration marker. Using a circular expression vector of cre and pac genes, Cre-mediated mutant cells were effectively enriched by pulse treatment of puromycin without stable integration of their genes. We have thus demonstrated the first application of pac as a transient gene-integration marker for ES cells.     

High sensitivity of embryonic stem cells to proteasome inhibitors correlates with low expression of heat shock protein and decrease of pluripotent cell marker expression

The ubiquitin-proteasome system is a major proteolytic system for nonlysosomal degradation of cellular proteins. Here, we investigated the response of mouse embryonic stem (ES) cells under proteotoxic stress. Proteasome inhibitors induced expression of heat shock protein 70 (HSP70) in a concentration- and time-dependent manner, and also induced apoptosis of ES cells. Importantly, more apoptotic cells were observed in ES cells compared with other somatic cells. To understand this phenomenon, we further investigated the expression of HSP70 and pluripotent cell markers. HSP70 expression was more significantly increased in somatic cells than in ES cells, and expression levels of pluripotent cell markers such as Oct4 and Nanog were decreased in ES cells. These results suggest that higher sensitivity of ES cells to proteotoxic stress may be related with lower capacity of HSP70 expression and decreased pluripotent cell marker expression, which is essential for the survival of ES cells.     

Development of reconstituted embryos derived from transgenic embryonic stem cell nuclei

This study was carried out to transform embryonic stem (ES) cells and to produce the reconstituted embryos derived from transgenic ES cell nuclei. Then, in vitro/vivo developmental potency of transgenic ES cells were compared to that of control ES cells (non-transgenic ES cells) in the reconstituted embryos. Unfertilized B6D2F1 ooplasm at metaphase II (M II) and two kinds of ES cell lines, 129SV and transgenic (tg) 129SV transformed by EGFP gene, were used as nuclear recipients and nuclear donors, respectively. The M II chromosome-spindle complex was aspirated into the pipette with a minimal volume of ooplasm. After enucleation, the ES cell nuclei was injected into the enucleated ooplasm directly. Then, reconstituted embryos were activated in SrCl2, and they were cultured in HTF medium. There was no difference of developmental rate between reconstituted embryos derived from the control (non-transgenic) and the tg ES cells. From this result, we indicated that transgenic ES cells might not change the property of peculiarity of the ES cell by gene transfer. The expression of GFP gene in the embryos was observed by fluorescence microscope at the 4-cell and more stage. As comparison with development of the embryos derived from the control and tg ES cells, the difference of the development could not be confirmed between the two cell groups. When the reconstituted embryos derived from the control and tg ES cells were transferred into oviduct or uterus of pseudopregnant females, fetuses were observed 13.5 days post coitum. However, in all fetuses, developmental arrest and regression were seen 19.5 days post coitum.     

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.     

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and α1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.     

Cdk1 is required for the self-renewal of mouse embryonic stem cells

Cyclin-dependent kinase 1 (Cdk1) is indispensible for the early development of the embryo. However, its role in maintaining the undifferentiated state of the embryonic stem (ES) cells remains unknown. In this study, we dissected the function of Cdk1 in mouse ES cells by RNA-interference and gene expression analyses. Cdk1 expression is tightly correlated with the undifferentiated state of the ES cells. Upon differentiation, Cdk1 expression reduced drastically. Cdk1 knock-down by RNA interference resulted in the loss of proliferation and colony formation potential of the ES cells. Consequentially, expression of self-renewal genes was reduced while differentiation markers such as Cdx2 were induced. Our results suggest a role for Cdk1 in maintaining the unique undifferentiated and self-renewing state of the mouse ES cells.     

Apoptosis of mouse embryonic stem cells induced by single cell suspension

Embryonic stem cells (ES cells) are pluripotential, and are therefore used to construct gene knock-out mice. We found that the apoptosis of mouse ES cells was induced when the cells were dispersed as single cells, whereas this process was suppressed when they proliferated in aggregates. The apoptosis of ES cells was repressed when the cells were cultured on feeders prepared from STO cells, a cell line established from embryonic fibroblasts. Culture supernatants from STO cells did not block the apoptosis of ES cells, which suggests that a direct interaction between ES cells and STO cells is required for the suppression of apoptosis. The viability of ES cells examined by the trypan blue exclusion test or by the MTT ((3-4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay decreased dramatically when the cells were dispersed in phosphate-buffered saline PBS. Cellular activity was restored by the addition of culture medium for ES cells. Glucose in the medium was found to be a major factor responsible for the restoration. Amino acids also restored the decrease in reduction of MTT. Suspension of the ES cells in PBS(-) caused leakage of the nucleosome into cytoplasm. Results indicate that the single cell suspension of ES cells leads to leakage of substrates for oxidative phosphorylation from the mitochondria, and that these cells finally become committed to apoptosis.     

Passage number affects the pluripotency of mouse embryonic stem cells as judged by tetraploid embryo aggregation

The aim of this study was to determine whether the number of passages affected the developmental pluripotency of embryonic stem (ES) cells as measured by the attainment of adult fertile mice derived from embryonic stem (ES) cell/tetraploid embryo complementation. Thirty-six newborns were produced by the aggregation of tetraploid embryos and hybrid ES cells after various numbers of passages. These newborns were entirely derived from ES cells as judged by microsatellite DNA, coat-color phenotype, and germline transmission. Although 15 survived to adulthood, 17 died of respiratory failure, and four were eaten by their foster mother. From the 15 mice that reached adulthood and that could reproduce, none arose from ES cells at passage level 15 or more. All 15 arose from cells at passages 3–11. Our results demonstrate that the number of passages affects the developmental pluripotency of ES cells. This work was supported by the National Natural Science Foundation of China (grant no. 30571336) and the President Foundation of the Agricultural University of Hebei.     

Fine needle aspiration cytology of the Ewing's sarcoma family of tumors

OBJECTIVE: To study the cytomorphologic features of the Ewing's sarcoma (ES) family of tumors. STUDY DESIGN: During a period of eight years (1990-1997), 123 soft tissue tumors and 65 bone tumors were evaluated by fine needle aspiration cytology (FNAC); 14 cases were diagnosed as in the ES family of tumors. The ages of the patients ranged from 8 to 30 years. All the cases were histologically confirmed. RESULTS: Of 14 cases of the ES family of tumors, 7 were ES, 3 extraosseous ES (EOE), 2 peripheral primitive neuroectodermal tumor (PPNET) and 2 Askin tumor. Cytologically, smears from all the cases showed round tumor cells with a high nuclear/cytoplasmic ratio. On detailed examination, subtle differentiating features were observed. The cells in ES had finer nuclear chromatin in comparison to those of PPNET and Askin tumor, and punched-out clear cytoplasmic vacuoles were present. PPNET showed nuclear molding, unipolar cytoplasmic tags and Homer-Wright rosettes. Histologically, all cases of ES and EOE and one case of Askin tumor showed periodic acid-Schiff-positive inclusions. CONCLUSION: FNAC features coupled with clinical findings enable a rapid diagnosis of the ES family of tumors, from which treatment modalities can be determined.