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1.
Human embryonic stem (ES) cells and embryonic germ (EG) cells are pluripotent and are invaluable material for in vitro studies of human embryogenesis and cell therapy. So far, only two groups have reported the establishment of human EG cell lines, whereas at least five human ES cell lines have been established. To see if human EG cell lines can be reproducibly established, we isolated primordial germ cells (PGCs) from gonadal ridges and mesenteries (9 weeks post-fertilization) and cultured them on mouse STO cells. As with mouse ES colonies, the PGC-derived cells have given rise to multilayered colonies without any differentiation over a year of continuous culture. They are karyotypically normal and express high levels of alkaline phosphatase, Oct-4, and several cell-surface markers. Histological and immunocytochemical analysis of embryoid bodies (EBs) formed from floating cultures of the PGC-derived cell colonies revealed ectodermal, endodermal, and mesodermal tissues. When the EBs were cultured in the presence of insulin, transferrin, sodium selenite, and fibronectin for 1 week, markers of primitive neuroectoderm were expressed in cells within the EBs as well as in cells growing out from the EBs. These observations indicate that our PGC-derived cells satisfy the criteria for pluripotent stem cells and hence may be EG cells.  相似文献   

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Pluripotent stem cells, termed embryonic germ (EG) cells, have been generated from both human and mouse primordial germ cells (PGCs). Like embryonic stem (ES) cells, EG cells have the potential to differentiate into all germ layer derivatives and may also be important for any future clinical applications. The development of PGCs in vivo is accompanied by major epigenetic changes including DNA demethylation and imprint erasure. We have investigated the DNA methylation pattern of several imprinted genes and repetitive elements in mouse EG cell lines before and after differentiation. Analysed cell lines were derived soon after PGC specification, “early”, in comparison with EG cells derived after PGC colonisation of the genital ridge, “late” and embryonic stem (ES) cell lines, derived from the inner cell mass (ICM). Early EG cell lines showed strikingly heterogeneous DNA methylation patterns, in contrast to the uniformity of methylation pattern seen in somatic cells (control), late EG cell and ES cell lines. We also observed that all analysed XX cell lines exhibited less methylation than XY. We suggest that this heterogeneity may reflect the changes in DNA methylation taking place in the germ cell lineage soon after specification.  相似文献   

4.
We report herein the establishment of three bovine pluripotent embryonic cell lines derived from 8-16-cell precompacting embryos. Two cell lines were cultured for 10 passages and underwent spontaneous differentiation. One cell line (Z2) has been cultured continuously for over 3 years and has remained undifferentiated. These cells express cell surface markers that have been used routinely to characterize embryonic stem (ES) and embryonic germ (EG) cells in other species such as stage-specific embryonic antigens SSEA-1, SSEA-3, and SSEA-4, and c-Kit receptor. In the absence of a feeder layer, these cells differentiated into a variety of cell types and formed embryoid bodies (EBs). When cultured for an extended period of time, EBs differentiated into derivatives of three EG layers - mesoderm, ectoderm, and endoderm - which were characterized by detection of specific cell surface markers. Our results indicate that the Z2 cell line is pluripotent and resembles an ES cell line. To our knowledge, this is the first bovine embryonic cell line that has remained pluripotent in culture for more than 150 passages.  相似文献   

5.
BMP4 initiates human embryonic stem cell differentiation to trophoblast   总被引:27,自引:0,他引:27  
The excitement and controversy surrounding the potential role of human embryonic stem (ES) cells in transplantation therapy have often overshadowed their potentially more important use as a basic research tool for understanding the development and function of human tissues. Human ES cells can proliferate without a known limit and can form advanced derivatives of all three embryonic germ layers. What is less widely appreciated is that human ES cells can also form the extra-embryonic tissues that differentiate from the embryo before gastrulation. The use of human ES cells to derive early human trophoblast is particularly valuable, because it is difficult to obtain from other sources and is significantly different from mouse trophoblast. Here we show that bone morphogenetic protein 4 (BMP4), a member of the transforming growth factor-beta (TGF-beta) superfamily, induces the differentiation of human ES cells to trophoblast. DNA microarray, RT-PCR, and immunoassay analyses demonstrate that the differentiated cells express a range of trophoblast markers and secrete placental hormones. When plated at low density, the BMP4-treated cells form syncytia that express chorionic gonadotrophin (CG). These results underscore fundamental differences between human and mouse ES cells, which differentiate poorly, if at all, to trophoblast. Human ES cells thus provide a tool for studying the differentiation and function of early human trophoblast and could provide a new understanding of some of the earliest differentiation events of human postimplantation development.  相似文献   

6.
Derivation and characterization of pluripotent embryonic germ cells in chicken   总被引:24,自引:0,他引:24  
Embryonic germ (EG) cell lines established from primordial germ cells (PGCs) are undifferentiated and pluripotent stem cells. To date, EG cells with proven germ-line transmission have been completely established only in the mouse with embryonic stem (ES) cells. We isolated PGCs from 5.5-day-old (stage 28) chicken embryonic gonads and established a putative chicken EG cell line with EG culture medium supplemented with stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), interleukin-11 (IL-11), and insulin-like growth factor-I (IGF-I). These cells grew continuously for ten passages (4 months) on a feeder layer of mitotically active chicken embryonic fibroblasts. After several passages, these cells were characterized by screening with the periodic acid-Schiff reaction, anti-SSEA-1 antibody, and a proliferation assay. The chicken EG cells maintained characteristics of gonadal PGCs and undifferentiated stem cells. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types. The chicken EG cells were injected into stage X blastodermal layer and produced chimeric chickens with various differentiated tissues derived from the EG cells. Chicken EG cells will be useful for the production of transgenic chickens and for studies of germ cell differentiation and genomic imprinting.  相似文献   

7.
Induction of hematopoiesis in an embryonic germ (EG) cell line derived from mouse primordial germ cells (PGCs) was examined. When single undifferentiated EG-1 cells were inoculated directly into the methylcellulose medium, both primitive and definitive erythropoiesis were seen in embryoid bodies derived from the EG cells as observed in ES cells, and production of myeloid cell lineages was stimulated by IL-3. These results indicate that EG cells acquired in vitro potency to differentiate toward hematopoietic cells, although they were derived from PGC and are distinct from inner cell mass-derived ES cells with regard to gene expression and patterns of DNA methylation corresponding to genomic imprinting. It turns out that they are useful for study of cell differentiation in the animals whose ES cells are not available.  相似文献   

8.
Serum-free culture of murine primordial germ cells and embryonic germ cells   总被引:7,自引:0,他引:7  
Horii T  Nagao Y  Tokunaga T  Imai H 《Theriogenology》2003,59(5-6):1257-1264
Fetal calf serum (FCS) has usually been used for culture of embryonic stem (ES) cell as a component of the culture medium. However, FCS contains undefined factors, which promote cell proliferation and occasionally stimulate differentiation of ES cells. Recently, a chemically-defined serum replacement, Knockout Serum Replacement (KSR), was developed to maintain ES cells in an undifferentiated state. In this experiment, we examined the effects of KSR on the growth and differentiation of primordial germ cells (PGCs) and embryonic germ (EG) cells. PGCs were collected 8.5 days postcoitum (dpc) from B6D2F1 (C57BL/6JxDBA/2J) female mice mated with B6D2F1 males. Most of the PGCs that were cultured in FCS-supplemented medium (FCS medium) had alkaline phosphatase (AP) activity and acquired a fibroblast cell shape. In contrast, PGCs in KSR-supplemented medium (KSR medium) proliferated, maintaining round and stem cell-like morphology. In addition, EG cells were established more easily from PGCs cultured in KSR medium than from PGCs cultured in FCS medium. The percentage of undifferentiated colonies of EG cells was significantly higher in KSR medium than in FCS medium. The germ line chimera was also produced from EG cells established in KSR medium. These results suggest that KSR can be used for sustaining an undifferentiated state of PGCs and EG cells in vitro.  相似文献   

9.
潘光锦  裴端卿 《生命科学》2007,19(4):372-377
胚胎干细胞(ES细胞)来源于早期发育的胚胎,具有分化为任何细胞类型的多能性,因此具有巨大的基础研究及潜在的应用前景.目前认为ES细胞主要通过一些外源性信号分子的作用及某些重要的内源性转录因子的表达共同起作用来达到其维持多能性的目的.外源性信号分子LIF、BMP4以及Wnt等介导的信号传导通路与内源性转录因子Oct4、Nanog、Sox2、FoxD3等共同起作用来抑制那些促进ES细胞分化的基因表达和激活那些有助于维持ES细胞多能性维持的基因表达,进而形成一个相互调控和依存的基因调控网络共同维持ES细胞的多能性.  相似文献   

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人胚胎干细胞建系的研究现状与存在的问题   总被引:2,自引:0,他引:2  
人胚胎干细胞系的建立,对人类胚胎发生和人类发育生物学研究、人类新基因的发现和功能研究以及基因治疗、细胞和组织的移植治疗等领域的突破性进展具有重大意义;回顾了人胚胎干细胞建系研究的历程,就建系的几种方案、路线、意义和可行性进行了探讨;详细系统地说明了迄今为止建立人胚胎干细胞系所需要的饲养层类型、培养基组成、添加细胞因子种类及其作用;分析了建立和维持人胚胎干细胞系所需消化酶的种类及其作用以及目前常用的几种传代方法;从若干方面总结了人胚胎干细胞系的鉴定方法,并对建立和维持人胚胎干细胞系中存在的若干问题进行了剖析,提出了目前急待解决的问题。  相似文献   

12.
13.
Epigenotype switching of imprintable loci in embryonic germ cells   总被引:8,自引:0,他引:8  
 Expression of imprinted genes is dependent on their parental origin. This is reflected in the heritable differential methylation of parental alleles. The gametic imprints are however reversible as they do not endure for more than one generation. To investigate if the epigenetic changes in male and female germ line are similar or not, we derived embryonic germ (EG) cells from primordial germ cells (PGCs) of day 11.5 and 12.5 male and female embryos. The results demonstrate that they have an equivalent epigenotype. First, chimeras made with EG cells derived from both male and female embryos showed comparable fetal overgrowth and skeletal abnormalities, which are similar to but less severe than those induced by androgenetic embryonic stem (ES) cells. Thus, EG cells derived from female embryos resemble androgenetic ES cells more than parthenogenetic cells. Furthermore, the methylation status of both alleles of a number of loci in EG cells was similar to that of the paternal allele in normal somatic cells. Hence, both alleles of Igf2r region 2, Peg1/Mest, Peg3, Nnat were consistently unmethylated in EG cells as well as in the primary embryonic fibroblasts (PEFs) rescued from chimeras. More strikingly, both alleles of p57kip2 that were also unmethylated in EG cells, underwent de novo methylation in PEFs to resemble a paternal allele in somatic cells. The exceptions were the H19 and Igf2 genes that retained the methylation pattern in PEFs as seen in normal somatic tissues. These studies suggest that the initial epigenetic changes in germ cells of male and female embryos are similar. Received: 1 September 1997 / Accepted: 15 October 1997  相似文献   

14.
Bovine embryonic stem (ES) cell lines reported to date vary in morphology and marker expression (e.g., alkaline phosphatase [ALPL], stage-specific embryonic antigen 4 [SSEA4], and OCT4) that normally are associated with the undifferentiated, pluripotent state. These observations suggest that the proper experimental conditions for consistently producing bovine ES cells have not been identified. Here, we report three bovine ES cell lines, one from in vitro-fertilized and two from nuclear transfer embryos. These bovine ES cells grew in large, multicellular colonies resembling the mouse ES and embryonic germ (EG) cells and human EG cells. Throughout the culture period, most of the cells within the colonies stained positive for ALPL and the cell surface markers SSEA4 and OCT4. The staining patterns of nuclear transfer ES cells were identical to those of the blastocysts generated in vitro yet different from most previously reported bovine ES cell lines, which were either negative or not detected. After undifferentiated culture for more than 1 yr, these cells maintained the ability to differentiate into embryoid bodies and derivatives of all three EG layers, thus demonstrating their pluripotency. However, unlike the mouse and human ES cells, following treatment with trypsin, type IV collagenase, or protease E, our bovine ES cells failed to self-renew and became spontaneously differentiated. Presumably, this resulted from an interruption of the self-renewal pathway. In summary, we generated pluripotent bovine ES cells with morphology similar to those of established ES cells in humans and mice as well as marker-staining patterns identical to those of the bovine blastocysts.  相似文献   

15.
Porcine embryonic germ (EG) cells share common features with porcine embryonic stem (ES) cells, including morphology, alkaline phosphatase activity and capacity for in vitro differentiation. Porcine EG cells are also capable of in vivo development by producing chimeras after blastocyst injection; however, the proportion of injected embryos that yield a chimera and the proportion of cells contributed by the cultured cells in each chimera are too low for practical use in genetic manipulation. Moreover, somatic, but not germ-line chimerism, has been reported from blastocyst injection using porcine ES or EG cells. To test whether efficiency of chimera production from blastocyst injection can be improved upon by changing the host embryo, we used as host embryos four groups according to developmental stage or length in culture: fresh 4-cell and 8-cell stage embryos subsequently cultured into blastocysts, fresh morulae, fresh blastocysts, and cultured blastocysts. Injection and embryo transfer of fresh and cultured blastocysts produced similar percentages of live piglets (17% versus 19%). Four piglets were judged to have a small degree of pigmentation chimerism, but microsatellite analysis failed to confirm chimerism in these or other piglets. Polymerase chain reaction analysis for detection of the porcine SRY gene in female piglets born from embryos injected with male EG cells identified six chimeras, at least one, but not more than two, from each treatment. Chimerism was confirmed in two putative pigmentation chimeras and in four piglets without overt signs of chimerism. The low percentage of injected embryos that yielded a chimera and the small contribution by EG cells to development of each confirmed chimera indicated that procedural changes in how EG cells were combined with host embryos were unsuccessful in increasing the likelihood that porcine EG cells will participate in embryonic development. Alternatively, our results suggested that improvements are needed in EG cell isolation and culture procedures to ensure in vitro maintenance of EG cell developmental capacity.  相似文献   

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17.
Potential of embryonic stem cells   总被引:29,自引:0,他引:29  
Embryonic stem (ES) cells are pluripotent cell lines established from undifferentiated embryonic cells characterized by nearly unlimited self-renewal and differentiation capacity. During differentiation in vitro, ES cells were found to be able to develop into specialized somatic cells types and to recapitulate processes of early embryonic development. These properties allow to use ES cells as model system for studying early embryonic development by gain- or loss-of-function approaches, or to investigate the effects of drugs and environmental factors on differentiation and cell function in embryotoxicity and pharmacology. Now, ES cells derived of human blastocysts may be used for the generation of somatic precursor or differentiated cells in cell and tissue therapy. The review presents data of mouse ES cell differentiation and gives an outlook on future perspectives and problems of using human ES cells in regenerative medicine.  相似文献   

18.
Embryonic stem (ES) cells are capable of differentiating into all embryonic and adult cell types following mouse chimera production. Although injection of diploid ES cells into tetraploid blastocysts suggests that tetraploid cells have a selective disadvantage in the developing embryo, tetraploid hybrid cells, formed by cell fusion between ES cells and somatic cells, have been reported to contribute to mouse chimeras. In addition, other examples of apparent stem cell plasticity have recently been shown to be the result of cell fusion. Here we investigate whether ES cells contribute to mouse chimeras through a cell fusion mechanism. Fluorescence in situ hybridization (FISH) analysis for X and Y chromosomes was performed on dissociated tissues from embryonic, neonatal, and adult wild-type, and chimeric mice to follow the ploidy distributions of cells from various tissues. FISH analysis showed that the ploidy distributions in dissociated tissues, notably the tetraploid cell number, did not differ between chimeric and wild-type tissues. To address the possibility that early cell fusion events are hidden by subsequent reductive divisions or other changes in cell ploidy, we injected Z/EG (lacZ/EGFP) ES cells into ACTB-cre blastocysts. Recombination can only occur as the result of cell fusion, and the recombined allele should persist through any subsequent changes in cell ploidy. We did not detect evidence of fusion in embryonic chimeras either by direct fluorescence microscopy for GFP or by PCR amplification of the recombined Z/EG locus on genomic DNA from ACTB-cre::Z/EG chimeric embryos. Our results argue strongly against cell fusion as a mechanism by which ES cells contribute to chimeras.  相似文献   

19.
Embryonic stem (ES) cell lines derived from human blastocysts have the developmental potential to form derivatives of all three embryonic germ layers even after prolonged culture. Here we describe the clonal derivation of two human ES cell lines, H9.1 and H9.2. At the time of the clonal derivation of the H9.1 and H9.2 ES cell lines, the parental ES cell line, H9, had already been continuously cultured for 6 months. After an additional 8 months of culture, H9.1 and H9.2 ES cell lines continued to: (1) actively proliferate, (2) express high levels of telomerase, and (3) retain normal karyotypes. Telomere lengths, while somewhat variable, were maintained between 8 and 12 kb in high-passage H9.1 and H9.2 cells. High-passage H9.1 and H9.2 cells both formed teratomas in SCID-beige mice that included differentiated derivatives of all three embryonic germ layers. These results demonstrate the pluripotency of single human ES cells, the maintenance of pluripotency during an extended period of culture, and the long-term self-renewing properties of cultured human ES cells. The remarkable developmental potential, proliferative capacity, and karyotypic stability of human ES cells distinguish them from adult cells.  相似文献   

20.
In contrast to the highly developed genetic modification systems available for manipulating the mouse genome, at this time only simple gain of function modifications can be undertaken in domestic species. Clearly, the greatest barrier to gene targeting in domestic species has been the unavailability of cell lines that can be modified in vitro and still be used to generate a living organism. In the mouse, the embryonic stem (ES) cells and embryonic germ (EG) cells have fulfilled that role. While the nuclear transfer procedures have solved this problem in sheep and cattle, in swine ES and EG cells are still needed. In addition, targeting in domestic species is affected by the need to develop targeting constructs containing isogenic DNA regions. As a result, it is necessary to isolate the gene of interest, sequence required regions, and develop isogenic targeting constructs by technologies such as long-range PCR. On the positive side, enrichment protocols developed in the mouse can be applied to domestic species, thus facilitating the identification of correctly modified cell lines. Hence, progress in mammalian cloning, the development of EG cell lines, and advances in gene targeting presently allows the introduction of precise genetic modifications into the domestic animal genome.  相似文献   

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