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1.
Embryonic stem cells (ES) can self-replicate and differentiate into all cell types including insulin-producing, beta-like cells and could, therefore, be used to treat diabetes mellitus. To date, results of stem cell differentiation into beta cells have been debated, largely due to difficulties in defining the identity of a beta cell. We have recently differentiated non-human primate (rhesus) embryonic stem (rES) cell lines into insulin producing, beta-like cells with the beta cell growth factor, Exendin-4 and using C-peptide as a phenotype marker. Cell development was characterized at each stage by gene and protein expression. Insulin, NKX6.1 and glucagon mRNA were expressed in stage 4 cells but not in early undifferentiated cells. We concluded that rES cells could be differentiated ex vivo to insulin producing cells. These differentiated rES cells could be used to develop a non-human primate model for evaluating cell therapy to treat diabetes. To facilitate the identification of beta-like cells and to track the cells post-transplantation, we have developed a marker gene construct: fusing the human insulin promoter (HIP) to the green fluorescent protein (GFP) gene. This construct was transfected into stage 3 rES derived cells and subsequent GFP expression was identified in C-peptide positive cells, thereby substantiating endogenous insulin production by rES derived cells. Using this GFP detection system, we will enrich our population of insulin producing rES derived cells and track these cells post-transplantation in the non-human primate model.  相似文献   

2.
Human pluripotent embryonic stem cells (hESC) have great promise for research into human developmental biology and the development of cell therapies for the treatment of diseases. To meet the increased demand for characterized hESC lines, we present the derivation and characterization of five hESC lines on mouse embryonic fibroblast cells. Our stem cell lines are characterized by morphology, long-term expansion, and expression profiles of a number of specific markers, including TRA-1-60, TRA-1-81, alkaline phosphatase, connexin 43, OCT-4, NANOG, CXCR4, NODAL, LEFTY2, THY-1, TDGF1, PAX6, FOXD3, SOX2, EPHA2, FGF4, TAL1, AC133 and REX-1. The pluripotency of the cell line was confirmed by spontaneous differentiation under in vitro conditions. Whereas all of the cell lines expressed all the characteristics of undifferentiated pluripotent hESC, two of the cell lines carried a triploid karyotype.  相似文献   

3.
The rat is an important animal model for human disease research. Using inhibitors of glycogen synthase kinase 3 and MAPK signaling pathways, rat embryonic stem cells and rat induced pluripotent stem cells (riPSCs) have been derived. However, unlike rat embryonic stem cells, germ line competent riPSCs have only been derived from Wistar rats at low efficiency. Here, we found that an optimized induction medium containing knock-out serum replacement and vitamin C improved the rate and efficiency of riPSCs generation from Dark Agouti rat fibroblasts and Sertoli cells. riPSCs maintained an undifferentiated status for >30 passages and could differentiate into various cells types including germ cells when injected into rat blastocysts. Moreover, transgenic riPSCs could be generated through the PiggyBac transposon, which could be used to generate transgenic rats through germ line transmission. riPSCs can be used as a novel tool in genetic and genomic studies of the rat.  相似文献   

4.
Human embryonic stem (ES) cells are pluripotent cell lines that have been derived from the inner cell mass (ICM) of blastocyst stage embryos [1--3]. They are characterized by their ability to be propagated indefinitely in culture as undifferentiated cells with a normal karyotype and can be induced to differentiate in vitro into various cell types [1, 2, 4-- 6]. Thus, human ES cells promise to serve as an unlimited cell source for transplantation. However, these unique cell lines tend to spontaneously differentiate in culture and therefore are difficult to maintain. Furthermore, colonies may contain several cell types and may be composed of cells other than pluripotent cells [1, 2, 6]. In order to overcome these difficulties and establish lines of cells with an undifferentiated phenotype, we have introduced a reporter gene that is regulated by a promoter of an ES cell-enriched gene into the cells. For the introduction of DNA into human ES cells, we have established a specific transfection protocol that is different from the one used for murine ES cells. Human ES cells were transfected with enhanced green fluorescence protein (EGFP), under the control of murine Rex1 promoter. The transfected cells show high levels of GFP expression when in an undifferentiated state. As the cells differentiate, this expression is dramatically reduced in monolayer cultures as well as in the primitive endoderm of early stage (simple) embryoid bodies (EBs) and in mature EBs. The undifferentiated cells expressing GFP can be analyzed and sorted by using a Fluorescence Activated Cell Sorter (FACS). Thus, we have established lines of human ES cells in which only undifferentiated cells are fluorescent, and these cells can be followed and selected for in culture. We also propose that the pluripotent nature of the culture is made evident by the ability of the homogeneous cell population to form EBs. The ability to efficiently transfect human ES cells will provide the means to study and manipulate these cells for the purpose of basic and applied research.  相似文献   

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One of the challenges in studying early differentiation of human embryonic stem cells (hESCs) is being able to discriminate the initial differentiated cells from the original pluripotent stem cells and their committed progenies. It remains unclear how a pluripotent stem cell becomes a lineage-specific cell type during early development, and how, or if, pluripotent genes, such as Oct4 and Sox2, play a role in this transition. Here, by studying the dynamic changes in the expression of embryonic surface antigens, we identified the sequential loss of Tra-1-81 and SSEA4 during hESC neural differentiation and isolated a transient Tra-1-81(-)/SSEA4(+) (TR-/S4+) cell population in the early stage of neural differentiation. These cells are distinct from both undifferentiated hESCs and their committed neural progenitor cells (NPCs) in their gene expression profiles and response to extracellular signalling; they co-express both the pluripotent gene Oct4 and the neural marker Pax6. Furthermore, these TR-/S4+ cells are able to produce cells of both neural and non-neural lineages, depending on their environmental cues. Our results demonstrate that expression of the pluripotent factor Oct4 is progressively downregulated and is accompanied by the gradual upregulation of neural genes, whereas the pluripotent factor Sox2 is consistently expressed at high levels, indicating that these pluripotent factors may play different roles in the regulation of neural differentiation. The identification of TR-S4+ cells provides a cell model for further elucidation of the molecular mechanisms underlying hESC neural differentiation.  相似文献   

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8.
吴昭  成璐  肖磊 《生命科学》2009,(5):658-662
胚胎干细胞(embryonic stem cells,ESC)在人类遗传病学研究、疾病模型建立、器官再生以及动物物种改良和定向变异等方面的地位是其他类型的细胞不可取代的。但是,由于实验技术和体外培养条件的限制,除了小鼠、恒河猴和人之外,大鼠、猪、牛、羊等其他哺乳动物的ES细胞系被证明很难获得。先后有多个研究小组报道了他们利用新兴的诱导多能干细胞(induced pluripotent stem cells,iPS细胞)技术成功建立大鼠和猪的iPS细胞系的研究成果。迄今为止,这两个物种是在未成功建立ES细胞系之前利用iPS技术建立多能干细胞系的成功范例。这些研究对于那些还未建立ES细胞的物种建立多能干细胞系提供了一种新的方案,也将给这些物种的胚胎干细胞的建立、基因修饰动物的产生以及人类医疗事业的促进和发展带来新的希望。  相似文献   

9.
Human embryonic stem cells express a unique set of microRNAs   总被引:41,自引:0,他引:41  
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10.
Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. These cells have, therefore, potential for in vitro differentiation studies, gene function, and so on. The aim of this study was to produce a human embryonic stem cell line. An inner cell mass of a human blastocyst was separated and cultured on mouse embryonic fibroblasts in embryonic stem cell medium with related additives. The established line was evaluated by morphology; passaging; freezing and thawing; alkaline phosphatase; Oct-4 expression; anti-surface markers including Tra-1-60 and Tra-1-81; and karyotype and spontaneous differentiation. Differentiated cardiomyocytes and neurons were evaluated by transmission electron microscopy and immunocytochemistry. Here, we report the derivation of a new embryonic stem cell line (Royan H1) from a human blastocyst that remains undifferentiated in morphology during continuous passaging for more than 30 passages, maintains a normal XX karyotype, is viable after freezing and thawing, and expresses alkaline phosphatase, Oct-4, Tra-1-60, and Tra-1-81. These cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers in the presence or absence of recombinant human leukemia inhibitory factor. Royan H1 cells can differentiate in vitro in the absence of feeder cells and can produce embryoid bodies that can further differentiate into beating cardiomyocytes as well as neurons. These results define Royan H1 cells as a new human embryonic stem cell line.  相似文献   

11.
Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model.  相似文献   

12.
In vitro neuronal differentiation of cultured human embryonic germ cells   总被引:8,自引:0,他引:8  
Human embryonic germ (hEG) cells, which have been advanced as one of the most important sources of pluripotent stem cells [the other one being human embryonic stem cells], can be propagated in vitro indefinitely in the primitive undifferentiated state while being capable of developing into all three germ layer derivatives, hence have become anticipated developing novel strategies of tissue regeneration and transplantation in the treatment of degenerative diseases. In the experiments here, we derived hEG cells from cultured human primordial germ cells (PGCs) of 6- to 9-week-post-fertilization embryos. They satisfied the criteria previously used to define hEG cells, including the expression of markers characteristic of pluripotent cells-abundant alkaline phosphatase (AP) activity, stage specific embryonic antigen (SSEA)-1(+), SSEA-3(-), SSEA-4(+), TRA-1-60(+), TRA-1-81(+), Oct-4(+), and hTERT(+), the retention of normal karyotypes, and possessing pluripotency by forming embryoid bodies (EBs) in vitro. Furthermore, these derived cells tended to neurally differentiate in vitro, especially under high-density culture conditions. We successfully isolated neural progenitor cells from differentiating hEG cultures and about 10% cells induced by 2microM all-trans-retinoic acid (RA) or 0.1mM dibutyryl cyclic AMP (dbcAMP)/1mM forskolin to mature neurons expressing microtubule-associated protein 2ab (MAP2ab), synaptophysin, beta-tubulin III, neuron-specific enolase (NSE), tyrosine hydroxylase (TH), but no glial fibrillary acid protein (GFAP) and choline acetyl transferase (ChAT). The data suggested that hEG cells may provide a potential source of cells for use in transplantation therapy for neurological degenerative diseases.  相似文献   

13.
Pluripotent stem cells are able to proliferate indefinitely and differentiate in vitro into various cell types. However, in most cases in vitro differentiation of the pluripotent stem cells is asynchronous and incomplete, and the residual undifferentiated cells can initiate teratoma development after transplantation into recipients. These features of the pluripotent stem cells are the major issue for development of safe cell therapy technologies based on pluripotent stem cells. Considering significant resemblance of growth rates of pluripotent stem and cancer cells we investigated antiproliferative and cytotoxic effects of different type cytostatics (mitomycin C, etoposide, vinblastine and cycloheximide) on the undifferentiated and differentiating mouse embryonic stem cells, embryonic germ cells, blastocyst and on mouse embryonal teratocarcinoma cells and mouse embryonic fibroblasts. The findings showed that all cytostatics used induced both antiproliferative effects and acute toxic processes in undifferentiated pluripotent stem cells and embryonal teratocarcinoma cells whereas these effects were less in differentiating embryonic stem cells and embryonic fibroblast. Moreover, the trophoblast cells of mouse blastocysts were less sensitive to damaging effects of cytostatics than inner cell mass cells. The examination of deferred effects of cytostatics revealed that the effects of mitomycin C, etoposide and vinblastine, but not cycloheximide, were irreversible because survived cells were not able to proliferate. Nevertheless, the numbers of embryonic fibroblasts exposed to etoposide or vinblastine remained unchanged while vast majority of undifferentiated pluripotent cells treated underwent apoptosis. Thus, diverse effects of etoposide and vinblastine on the undifferentiated pluripotent stem cells and differentiated embryonic cells allow us to consider these cytostatics and their analogs as drug-candidates for selective elimination of the residual undifferentiated pluripotent stem cells from population of differentiating cells. These findings demonstrate for the first time the possibility of selective elimination of undifferentiated pluripotent stem cells using cytostatic drugs approved for clinic practice. However, to improve effectiveness and safety of this approach and to prevent mutagenic, carcinogenic and teratogenic effects on undifferentiated pluripotent stem cells and their differentiated cell derivatives large-scale studies of cytostatic effects using different experimental design and active doses must be performed.  相似文献   

14.
Embryonic stem (ES) cell lines represent a population of undifferentiated pluripotent cells capable of multilineage differentiation in vitro. Although very useful for studying developmental processes, human ES cell lines have also been suggested as a potential and unlimited source for cellular transplantation and the treatment of human disease. The proteomic basis of embryonic stemness (pluripotentiality and multilineage differentiation) and the transitions that lead to specific cell lineages however, remain to be defined. As an important first step in defining these processes, we have performed a proteomic analysis of undifferentiated mouse R1 ES cell lines using pH 3-10, 4-7 and 6-11 two-dimensional electrophoresis gels, matrix-assisted laser desorption/ionization and tandem mass spectrometry. Of the 700 gel spots analyzed, 241 distinct protein species were identified corresponding to 218 unique proteins, with a significant proportion functionally related to protein expression.  相似文献   

15.
16.
Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.  相似文献   

17.
Control of the embryonic stem cell state   总被引:2,自引:0,他引:2  
Young RA 《Cell》2011,144(6):940-954
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20.
An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti-stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.  相似文献   

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