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目的 应用旋转生物反应器(RCCS)和微载体培养体系尝试建立一种实现批量培养干细胞的新方法.方法 应用RCCS和微载体培养体系对小鼠胚胎干细胞(mESCs)进行体外培养扩增,定期收集细胞样品,镜下观察mESCs在RCCS生长的形态特征,并定量绘制细胞生长曲线,利用MATLAB软件计算细胞生长参数并对照平面培养体系,利用H&E染色、免疫荧光及RT-PCR技术对RCCS内培养的mESCs的细胞形态,未分化标志蛋白(SSEA-1)和标志基因(oct-4)的表达进行定性或半定量分析.结果 mESCs可在RCCS内以贴附于微载体表面的形式实现三维生长,其生长增殖状态良好,且伴随培养时间的延长,SSEA-1蛋白及oct-4 基因的表达水平逐渐降低.这表明RCCS内培养扩增的mESCs逐渐走向分化,该分化进程同步于平面对照培养体系.结论 RCCS可以为mESCs的体外规模化扩增培养提供良好的培养体系.  相似文献   

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Understanding endothelial cell (EC) differentiation is a step forward in tissue engineering, controlling angiogenesis, and endothelial dysfunction. We hypothesized that epigenetic activation of EC lineage specification genes is an important mediator of embryonic stem cell (ESC) differentiation into EC. Mouse ESC was differentiated by removing leukemia inhibitory factor (LIF) from the maintenance media in the presence or absence of the specific DNA methyltransferase (DNMT) inhibitor 5′-aza-2′-deoxycytidine (aza-dC). Expression of EC specification and marker genes was monitored by quantitative PCR, western, immunocytochemistry, and flow cytometry. Functionality of differentiated EC was assessed by angiogenesis assay. The methylation status in the proximal promoter CpGs of the mediators of EC differentiation VEGF-A, BMP4, and EPAS-1 as well as of the mature EC marker VE-cadherin was determined by bisulfite sequencing. ESC differentiation resulted in repression of OCT4 expression in both the absence and presence of aza-dC treatment. However, significant increase in angiogenesis and expression of the mediators of EC differentiation and EC-specific genes was only observed in aza-dC-treated cells. The DNMT inhibition-mediated increase in EC specification and marker gene expression was not associated with demethylation of these genes. These studies suggest that DNMT inhibition is an efficient inducer of EC differentiation from ESC.  相似文献   

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Directed differentiation of dendritic cells from mouse embryonic stem cells   总被引:14,自引:0,他引:14  
Dendritic cells (DCs) are uniquely capable of presenting antigen to naive T cells, either eliciting immunity [1] or ensuring self-tolerance [2]. This property identifies DCs as potential candidates for enhancing responses to foreign [3] and tumour antigens [4], and as targets for immune intervention in the treatment of autoimmunity and allograft rejection [1]. Realisation of their therapeutic potential would be greatly facilitated by a fuller understanding of the function of DC-specific genes, a goal that has frequently proven elusive because of the paucity of stable lines of DCs that retain their unique properties, and the inherent resistance of primary DCs to genetic modification. Protocols for the genetic manipulation of embryonic stem (ES) cells are, by contrast, well established [5], as is their capacity to differentiate into a wide variety of cell types in vitro, including many of hematopoietic origin [6]. Here, we report the establishment, from mouse ES cells, of long-term cultures of immature DCs that share many characteristics with macrophages, but acquire, upon maturation, the allostimulatory capacity and surface phenotype of classical DCs, including expression of CD11c, major histocompatibility complex (MHC) class II and co-stimulatory molecules. This novel source should prove valuable for the generation of primary, untransformed DCs in which candidate genes have been overexpressed or functionally ablated, while providing insights into the earliest stages of DC ontogeny.  相似文献   

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Presence of specific growth factors and feeder layers are thought to be important for in vitro embryonic stem cell (ESCs) differentiation. In this study, the effect of bone morphogenetic protein 4 (BMP4) and mouse embryonic fibroblasts (MEFs) co-culture system on germ cell differentiation from mouse ESCs was evaluated. One-day-old embryoid body was cultured for 4?d in simple culture systems or on top of the MEFs, both in the presence or absence of BMP4. Data showed significant higher viability percent and proliferation rate in simple culture media compared to co-culture systems. Analysis of gene expression indicated that the germ cell-specific genes (VASA and Stra8) were expressed in a significant higher ratio in BMP4-treated cells in simple culture system. Also, the results of immunocytochemistry in simple culture systems showed that the mean percentage of immunostaining cells of VASA, the primordial germ cell (PGC) marker, was increased significantly in BMP4-treated cells compared with BMP4-free group. Meanwhile, CDH1, the late premiotic germ cell marker, showed no significant difference between these two groups. The results suggest that BMP4 is an efficient inducer in PGC derivation from mouse ESC. However, the employment of MEFs as feeder has no apparent effect on PGC derivation.  相似文献   

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Embryonic stem (ES) cells are typically derived from the inner cell mass of the preimplantation blastocyst and can both self-renew and differentiate into all the cells and tissues of the embryo. Because they are pluripotent, ES cells have been used extensively to analyze gene function in development via gene targeting. The embryonic stem cell is also an unsurpassed starting material to begin to understand a critical, largely inaccessible period of development. If their differentiation could be controlled, they would also be an important source of cells for transplantation to replace cells lost through disease or injury or to replace missing hormones or genes. Traditionally, ES cells have been differentiated in suspension culture as embryoid bodies, named because of their similarity to the early postimplantation-staged embryo. Unlike the pristine organization of the early embryo, differentiation in embryoid bodies appears to be largely unpatterned, although multiple cell types form. It has recently been possible to separate the desired cell types from differentiating ES cells in embryoid bodies by using cell-type-restricted promoters driving expression of either antibiotic resistance genes or fluorophores such as EGFP. In combination with growth factor exposure, highly differentiated cell types have successfully been derived from ES cells. Recent technological advances such as RNA interference to knock down gene expression in ES cells are also producing enriched populations of cells and elucidating gene function in early development.  相似文献   

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In this study, we selected gelatin as ECM (extracellular matrix) to support differentiation of mES (mouse embryonic stem) cells into TE (trophectoderm), as gelatin was less expensive and widely used. We found that 0.2% and 1.5% gelatin were the suitable concentrations to induce TE differentiation by means of detecting Cdx2 expression using real-time PCR. Moreover, about 15% cells were positive for Cdx2 staining after 6 days differentiation. We discovered that the expressions of specific markers for TE, such as Cdx2, Eomes, Hand1 and Esx1 were prominently increased after gelatin induction. Meanwhile, the expression of Oct4 was significantly decreased. We also found that inhibition of the BMP (bone morphogenetic protein) signalling by Noggin could promote mES cells differentiation into TE, whereas inhibition of the Wnt signalling by Dkk1 had the contrary effect. This could be used as a tool to study the differentiation and function of early trophoblasts as well as further elucidating the molecular mechanism during abnormal placental development.  相似文献   

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Embryonic stem cells (ESCs) are potentially powerful tools for regenerative medicine and establishment of disease models. The recent progress in ESC technologies is noteworthy, but ESC differentiation into renal lineages is relatively less established. The present study aims to differentiate mouse ESCs (mESCs) into a renal progenitor pool, the intermediate mesoderm (IM), without addition of exogenous cytokines and embryoid formation. First, we treated mESCs with a combination of small molecules (Janus-associated tyrosine kinase inhibitor 1, LY294002, and CCG1423) and differentiated them into BMP7-positive cells, BMP7 being the presumed inducing factor for IM. When these cells were cultured with adding retinoic acid, expression of odd-skipped related 1 (Osr1), which is essential to IM differentiation, was enhanced. To simplify the differentiation protocol, the abovementioned four small molecules (including retinoic acid) were combined and added to the culture. Under this condition, more than one-half of the cells were positive for Osr1, and at the same time, Pax2 (another IM marker) was detected by real-time PCR. Expressions of ectodermal marker and endodermal marker were not enhanced, while mesodermal marker changed. Moreover, expression of genes indispensable to kidney development, i.e., Lim1 and WT1, was detected by RT-PCR. These results indicate the establishment of a specific, effective method for differentiation of the ESC monolayer into IM using a combination of small molecules, resulting in an attractive cell source that could be experimentally differentiated to understand nephrogenic mechanisms and cell-to-cell interactions in embryogenesis.  相似文献   

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An improved cryopreservation method for a mouse embryonic stem cell line   总被引:1,自引:1,他引:1  
Embryonic stem (ES) cell lines including the C57BL/6 genetic background are central to projects such as the Knock-Out Mouse Project, North American Conditional Mouse Mutagenesis Program, and European Conditional Mouse Mutagenesis Program, which seek to create thousands of mutant mouse strains using ES cells for the production of human disease models in biomedical research. Crucial to the success of these programs is the ability to efficiently cryopreserve these mutant cell lines for storage and transport. Although the ability to successfully cryopreserve mouse ES cells is often assumed to be adequate, the percent post-thaw recovery of viable cells varies greatly among genetic backgrounds and individual cell lines within a genetic background. Therefore, there is a need to improve the efficiency and reduce the variability of current mouse ES cell cryopreservation methods. To address this need, we employed the principles of fundamental cryobiology to improve the cryopreservation protocol of a C57BL/6 mouse ES cell line by characterizing the membrane permeability characteristics and osmotic tolerance limits. These values were used to predict optimal cooling rates, warming rates, and type of cryoprotectant, which were then verified experimentally. The resulting protocol, generated through this hypothesis-driven approach, resulted in a 2-fold increase in percent post-thaw recovery of membrane-intact ES cells as compared to the standard freezing protocol, as measured by propidium iodide exclusion. Additionally, our fundamental cryobiological approach to improving cryopreservation protocols provides a model system by which additional cryopreservation protocols may be improved in future research for both mouse and human ES cell lines.  相似文献   

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The use of small specific molecules has been instrumental in the modulation of stem cell proliferation and differentiation to obtain insulin-containing cells. Examples include nutrients (glucose, nicotinamide and retinoic acid), acids (butyrate), alkaloids (cyclopamine and conophylline) and pharmacological agents (LY294002 and wortmannin). These molecules, alone or in combination with specific growth factors and hormones, will likely provide key information to design specific culture media in order to obtain customized cells for implantation in diabetes. In addition, the study of such molecules will help to understand the mechanisms involved in stem cell biology as well as contribute to the design of specific drugs for islet repair and regeneration in diabetes.  相似文献   

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Proteomic analysis of neural differentiation of mouse embryonic stem cells   总被引:4,自引:0,他引:4  
Wang D  Gao L 《Proteomics》2005,5(17):4414-4426
Mouse embryonic stem cells (mESCs) can differentiate into different types of cells, and serve as a good model system to study human embryonic stem cells (hESCs). We showed that mESCs differentiated into two types of neurons with different time courses. To determine the global protein expression changes after neural differentiation, we employed a proteomic strategy to analyze the differences between the proteomes of ES cells (E14) and neurons. Using 2-DE plus LC/MS/MS, we have generated proteome reference maps of E14 cells and derived dopaminergic neurons. Around 23 proteins with an increase or decrease in expression or phosphorylation after differentiation have been identified. We confirmed the downregulation of translationally controlled tumor protein (TCTP) and upregulation of alpha-tubulin by Western blotting. We also showed that TCTP was further downregulated in derived motor neurons than in dopaminergic neurons, and its expression level was independent of extracellular Ca(2+) concentration during neural differentiation. Potential roles of TCTP in modulating neural differentiation through binding to Ca(2+), tubulin and Na,K-ATPase, as well as the functional significance of regulation of other proteins such as actin-related protein 3 (Arp3) and Ran GTPase are discussed. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.  相似文献   

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Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb9. Using this robust protocol, we achieved 51 ± 0.8% of differentiation efficiency (n = 3; p < 0.01, Student's t-test). Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.  相似文献   

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Neural differentiation of mouse embryonic stem cells grown in monolayer   总被引:5,自引:0,他引:5  
To drive neural differentiation of mouse embryonic stem (ES) cells, various culture protocols have been previously developed that all require the formation of embryoid bodies, usually combined with a treatment by all-trans retinoic acid (aRA). Here, we investigated whether or not neural differentiation can also occur in a simplified monolayer culture. Mouse ES cells were plated in serum-containing DMEM media with and without aRA and grown under these conditions for 2 days. Then, the cells were transferred to fresh serum-containing DMEM media and/or to serum-free DMEM/F12 media supplemented with a mixture of insulin, transferrin, selenium, and fibronectin (ITSF) for further culture. The changes in cell morphology and in the expression of selected molecular markers were monitored. Generally, in contrast to all the others, the protocol consisting of a 2-day culture in serum-containing DMEM followed by continuous exposure to the ITSF supplement in DMEM/F12 drove a vast majority of ES cells to generate phenotypic signs of neural lineage. Altogether, neural differentiation can be achieved in vitro without the step involving the formation of embryoid bodies as well as the treatment by aRA.  相似文献   

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