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1.
Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells 总被引:28,自引:0,他引:28
Human embryonic stem (hES) cells are typically derived and serially propagated on inactivated murine embryonic fibroblast (MEF) feeders. The use of MEFs and other components of animal origin in the culture media for hES cell support substantially elevates the risk of contaminating these cell lines with infectious agents of animal origin thereby severely limiting their potential for clinical application. We have previously shown that it is possible to derive and establish new hES cell lines in a xeno-free culture system using human fetal muscle fibroblast feeders. In this report, we have comparatively evaluated a panel of 11 different human adult, fetal, and neonatal feeders for hES cell support and have ranked them as supportive and non-supportive. We report that two adult skin fibroblast cell lines established in-house from abdominal skin biopsies supported prolonged undifferentiated hES cell growth for over 30 weekly passages in culture. Furthermore, hES cell lines cultured on adult skin fibroblast feeders retain hES cell morphology and remain pluripotent. Also, differences in feeder support exist between human cell types and sources. The use of human adult skin feeders is convenient for hES cell support given the ease of obtaining skin biopsies. 相似文献
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MicroRNA expression pattern of undifferentiated and differentiated human embryonic stem cells 总被引:4,自引:0,他引:4
Lakshmipathy U Love B Goff LA Jörnsten R Graichen R Hart RP Chesnut JD 《Stem cells and development》2007,16(6):1003-1016
Many of the currently established human embryonic stem (hES) cell lines have been characterized extensively in terms of their gene expression profiles and genetic stability in culture. Recent studies have indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Using both microarrays and quantitative PCR, we report here the differences in miRNA expression between undifferentiated hES cells and their corresponding differentiated cells that underwent differentiation in vitro over a period of 2 weeks. Our results confirm the identity of a signature miRNA profile in pluripotent cells, comprising a small subset of differentially expressed miRNAs in hES cells. Examining both mRNA and miRNA profiles under multiple conditions using cross-correlation, we find clusters of miRNAs grouped with specific, biologically interpretable mRNAs. We identify patterns of expression in the progression from hES cells to differentiated cells that suggest a role for selected miRNAs in maintenance of the undifferentiated, pluripotent state. Profiling of the hES cell "miRNA-ome" provides an insight into molecules that control cellular differentiation and maintenance of the pluripotent state, findings that have broad implications in development, homeostasis, and human disease states. 相似文献
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Human embryonic stem cells (hESCs) possess unique properties for studying mechanisms controlling cell fate commitment during early mammalian development. Gain of function is a common strategy to study the function of specific genes involved in these mechanisms. However, transgene toxicity can be a major limitation, especially with factors influencing proliferation or differentiation. Here, we describe an efficient method based on the inducible recombinase Cre-ERT2 for conditional gene expression in hESCs and their differentiated derivatives. Using this approach, we have established several hESC sublines inducible for the expression of the enhanced green fluorescent protein and the transforming growth factor beta family member Nodal. Together, these results demonstrate that Cre-ERT2 can be used to control gene expression in undifferentiated and differentiated cells, thereby providing the first conditional transgene expression system that works effectively in hESCs. Disclosure of potential conflicts of interest is found at the end of this article. 相似文献
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Fiegel HC Lioznov MV Cortes-Dericks L Lange C Kluth D Fehse B Zander AR 《Stem cells (Dayton, Ohio)》2003,21(1):98-104
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Park Y Kim JH Lee SJ Choi IY Park SJ Lee SR Sung HJ Yoo YD Geum DH Choi CW Kim SH Kim BS 《Stem cells and development》2011,20(11):1901-1910
In the culture system using human feeder cells, the mechanism through which these cells support undifferentiated growth of embryonic stem cells (ESCs) has not been well investigated. Here, we explored the mechanisms of 3 kinds of human feeder cells, including human placental cells from the chorionic plate, human bone marrow stromal cells, and human foreskin fibroblasts. First, we determined that undifferentiated growth of 2 kinds each of human (H1 and HSF6) and mouse (D3 and CE3) ESCs was possible in all human feeder cell types tested (human placental cells, human bone marrow stromal cells, and human foreskin fibroblasts), without the need for exogenous cytokine supplementation including basic fibroblast growth factor (bFGF) and leukemia inhibitory factor. We then prepared their corresponding endogenous bFGF-knockout feeders using siRNA and tried to maintain human and mouse ESCs in their undifferentiated state; however, neither human nor mouse ESCs could be maintained in bFGF-knockout human feeder cells. The expressions of stemness markers such as Oct-4 and Nanog were significantly decreased in the bFGF-knockout group compared with those in the controls, and differentiation had already occurred, despite the undifferentiated morphologic appearance of the ESCs. In conclusion, human feeder cells are able to support the undifferentiated growth of human and mouse ESCs via bFGF synthesis. Further, a bFGF-dependent pathway might be crucial for maintaining the undifferentiated characteristics of mouse and human ESCs. 相似文献
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Homologous feeder cells support undifferentiated growth and pluripotency in monkey embryonic stem cells 总被引:5,自引:0,他引:5
Li T Wang S Xie Y Lu Y Zhang X Wang L Yang S Wolf D Zhou Q Ji W 《Stem cells (Dayton, Ohio)》2005,23(8):1192-1199
In the present study, five homologous feeder cell lines were developed for the culture and maintenance of rhesus monkey embryonic stem cells (rESCs). Monkey ear skin fibroblasts (MESFs), monkey oviductal fibroblasts (MOFs), monkey follicular granulosa fibroblast-like (MFG) cells, monkey follicular granulosa epithelium-like (MFGE) cells, and clonally derived fibroblasts from MESF (CMESFs) were established and compared with the ability of mouse embryonic fibroblasts (MEFs) to support rESC growth. MESF, MOF, MFG, and CMESF cells, but not MFGE cells, were as good as or better than MEFs in supporting undifferentiated growth while maintaining the differentiation potential of the rESCs. In an effort to understand the unique properties of supportive feeder cells, expression levels for a number of candidate genes were examined. MOF, MESF, and MEF cells highly expressed leukemia inhibitory factor, ciliary neurotrophic factor, basic fibroblast growth factor, stem cell factor, transforming growth factor beta1, bone morphogenetic protein 4, and WNT3A, whereas WNT2, WNT4, and WNT5A were downregulated, compared with MFGE cells. Additionally, all monkey feeder cell lines expressed Dkk1 and LRP6, antagonists of the WNT signaling pathway, but not WNT1, WNT8B, or Dkk2. rESCs grown on homologous feeders maintained normal karyotypes, displayed the characteristics of ESCs, including morphology, alkaline phosphatase, Oct4, the cell surface markers stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor-related antigen (TRA)-1-60, and TRA-1-81, and formed cystic embryoid bodies in vitro that included differentiated cells representing the three major germ layers. These results indicate that the four homologous feeder cell lines can be used to support the undifferentiated growth and maintenance of pluripotency in rESCs. 相似文献
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目的: 探讨血红素氧合酶-1(HO-1)是否提高骨髓间充质干细胞(MSCs) 在无氧无血清条件下的存活率。方法:取大鼠骨髓,体外分离扩增培养 MSCs;HO-1腺病毒转染 MSCs,荧光显微镜下观察绿色荧光蛋白 (GFP) 的表达;DAPI 染色观察无氧无血清条件下 HO-1-MSCs、MSCs 细胞核变化;流式细胞仪检测无氧无血清条件下HO-1-MSCs、MSCs 的凋亡率;ELISA 检测无氧无血清条件下 HO-1-MSCs、MSCs 细胞因子的分泌。用无氧无血清条件下 HO-1-MSCs、MSCs 的培养上清液对无氧无血清条件下的心肌细胞进行培养,Western blotting 检测凋亡蛋白 caspase-3 的变化,分光光度计检测 caspase-3 酶活性。结果: HO-1 转染的 MSCs 可稳定高效地表达 GFP; DAPI 染色后,MSCs 细胞核为典型的凋亡形态,HO-1-MSCs 为较均匀蓝色荧光;HO-1-MSCs 在无氧无血清条件下的凋亡率显著低于 MSCs (P<0.01),且在无氧无血清条件下分泌肝细胞生长因子(HGF)、碱性成纤维细胞生长因子 (bFGF)、血管内皮生长因子 (VEGF) 水平显著增高 (P<0.01);HO-1-MSCs、MSCs 培养上清液在无氧无血清条件下分别培养心肌细胞,HO-1-MSCs 组caspase-3 蛋白和 caspase-3 活性都显著降低 (P<0.01)。结论:无氧无血清条件下,HO-1 提高 MSCs 的存活率;HO-1-MSCs 分泌更多的细胞因子,且对无氧无血清条件下的心肌细胞有保护作用。 相似文献
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人胚胎干细胞在血清和无血清培养体系中的特性比较 总被引:2,自引:0,他引:2
目的 探讨血清培养体系和无血清培养体系对人胚胎干细胞(hES cells)生长特性的影响。 方法 将人胚胎干细胞株BG02接种在丝裂霉素C处理灭活的小鼠胚胎成纤维细胞饲养层上,分别在含血清hES完全培养基或无血清培养基中连续培养25~30代。在不同培养体系下比较人胚胎干细胞形态、集落贴壁率;运用BrdU掺入法检测人胚胎干细胞增殖,用细胞计数法计算细胞倍增时间;采用免疫荧光染色检测人胚胎干细胞特异性分子标志的表达;用流式细胞仪检测人胚胎干细胞Oct-4,Nanog阳性的比例;RT-PCR检测成纤维细胞生长因子(FGFs)家族基因的表达。 结果 在两种培养体系的BG02细胞都具有人胚胎干细胞的形态特征。在含血清培养体系下,BG02细胞集落贴壁率和表达OCT-4,Nanog的阳性细胞明显高于无血清培养体系( P<0.05)。无血清培养体系中,BG02细胞生长速度明显高于含血清培养体系,细胞倍增时间分别为(33.8±4.3) h、(45.9±5.7) h,( P<0.05)。无血清培养体系的BG02细胞高表达 FGF2 、 FGFR2 、 FGFR4 。 结论 两种不同培养体系中人胚胎干细胞的体外培养特性存在一定的差异,可能与BG02细胞 FGFs 家族基因激活有关。 相似文献
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背景:人类胚胎干细胞的修饰和操作技术多种多样,它们在效率、可靠性及安全性方面各有不同。
目的:对比观察两种化学转染试剂转染含不同启动子的增强型绿色荧光蛋白表达载体在人胚胎干细胞中的表达效率。
方法:采用Fugene HD和lipofectamine2000分别转染无滋养层培养的H9细胞,荧光显微镜下观察阳性克隆,流式细胞术分析不同载体pCAG-EGFP、pEGFP-N3在H9细胞中的表达效率。
结果与结论:Fugene HD转染的pCAG-EGFP、pEGFP-N3两种载体在H9细胞中的表达效率分别为(42.45±3.32)%、 (25.95±1.91)%,差异有显著性意义(P < 0.05)。lipofectamine2000转染的pCAG-EGFP、pEGFP-N3两种载体在H9细胞中的表达效率分别为(1.94±0.18)%、(1.49±0.33)%。采用Fugene HD转染的表达效率均高于lipofectamine2000转染的表达效率(P < 0.05)。结果显示在人胚胎干细胞的H9细胞系中,Fugene HD的转染效率显著高于lipofectamine2000;CAG启动子驱动的增强型绿色荧光蛋白表达效率显著高于CMV启动子。 相似文献
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Lipidomics is an emerging research field that comprehensively characterizes lipid molecular species and their metabolic regulation and biological roles. We performed the first lipidomics study on glycerophospholipids (GPLs) in adult mammalian retinal stem cells (RSCs) and non-RSC control cells. A unique GPL signature identified by electrospray ionization tandem mass spectrometry showed new prominent peaks of 16:0 (sn-1)-18:0 (sn-2) or 16:0-16:0 saturated fatty acids, instead of 18:0-20:4 or 18:0-22:6 polyunsaturated essential fatty acids, at 720 m/z of phosphatidylethanolamine, 764 m/z of phosphatidylserine, and 809 m/z of phosphatidylinositol in RSCs (sphere colony RSCs and enriched RSCs), but not in non-RSCs (retinal cells, ciliary cells, sphere colony-derived retinal cells, and nonretinal cells). To seek whether the GPL signature was associated with long-chain acyl-CoA synthetase (LACS), a potential modulator of fatty acid profiles in de novo GPL synthesis, we analyzed gene expression, catabolic activity, substrate selectivity, and inhibitor sensitivity of diverse LACSs. LACSs in RSCs mediated less utilization by GPLs of polyunsaturated essential fatty acids, including arachidonic acid (20:4 [n-6], a second messenger in cell signaling), which was accompanied by lower plasma membrane fluidity in proliferating RSCs compared with differentiated non-RSCs. These novel findings suggest that LACS-associated GPL signature and cell membrane fluidity may participate in regulating proliferation versus differentiation in RSCs and, perhaps, other types of stem cells. 相似文献
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Clements MO Godfrey A Crossley J Wilson SJ Takeuchi Y Boshoff C 《Tissue engineering》2006,12(7):1741-1751
Human stem cells could revolutionize the field of medicine by providing a diverse range of cell types for tissue replacement therapies and drug discovery. To achieve this goal, genetic tools need to be optimized and developed for controlling and manipulating stem cells ex vivo. Here we describe a lentiviral delivery system capable of high infection rates in human mesenchymal and embryonic stem cells. The lentiviral backbone was modified to express mono- and bi-cistronic transgenes and was also used to deliver short hairpin ribonucleic acid for specific silencing of gene expression in human stem cells. We show that lentiviral transduction can be used to alter gene expression without altering the genes' ability to differentiate in vitro. These vectors will enable rapid analysis of gene function in stem cells and permit the generation of knock-in / knock-out models of human disease in the rapidly developing field of gene therapy. 相似文献
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Stable plasmid-based siRNA silencing of gene expression in human embryonic stem cells 总被引:2,自引:0,他引:2
Liu YP Dambaeva SV Dovzhenko OV Garthwaite MA Golos TG 《Stem cells and development》2005,14(5):487-492
RNA interference (RNAi) using short inhibitory RNAs (siRNAs) has been widely explored for the suppression of cellular mRNA levels to investigate the function of specific genes, including gene function in differentiation and development. The establishment of human embryonic stem cell (hESC) models for differentiation of selected lineages is an area of intense interest and activity. On the basis of our previous work with stable overexpression of enhanced green fluorescent protein (EGFP) in hESC, we used plasmid vector-based siRNA expression to silence EGFP expression in stably-transfected hESC. After hygromycin selection, we derived several cell lines in which EGFP expression was significantly reduced. At the genomic DNA level, there was no difference between the two cell lines and the parental H1EGFP cell line when analyzed with quantitative PCR; however, there were significant differences among the three cell lines at the RNA and protein levels as analyzed with real-time RT-PCR and Western blotting. From these data, we conclude that the decrease in EGFP expression was caused by RNAi, not by genomic DNA loss. Down-regulation of EGFP expression was sustained through multiple passages of both siEGFP cell lines. This simple silencing system will allow novel investigations of target gene function in hESC self-renewal or differentiation, as well as differentiated function in other cell types. 相似文献
17.
Jiaqiang Ren Ping Jin Ena Wang Francesco M Marincola David F Stroncek 《Journal of translational medicine》2009,7(1):20-17
Background
The unique features of human embryonic stem (hES) cells make them the best candidate resource for both cell replacement therapy and development research. However, the molecular mechanisms responsible for the simultaneous maintenance of their self-renewal properties and undifferentiated state remain unclear. Non-coding microRNAs (miRNA) which regulate mRNA cleavage and inhibit encoded protein translation exhibit temporal or tissue-specific expression patterns and they play an important role in development timing. 相似文献18.
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High-efficiency genetic modification of human embryonic stem (hES) cells would enable manipulation of gene activity, routine gene targeting, and development of new human disease models and treatments. Chemical transfection, nucleofection, and electroporation of hES cells result in low transfection efficiencies. Viral transduction is efficient but has significant drawbacks. Here we describe techniques to transiently and stably express transgenes in hES cells with high efficiency using a widely available vector system. The technique combines nucleofection of single hES cells with improved methods to select hES cells at clonal density. As validation, we reduced Oct4 and Nanog expression using siRNAs and shRNA vectors in hES cells. Furthermore, we derived many hES cell clones with either stably reduced alkaline phosphatase activity or stably overexpressed green fluorescent protein. These clones retained stem cell characteristics (normal karyotype, stem cell marker expression, self-renewal, and pluripotency). These studies will accelerate efforts to interrogate gene function and define the parameters that control growth and differentiation of hES cells. Disclosure of potential conflicts of interest is found at the end of this article. 相似文献
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含不同启动子的GFP载体在不同种属的胚胎干细胞系中表达效率的研究 总被引:1,自引:0,他引:1
目的 对比含不同启动子的GFP表达载体在hESC及小鼠胚胎干细胞(mouse embryonic stem cell, mESC)的表达效率,为探索ESC及其衍生细胞移植在体内的存活、迁移、分化及整合提供细胞模型.方法 阳离子脂质体转染hESC及mESC ES-D3,荧光显微镜下观察阳性克隆,流式细胞术(flow cytometry, FCM)计算不同载体pCX-hrGFP、pIRES-hrGFP在不同种属细胞中的表达效率.结果 两种载体在mESC的表达效率分别为pCX-hrGFP 90±2.5%, pIRES-hrGFP 0.67±0.02%, 两组间比较差异有统计学意义(P<0.05).在hESC的表达效率分别为pCX-hrGFP 0.8±0.1%, pIRES-hrGFP 0.62±0.08%,两组间比较有统计学差异(P<0.05).pCX-hrGFP在mESC及hESC间的表达效率差异有统计学意义 (P<0.01),pIRES-hrGFP在mESC及hESC间的表达效率差异无统计学意义 (P>0.05).结论 (1)CBA启动子引导的GFP表达效率高于CMV启动子.(2)同一载体在不同种属细胞内表达效率不同. 相似文献