胚胎体外培养及胚胎干细胞系的建立

背景：人类胚胎干细胞体外建系成功,对人类胚胎发育机制和发育生物学研究、细胞和组织移植治疗某些疾病等领域都有重大意义。目的：综述近年来关于胚胎体外培养及建立胚胎干细胞系的研究进展,重点探讨胚胎体外培养影响因素、人废弃胚胎培养分离内细胞团建立胚胎干细胞系的方法及建立胚胎干细胞系的条件。方法：以“胚胎(embryo),胚胎干细胞(embryonic stem cell),共培养(co culture),序贯培养(sequential culture)”为检索词,由第一作者检索2000至2014年CNKI数据库和SCI数据库,获取有关胚胎体外培养、移植及胚胎干细胞建系的相关文献,并进行系统评价,最终保留58篇文献进行分析。结果与结论：胚胎体外培养条件是影响胚胎移植结局的重要因素,其中包括培养液的成分和培养体系。在过去的研究过程中,培养液的构成及应用已经发生了很大的变化,培养体系也从单一培养发展到共培养、序贯培养。伦理问题及胚胎来源的限制束缚着人胚胎干细胞系的建立,利用临床废弃的低质量的胚胎可作为建立人胚胎干细胞系的材料来源之一,有效的缓解了建立人胚胎干细胞系过程中胚胎缺乏的问题,并减少其中的伦理学纷争。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

人胚胎生殖细胞（hEG）的体外培养、鉴定及其自发分化

人胚胎干细胞(hESC)、人胚胎原始生殖细胞(hEGC)以及人胚胎瘤细胞(hEC)统称为人胚胎干细胞。人胚胎干细胞属于多潜能干细胞，多潜能干细胞系的获得不仅可在体外研究早期人胚的发育，而更重要的是为移植和治疗退行性疾病提供细胞和组织来源，所以分离和获得足够量的多潜能干细胞一直是人们感兴趣的课题。     

慢病毒介导绿色荧光蛋白转染人胚胎干细胞及其培养

目的 稳定培养人胚胎干细胞,并通过慢病毒载体对其进行绿色荧光蛋白标记.方法 利用小鼠胚胎成纤维细胞作为饲养层或Matrigel作为基质培养人胚胎干细胞,包装带有GFP序列的慢病毒转染人胚胎干细胞.对转染前后的人胚胎干细胞进行了碱性磷酸酶和SSEA-3免疫组化鉴定.结果 在MEF饲养层和Matrigel上均可培养出呈克隆样生长,表达标志抗原的人胚胎干细胞,经慢病毒转染及抗生素筛选后仍可稳定表达GFP.结论 成功地培养了人胚胎干细胞系,并进行了GFP标记.     

人胚胎干细胞不同传代方法的比较

目的探讨不同传代方法对人胚胎干细胞生长和分化情况的影响。方法用1mg/mlⅣ型胶原酶和机械切割两种不同的方法传代人胚胎干细胞,比较这两种方法传代的人胚胎干细胞克隆生长和分化情况。结果 1.机械切割法传代的人胚胎干细胞克隆较传统酶消化法生长快速且不易分化。2.传代后的人胚胎干细胞仍然维持胚胎干细胞的特有形态：表达胚胎干细胞表面标记,具有正常核型和多潜能分化能力（在体外形成拟胚体在体内形成畸胎瘤）。结论采用机械切割法传代人胚胎干细胞更利于细胞的培养扩增,且传代后细胞在继续培养中仍可保持干细胞的所有特性。     

人胚胎干细胞Pten基因表达及PI3K/Akt/mTOR信号通路下游蛋白的磷酸化

背景：各种磷酸化蛋白质表达水平对人胚胎干细胞维持未分化状态或定向分化的影响逐渐成为人胚胎干细胞的研究热点,研究发现磷酸化蛋白质表达水平可能决定着人胚胎干细胞的命运。 目的：对PI3K/Akt途径下游的关键蛋白磷酸化水平进行检测,寻找PI3K/Akt途径中能够维持人胚胎干细胞未分化状态的下游蛋白。 方法：用胎鼠成纤维细胞作为饲养层,二维培养的方法培养人胚胎干细胞,胶原酶消化后待测;以饲养层细胞、K562细胞株为对照。观察人胚胎干细胞生长状态;RT-PCR检测Pten基因表达;Western Blot检测p-PTEN、p-mTOR、p-P70S6K、p-4E-BP1 4种蛋白的表达。 结果与结论：人胚胎干细胞在未分化状态时Pten mRNA表达高于肿瘤细胞K562,而且Pten抑制的mTOR信号通路中关键蛋白表达均明显低于K562,尤其以p-4E-BP1表达最低。提示PI3K/Akt/mTOR信号通路下游磷酸化蛋白在人胚胎干细胞活性较低,如果抑制负调节蛋白PTEN或直接激活该通路正调节关键蛋白,可能会加快人胚胎干细胞增殖、减少凋亡,进而为定向分化、再生医学提供更多的细胞来源。     

人胚胎干细胞HuES17向造血干细胞的分化

背景：人类胚胎干细胞是来源于着床前囊胚的内细胞团,能在长期培养中无限增殖并保持未分化状态,且具有分化成人体组织各种细胞类型能力的细胞。 目的：进一步验证人胚胎干细胞HuES17细胞株向造血干细胞分化的能力。 方法：人胚胎干细胞HuES17采用与人包皮成纤维细胞二维共培养的方式培养,采用人胚胎干细胞与小鼠骨髓基质细胞(OP9) 二维共培养的方法诱导胚胎干细胞向造血干细胞分化。 结果与结论：人胚胎干细胞与小鼠骨髓基质细胞(OP9) 二维共培养诱导造血分化的第四五天即开始出现OP9细胞逐渐老化,很快死亡;可以观察到人胚胎干细胞分化,然而,随着OP9细胞死亡,分化的人胚胎干细胞亦死亡,不能诱导人胚胎干细胞向造血干细胞分化。提示人胚胎干细胞HuES17细胞株可能不能向造血干细胞分化,或向造血干细胞分化的能力较低。     

人胚胎成纤维细胞饲养层的制备

背景：极小胚胎样干细胞是近年来发现的一种具有类似胚胎干细胞生物学特性的非造血干细胞,但对其体外培养扩增的方法报道极少。有研究推测,人胚胎成纤维细胞能为人骨髓极小胚胎样干细胞体外培养扩增提供良好的微环境。 目的：从人胚胎躯干中分离、培养人胚胎成纤维细胞,制备人胚胎成纤维细胞饲养层用于人骨髓极小胚胎样干细胞的培养。 方法：利用胰酶消化法从孕5-9周龄人胚胎躯干中分离培养人胚胎成纤维细胞。制作饲养层,使用不同浓度丝裂霉素C处理后,用于培养分选后的人骨髓极小胚胎样干细胞,以细胞形态、生长曲线作为胚胎成纤维细胞和饲养层的评价指标。 结果与结论：从人胚胎中成功分离培养出人胚胎成纤维细胞,该细胞可传代24代以上,且经过传代及冻存复苏后生物学特性无改变。丝裂酶素C低于12 mg/L时,人胚胎成纤维细胞增殖不能完全抑制;高于14 mg/L,人胚胎成纤维细胞可能死亡。12 mg/L丝裂霉素C作用3 h后能较好地抑制人胚胎成纤维细胞的增殖,并且保持其活力约2周,可以在很长一段时间内用作人骨髓极小胚胎样干细胞的饲养层。     

人诱导性多潜能干细胞系的建立

背景：诱导性多潜能干细胞与肿瘤干细胞的发生过程极其相似,而且具有的干细胞特性极其接近人胚胎干细胞。因此,研究诱导性多潜能干细胞有利于人们进一步认识并了解人类发育以及肿瘤的发生过程。 目的：掌握建立人诱导性多潜能干细胞系的技术,以便为特异性疾病细胞的重编程建立技术平台,从而利用重编程技术研究疾病的发病机制。 方法：将含有Oct4、Sox2、Klf-4和c-Myc 4个转录因子的反转录病毒感染人皮肤成纤维细胞(HS27细胞),在人胚胎干细胞培养条件下诱导产生人胚胎干细胞样的克隆。挑取并进一步扩增,通过克隆形态、碱性磷酸酶活性、免疫荧光检测是否有人胚胎干细胞标记物Oct4、Sox2、c-Myc、Klf-4的表达,悬滴法检测HS27细胞来源的克隆形成畸胎瘤的能力和验证向3个胚层的分化能力。 结果与结论：经病毒感染诱导产生的胚胎干细胞样克隆呈绿色荧光蛋白阴性,克隆在细胞形态方面与人胚胎干细胞克隆相似,进一步扩增经碱性磷酸酶检测克隆呈阳性,免疫荧光检测克隆表达Oct4、Sox2、c-Myc、Klf-4,并且HS27细胞来源的克隆注入免疫缺陷小鼠体内可以形成畸胎瘤并经苏木精-伊红染色显示具有向三胚层分化能力。实验成功构建了人诱导性多潜能干细胞系,为下一步开展疾病细胞特异性重编程研究奠定了良好的实验基础。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

诱导人孤雌胚胎干细胞分化为胰岛样细胞团

背景:随着人类孤雌胚胎干细胞系的成功建立,对该细胞的进一步分化功能研究成为新的研究热点。目的:研究体外培养的人类孤雌胚胎干细胞诱导分化为胰岛样细胞团的潜能。方法:自主建系孤雌来源人胚胎干细胞和正常来源人胚胎干细胞各1株,运用基于胰腺体内发育规律的改良5阶段诱导法诱导人孤雌胚胎干细胞为胰岛样细胞团,加入不同生长因子及诱导试剂对人胚胎干细胞分5阶段序贯培养。结果与结论:终末分化细胞光镜下呈团状聚集,RT-PCR、免疫荧光染色检测分化细胞表达胰岛细胞特征性的基因与蛋白。胰岛素释放实验提示获得的细胞具有胰岛样生化功能。由人类孤雌胚胎干细胞来源的胰岛样细胞团具备胰岛的基本特征,是未来治疗1型糖尿病的可用材料。     

最大人胚胎干细胞遗传变异研究结果发布

据Amps K 2011年11月27日(Nat Biotechnol,2011 Nov 27.doi:10.1038/nbt.2051.)报道,在国际干细胞研究组织倡导下,来自中国、英国、新加坡、伊朗、美国、以色列、瑞士、瑞典、韩国、澳大利亚、捷克、巴西、印度、日本、俄罗斯、比利时、加拿大、荷兰和芬兰等19个国家的人胚胎干细胞科学家携手合作,开展了一项全世界最大规模的人胚胎干细胞遗传变异研究。     

CD133 expression by neural progenitors derived from human embryonic stem cells and its use for their prospective isolation

The ability to generate purified neural progenitors is critical to the development of embryonic stem cell-based therapies to alleviate human neurological disorders. While many cell culture protocols for directed differentiation of human embryonic stem (hES) cells into neural cells have been described, most yield mixed populations, some containing cells of different embryonic germ layer lineages, or even undifferentiated embryonic stem cells. In this study, we describe a method for single-cell dissociation, isolation by flow cytometry, and subsequent culture of neural progenitors from hES cells. As reported earlier, hES cells treated with the bone morphogenetic protein (BMP) antagonist noggin gave rise to neurospheres at a relatively high frequency. However, these noggin-treated embryonic stem cell cultures were heterogeneous, with cells expressing embryonic stem markers still detectable even following 14 days of differentiation. In order to isolate pure human neural progenitors, we fractionated noggin-treated ES cells on the basis of their expression of the putative neural stem cell marker, CD133, and the GCTM-2, and SSEA-1 antigens, which mark pluripotent stem cells and differentiated cells respectively from hES cell culture. CD133(+) cells formed larger spheres compared to CD133(-) cells. CD133(+)SSEA1(+) cells and CD133(+)SSEA-1(-) cells expressed similar levels of neural genes and formed neurospheres at similar frequencies. By contrast, CD133(+)GCTM-2(+) cells expressed high levels of OCT4 but not neural lineage genes and failed to form neurospheres. CD133(+)GCTM-2(-) cells formed neurospheres at the relative highest frequency. Thus, negative selection with GCTM-2 may be useful for the purification of specific cell types differentiated from hES cells.     

人胚胎干细胞建系、培养及体外诱导分化研究现状

胚胎干细胞具有体外无限增殖和分化成三胚层细胞的潜能,它已被视为治疗多种疾病的一种新兴策略。目前胚胎干细胞常规的建系和培养技术已很成熟,并有一套国际公认的鉴定标准。但常规方法存在异源病原体污染的可能,急需研究适于标准化、无动物源性污染及可大量培养胚胎干细胞的培养体系。在现阶段,通过不同的体外诱导途径可将胚胎干细胞诱导成为胚外和三胚层来源的各种细胞,但定向分化的问题仍亟需解决。     

Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells

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.     

Do we Still Need Human Embryonic Stem Cells for Stem Cell-Based Therapies? Epistemic and Ethical Aspects

While scientific community disagrees about similarities and differences between human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells, some politicians embrace translational hiPS cell research as a replacement for translational hES cell research. We examine the ethical relevance of the main differences between hES and hiPS cell-based therapies and discuss whether, given the current state of knowledge, certain differences are essential. We discuss whether well-founded preferences can be made in hypothetical scenarios with varying levels of patient safety, treatment efficacy, treatment accessibility and ethical controversy.     

Human embryonic stem cells have a unique epigenetic signature

Human embryonic stem (hES) cells originate during an embryonic period of active epigenetic remodeling. DNA methylation patterns are likely to be critical for their self-renewal and pluripotence. We compared the DNA methylation status of 1536 CpG sites (from 371 genes) in 14 independently isolated hES cell lines with five other cell types: 24 cancer cell lines, four adult stem cell populations, four lymphoblastoid cell lines, five normal human tissues, and an embryonal carcinoma cell line. We found that the DNA methylation profile clearly distinguished the hES cells from all of the other cell types. A subset of 49 CpG sites from 40 genes contributed most to the differences among cell types. Another set of 25 sites from 23 genes distinguished hES cells from normal differentiated cells and can be used as biomarkers to monitor differentiation. Our results indicate that hES cells have a unique epigenetic signature that may contribute to their developmental potential.     

Embryonic stem cells: similarities and differences between human and murine embryonic stem cells

The derivation of murine embryonic stem (mES) cell lines was reported for the first time in 1981 (Nature, 1981; 292:154-156; Proc Natl Acad Sci U S A, 1981; 78:7634-7638), and they have since proved to be a very useful tool with which to study mammalian development, which is characterized by pluripotency and differentiation. About 20 years later, the successful generation of human embryonic stem (hES) cell lines was described (Science, 1998; 282:1145-1147). Although mES and hES are derived from mammals, they cannot be looked at as being one and the same. While basic information for hES can be derived from mES, such information does not correspond on a one-to-one basis. This review gives an overview of the characteristics of embryonic stem cells with the main focus on the similarities and differences between human and mES cells.     

Improved efficiency and pace of generating induced pluripotent stem cells from human adult and fetal fibroblasts

It was reported recently that human fibroblasts can be reprogrammed into a pluripotent state that resembles that of human embryonic stem (hES) cells. This was achieved by ectopic expression of four genes followed by culture on mouse embryonic fibroblast (MEF) feeders under a condition favoring hES cell growth. However, the efficiency of generating human induced pluripotent stem (iPS) cells is low, especially for postnatal human fibroblasts. We started supplementing with an additional gene or bioactive molecules to increase the efficiency of generating iPS cells from human adult as well as fetal fibroblasts. We report here that adding SV40 large T antigen (T) to either set of the four reprogramming genes previously used enhanced the efficiency by 23-70-fold from both human adult and fetal fibroblasts. Discernible hES-like colonies also emerged 1-2 weeks earlier if T was added. With the improved efficiency, we succeeded in replacing MEFs with immortalized human feeder cells that we previously established for optimal hES cell growth. We further characterized individually picked hES-like colonies after expansion (up to 24 passages). The majority of them expressed various undifferentiated hES markers. Some but not all the hES-like clones can be induced to differentiate into the derivatives of the three embryonic germ layers in both teratoma formation and embryoid body (EB) formation assays. These pluripotent clones also differentiated into trophoblasts after EB formation or bone morphogenetic protein 4 induction as classic hES cells. Using this improved approach, we also generated hES-like cells from homozygous fibroblasts containing the sickle cell anemia mutation Hemoglobin Sickle. Disclosure of potential conflicts of interest is found at the end of this article.     

Culture and expansion of the human embryonic stem cell line HS181, evaluated in a double-color system

An approach of using RFP-transfected human foreskin fibroblasts (hFS-RFP) to support the growth of GFP expressing human embryonic stem cells (hES; HS181-GFP) is reported. The two-color system was applied to detect interactions between hFS and human embryonic stem cells (hES). After overnight culture, the hES cell colonies showed a behavior of "pushing away" the underlying feeder cells. This phenomenon occurred with both a low and high density of feeders. The density of the feeder cell layer, however, influenced the growth pattern of hES cell colonies. At a high feeder cell density, the hES colonies were more pointed and aligned with the direction of the fibroblasts, whereas less dense feeder layers allowed a more rounded and flat hES colony formation. Not surprisingly, a small fraction of mitotically inactivated feeder cells reattached after passage and remained viable in the cultures for up to four subsequent passages. The prospect of using the two-color system for detection of possible fusion events between hES cells and feeder cells was assessed by screening a large number of cell cultures for double RFP/EGFP expressing cells. The results indicate that fusion events are extremely rare (<10(-6)), or alternatively that after fusion the dual expression of both EGFP and RFP is not easily detected for other reasons. In summary, a two-color system allows analysis of colony formation and also helps to identify and follow the differentiation of cells.