Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.     

昆明白小鼠胚胎干细胞分离与体外培养

为探索昆明白小鼠胚胎干细胞建系方法,将受孕4.5天的昆明白小鼠囊胚用免疫手术法去除滋胚层,然后将内细胞团(ICM)接种于胎鼠成纤维细胞饲养层上培养,形成的胚胎干细胞样集落用胰蛋白酶-EDTA消化法传代,培养后进行相差显微镜观察及碱性磷酸酶染色。结果饲养层上生长的ICM细胞呈典型的ES样细胞集落,传至第8代碱性磷酸酶染色呈强阳性。实验表明免疫手术法适用于昆明白小鼠ES细胞建系,获得的细胞集落具有ES细胞的主要生物学性状。     

Derivation of Mouse Haploid Trophoblast Stem Cells

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Derivation and Characterization of Hepatic Progenitor Cells from Human Embryonic Stem Cells

The derivation of hepatic progenitor cells from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential hepatic progenitor cells from hES cells. These hES cell–derived hepatic progenitor cells could be effectively used as an in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.     

人胚胎干细胞建系和培养的研究进展

人胚胎干细胞(human embryonic stem cell,hESc)在再生医学、药物筛选和发育生物学等领域具有重要的研究和应用价值.本文对人胚胎干细胞建系方法的现状包括胚胎来源、内细胞团分离方法、以及人胚胎干细胞培养体系的改进作了介绍,讨论了与全能性维持和定向分化有关的信号通路的研究进展,以及胚胎干细胞研究中伦理问题的争议.     

免疫外科法分离克隆BALB/c小鼠胚胎干细胞

目的　使用免疫外科法分离克隆BALB c小鼠胚胎干细胞 (embryonicstemcells,ES细胞 ) ,为进一步建立BALB c小鼠ES细胞系打下基础。方法　用免疫外科法从 4 5枚BALB c小鼠囊胚中分离得到 2 0枚去除滋养层细胞的ICM ,接种在MEF饲养层上 ,使用DMEM(高糖 ) 15 ?S 0 1mmol Lβ 巯基乙醇 0 0 1mmol L非必需氨基酸 10 0 0IU mlLIF 10 0IU mL青霉素 10 0IU ml链霉素培养液 ,17枚形成典型的ICM集落 (85 0 % ) ,有一枚胚胎传至第 10代。用于全胚培养的BALB c小鼠胚胎共 10 2枚 ,使用与免疫外科相同的培养方法 ,6 6枚形成典型的ICM集落 (6 4 7% ) ,其中一枚胚胎传至第 8代。结果　免疫外科法较全胚培养法有利于小鼠ES细胞的分离与克隆 ;添加LIF(10 0 0IU ml)有利于小鼠ES细胞的分离与传代 (P <0 0 5 ) ;0 0 5 %胰酶 0 0 0 8?TA是较好的ES细胞消化液 ,对细胞综合损伤力小 ,且传代后ES细胞集落形成能力也较高 (P <0 0 5 )。结论　分离得到的ES细胞经形态学观察 ,AKP染色 ,体外分化实验 ,核型分析等证明其具有胚胎干细胞的诸多特性。     

分离和克隆小鼠ES细胞集落的主要影响因素

目的　研究不同培养条件分离和克隆小鼠ES细胞集落的效率。方法　以PMEF饲养层、NIH3T3细胞饲养层或培养液中加入LIF为培养条件 ,分离和克隆昆明小鼠ES细胞集落 ,比较其效率。结果　饲养层的培养条件明显优于培养液中加入LIF的培养条件 ;有饲养层的培养条件下 ,桑椹胚的ES细胞集落出现率显著低于囊胚 ;两种饲养层培养囊胚 ,其ES细胞集落的出现率差异无显著性。结论　以PMEF或NIH3T3细胞作饲养层 ,培养昆明小鼠的囊胚 ,适时离散ICM ,是比较理想的分离ES细胞集落的方法。     

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.Download video file.^{(116M, mp4)}     

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.     

615小鼠ES细胞集落的分离及初步鉴定

目的　分离和培养 6 15小鼠的ES细胞集落 ,为建系打下基础。方法　以PMEF为饲养层分离 6 15小鼠的ES细胞集落 ,进行无饲养层培养 ,并对其进行初步鉴定。结果　ES细胞集落的出现率和传代成功率为 2 2 6 %和 0 94 % ,其ALP染色阳性 ,具有稳定的二倍体核型 ,可自发分化为多种类型的细胞。结论　成功分离和培养了6 15小鼠的ES细胞集落     

对小鼠ES细胞进行特异性的标记

虽然ES细胞技术的应用十分广泛,对ES细胞多能性本质的研究还不是很深入,体外培养的ES细胞群体的不均一性加大了这方面研究的难度,报道了对ES细胞中特异表达的基因,将报告基因βgeo插入oct-基因转录元件中构建了标记载体pG18NG,转染ES细胞MESPU22和MESPU13后获得了稳定整合的细胞克隆,经体外培养、诱导分化、嵌入体制作等实验,证明利用该载体对ES细胞中的未分化细胞成功进行了标记,该标记在体内、体外都是有效的。     

无血清无饲养层条件下培养小鼠胚胎干细胞

目的研究在无血清无饲养层条件下小鼠胚胎干细胞的培养方法,为最终建立无血清无饲养层培养系统打下基础。方法比较小鼠胚胎干细胞ES-S8株在无血清培养体系和有血清培养体系中的生长情况,分析ES-S8细胞克隆形成效率,测定其生长速度;然后在撤去血清和饲养层的条件下培养ES-S8细胞,进行AKP染色和表面标记物SSEA-1免疫荧光检测。结果ES-S8细胞在无血清培养条件下细胞生长速度减缓,克隆形成率降低,但AKP染色、SSEA-1免疫荧光均显阳性;在无血清无饲养层条件下ES-S8细胞培养仍能形成克隆,且AKP染色、SSEA-1免疫荧光均显阳性。结论研究表明ES-S8细胞能够在无血清无饲养层的培养条件下生长,保持其良好的未分化特性。     

昆明小鼠胚胎成纤维细胞的分离与培养

目的:建立稳定高效的小鼠胚胎成纤维细胞饲养层培养体系.方法:取不同胎龄的小鼠分离原代胚胎成纤维细胞,观察不同胎龄小鼠对分离和培养效果的影响.结果:从不同胎龄小鼠均分离得到胚胎成纤维细胞,但最佳分离时间为13.5～14.5 d;传代时在室温下消化单层贴壁细胞可随时控制消化时间,效果良好.结论:从13.5～15.5 d胎龄小鼠胚胎分离培养胚胎成纤维细胞效果最佳.     

人类及小鼠胚胎干细胞的不同多能性状态

胚胎干细胞(embryonic stem cells,ESCs)是来源于早期胚胎的全能性细胞,在合适条件下具有分化为任何一类成体细胞的潜力。在小鼠中,根据细胞来源的胚胎发育时间,ESCs可以被分为原始态多能性(na(?)ve pluripotency)和始发态多能性(primed pluripotency)两种状态。这两种状态的细胞在发育上相互联系,具有不同的形态、信号依赖、发育性质、基因表达及表观遗传学性质,并且在特定的条件下可以相互转化。人类胚胎干细胞(human embryonic stem cells,hESCs)的发育潜能曾一度被认为低于小鼠胚胎干细胞(mouse embryonic stem cells,mESCs),直到人类原始态胚胎干细胞的发现证明了hESCs可以表现出与mESCs相似的性质。这对于人类胚胎发育的研究及ESCs在临床治疗上的实际应用都具有重要的意义。     

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of naïve macrophages in the peritoneal cavity makes it a preferred site for the collection of naïve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5⁺ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.