从原始生殖细胞分离克隆鸡胚胎生殖细胞的研究

从孵化 5 5天的鸡胚生殖腺中分离得到大量原始生殖细胞 (PGCs)集落 ,这些集落的细胞经多次克隆传代具有胚胎生殖细胞 (EG)的诸多特征 ,如有连续传代的能力 (传至第 9代 ) ,细胞集落有典型鸟巢状结构 ,PAS染色阳性 ,AKP染色阳性 ,在无饲养层无分化抑制因子LIF时可以自发分化成几种细胞类型 ,包括成纤维细胞、神经细胞、自律细胞等 ,悬浮培养时具有形成类胚体的能力。上述发现表明该细胞具EG细胞的诸多特性 ,为类EG细胞     

禽类胚胎生殖细胞的研究

胚胎干细胞有2种来源：一种来自于早期胚胎囊胚期内细胞团（inner cell mass,ICM）的胚胎干细胞（embryonic stem cells,ES细胞）,另一种是来自胚胎生殖腺原始生殖细胞（primordial germ cells,PGCs）的胚胎生殖细胞（embryonic germ cells,EG细胞）。PGCs是生殖母细胞的前体细胞,是精子或卵子的祖先细胞。自Matsui等证实了PGCs同样可以作为胚胎干细胞的原材料之后,现已在人、小鼠、猪和鸡等多种动物的PGCs进行了分离培养并获得了EG细胞。现从形态特征和迁移、分离培养及鉴定方面对禽类EG细胞的研究进展作一综述。     

胚胎干细胞与胚胎生殖细胞

西北农林科技大学、陕西省干细胞工程技术研究中心的徐小明、杨炜峰、窦忠英等先生对胚胎干细胞(embrgonicstemcells,ES)与胚胎生殖细胞(embryonicgermCells,EG)分别从附置前早期胚胎内细胞团(innercellmass,ICM)和早期胎儿生殖嵴原始生殖细胞(primordialgtrmcells,PGCs)分离克隆出来的一种具有自我更新、无限增殖能力,能分化成代表3个胚层组织细胞能力的干细胞、可对其进行遗传操作、选择和冻存不失其多能性。ES/EG应用于移植医学,在特定条件下可诱导其分化为特定细胞,并可在体外构建特定组织器官,如诱导分化为心肌细胞来治疗心肌梗…     

胚胎干细胞起源的探讨

目前胚胎干细胞(ESCs)建系的取材来源包括桑椹胚的卵裂球、囊胚的内细胞团(ICM)、上胚层细胞和原始生殖细胞(PGCs),甚至从新生鼠睾丸细胞也分离得到类ES样细胞系。这就提出了一个问题,什么是ESCs最接近的体内细胞来源。传统观念常常把ESCs等同于ICM细胞,也有学者认为ESCs更象上胚层细胞,而在已知的分子标记基因方面,ESCs所具有的特征更接近体内早期生殖细胞。不清楚ESCs最接近的体内细胞来源,可能是制约许多品系小鼠和大多哺乳类动物建系成功率提高的原因之一。ESCs系与EG细胞系的分离条件不同表明,加强对ESCs多能性维持基因调控研究具有重要意义。本文从ESCs的经典概念及其发展,早期胚胎细胞和生殖细胞发育规律,早期胚胎细胞、早期生殖细胞和ESCs的关系等方面进行综合分析,认为ESCs可能有多种接近的体内细胞来源。进一步应通过对ESCs建系不同的取材细胞和不同品系的ESCs间进行比较研究,以便弄清ESCs的来源和转化机制,为提高不同物种ESCs建系效率提供理论支持。     

胚胎干细胞起源与本质的探讨

胚胎干细胞(ESC)建系取材包括桑椹胚的卵裂球、囊胚的内细胞团(ICM)、上胚层细胞和原始生殖细胞(PGCs),甚至从新生鼠睾丸细胞也分离得到ES样细胞.传统观念常常把ESC等同于ICM细胞,也有学者认为ESC更像上胚层细胞,而在已知的基因标记方面,ESC所具有的特征更接近体内早期生殖细胞.不清楚ESC最接近的体内细胞类型与本质,可能是制约许多品系小鼠和大多哺乳类动物建系成功率提高的原因之一.综述了胚胎多能性细胞、早期生殖细胞和ESC的研究进展及相互关系,发现ESC可源于多种细胞类型.目前仍难以确定ESC最接近的体内细胞类型,进一步应通过研究不同体内细胞类型源ESC系间的差异,以弄清ESC本质,为提高ESC建系效率提供理论支持.     

小鼠原生殖细胞建系过程及其分化特性的研究

以小鼠8.5dpc、10.5dpc、12.5dpc胚胎为材料,分离其中包含PGC的胚胎组织,使其生长于饲养层细胞上,在生长因子LIF、SCF和bFGF的共同作用下存活增殖,形成PGC克隆,经过几次分散转移至新的饲养层细胞,产生稳定增殖的EG干细胞克隆,共建成5株EG细胞系,AKP染色以及oct-4基因表达产物的免疫荧光检测均显示阳性。EG1、EG2、EG3、EG4、EG5,分别来自8.5、10.5dpc的胚胎,没有得到长期培养的12.5dpc的EG细胞系。EG细胞系在有饲养层细胞或添加LIF的环境中可稳定传代,保持不分化状态,至少15代内正常核型细胞所占比例80%以上。去除抑制分化因素的前提下,悬浮培养的EG细胞形成胚体,分化出类似胚胎内胚层和外胚层的细胞结构;贴壁生长的胚体能产生不同类型的分化细胞,包括上皮细胞、成纤维细胞、神经细胞等。EG细胞在裸鼠体内形成畸胎瘤。以上结果证实我们建立的EG细胞系具发育多能性,为研究早期胚胎和生殖细胞生长分化提供了模型。     

鸡胚胎原始生殖细胞体外培养

以14-15期鸡胚血液为材料,采用Ficoll密度梯度离心方法,提取鸡胚胎原始生殖细胞(primordial germ cells,PGCs),在无基质细胞和基质细胞上分别进行体外培养。从实验结果可以看出：在含有胎牛血清(fetal bovine serum,FBS)、鸡血清(chicken serum,CS)、碱性成纤维细胞生长因子(bFGF)、人胰岛素样生长因子(hIGF-1)、小鼠白血病抑制因子(mLIF)和青,链霉素双抗的M199培养液中培养时,鸡PGCs最多能够存活4天：当采用细胞因子和5天鸡胚胎性腺基质细胞共培养时能存活23代且每代细胞增殖可达近10倍。提纯后的PGCs细胞冻存复苏后,经台盼蓝染色鉴定存活率可达80％左右。     

影响鸡原始生殖细胞分离克隆因素的研究（简报）

具有多向分化潜能的胚胎干细胞有两种来源：一是来自于早期胚胎内细胞团的胚胎干细胞(Em．bryonic Stem Cells,ESCs),另一种是来自于胚胎生殖腺原始生殖细胞(Primordial Germ Cells,PGCs)的胚胎生殖细胞(Embryonic Germ Cells,EGCs)。     

人多潜能胚胎生殖细胞的分离和培养

多潜能胚胎性干细胞来源有两条途经,从植入前的早期胚胎内细胞团(inner cell mass,ICM)分离出来的称胚胎干细胞(embryonic stem cells,ES);从原始生殖细胞(primordial germ cells,PGCs)分离得到的称胚胎生殖细胞(embryonic germ cells,EG)。这两种干细胞在小鼠嵌合体实验中,都证明具有参与生殖系传递的能力。这类干细胞在体外保持     

小鼠白血病抑制因子基因的克隆、表达及其对维持鸡原始生殖细胞未分化状态的影响

为了探索鸡原始生殖细胞(Primordial germ cells,PGCs)适合的培养体系,我们在已构建的分泌型真核表达载体pSecTag-mlif(sp-)的基础上,通过脂质体介导将mlif转染到鸡PGCs和鸡胚胎成纤维(Chicken embryonic fibroblast, CEF)细胞中,48 h后收集细胞上清液,蛋白印迹均检测到小鼠白血病抑制因子(mLIF)的表达.以CEF细胞作饲养层,分七组来培养鸡PGCs,结果发现四组和五组培养的PGCs生长状态最好,三组传代后开始2-3天生长状况较好,3天后克隆周围有明显的分化现象.本实验将已构建了含mLIF基因的分泌型真核表达载体成功地瞬时转染到PGCs和CEF细胞中,且表达的mLIF具有维持鸡PGCs未分化状态的功能.     

Derivation of Putative Porcine Embryonic Germ Cells and Analysis of Their Multi-Lineage Differentiation Potential

Embryonic germ (EG) cells are cultured pluripotent stem cells derived from the primordial germ cells (PGCs) that migrate from the dorsal mesentery of the hindgut to the developing genital ridge. In this study, the morphology of the porcine genital ridge was assessed in embryos harvested on days 22–30 of pregnancy. PGCs from embryos at these stages were cultured to obtain porcine EG cell lines, and EG-like cells were derived from PGCs from embryos harvested on days 24–28 of pregnancy. The EG-like cells expressed Oct4, Sox2, Nanog, SSEA-3, SSEA-4 and alkaline phosphatase (AP). These cells were able to form embryoid bodies (EBs) in suspension culture and differentiate into cells representative of the three germ layers as verified by a-fetoprotein (AFP), α-smooth muscle actin (α-SMA), and Nestin expression. Spontaneous differentiation from the porcine EG-like cells of delayed passage in vitro showed that they could differentiate into epithelial-like cells, mesenchymal-like cells and neuron-like cells. In vitro directed differentiation generated osteocytes, adipocytes and a variety of neural lineage cells, as demonstrated by alizarin red staining, oil red O staining, and immunofluorescence for neuronal class Ⅲ β-tubulin (Tuj1), glial fibrillary protein (GFAP) and galactosylceramidase (GALC), respectively. These results indicate that porcine EG-like cells have the potential for multi-lineage differentiation and are useful for basic porcine stem cell research.     

Isolation and characterization of EG-like cells from Chinese swamp buffalo (Bubalus bubalis)

There have been few studies done on the isolation and characterization of Chinese swamp buffalo embryonic germ cells (EG cells). Here, we first report on EG-like cells isolated from Chinese swamp buffalo fetuses. The results showed the cells grew in large, multilayered colonies, which were densely packed with an obvious border resembling mouse embryonic stem cells (ES cells) and EG cells. The buffalo EG-like cells expressed AP, SSEA-1, SSEA-3, SSEA-4 and OCT-4. By RT-PCR, we found that undifferentiated swamp buffalo EG-like cells expressed the OCT-4, NANOG, SOX2, FOXD3, GP130, STAT3, and HEB gene mRNA, but not Fgf4. When these cells were cultured for more than 2weeks without passage, they could differentiate into several types of cells including fibroblast-like, neuron-like, smooth muscle-like, and epithelial-like cells. Some cells formed simple embryoid bodies (EBs) and cystic EBs by suspension culture. By RT-PCR, we found cystic EBs expressed FOXD3, GP130, STAT3 and HEB gene mRNA, but not OCT-4, NANOG, and SOX2 gene mRNA, which could be detected in undifferentiated buffalo EG-like cells. At the same time, the expression of KERATIN-14 (Endoderm), GATA4, ACTA2 (Mesoderm) and TUBB3 (Ectoderm) gene mRNA were also detected in cystic EBs. The results suggested that these cells were capable of forming three germ layers in in vitro differentiation. The expression of OCT-4, NANOG and SOX2 might be essential for Chinese swamp buffalo EG-like cells in a pluripotent state. During the isolation and culture of Chinese swamp buffalo EG-like cells, we found the fetuses that were at 30-80days post-coitus were more efficient than others; and the mechanical method was better than trypsin digestion. The maximal passage of the mechanical method was eight, but the trypsin digestion was just three passages. So it seemed like that the buffalo EG-like cells were sensitive to trypsin. In summary, we were the first to isolate and characterize Chinese swamp buffalo EG-like cells that had morphology and characterization similar to those of established EG/EG-like cells in mouse and human.     

Heterogeneity in imprinted methylation patterns of pluripotent embryonic germ cells derived from pre-migratory mouse germ cells

Pluripotent stem cells, termed embryonic germ (EG) cells, have been generated from both human and mouse primordial germ cells (PGCs). Like embryonic stem (ES) cells, EG cells have the potential to differentiate into all germ layer derivatives and may also be important for any future clinical applications. The development of PGCs in vivo is accompanied by major epigenetic changes including DNA demethylation and imprint erasure. We have investigated the DNA methylation pattern of several imprinted genes and repetitive elements in mouse EG cell lines before and after differentiation. Analysed cell lines were derived soon after PGC specification, “early”, in comparison with EG cells derived after PGC colonisation of the genital ridge, “late” and embryonic stem (ES) cell lines, derived from the inner cell mass (ICM). Early EG cell lines showed strikingly heterogeneous DNA methylation patterns, in contrast to the uniformity of methylation pattern seen in somatic cells (control), late EG cell and ES cell lines. We also observed that all analysed XX cell lines exhibited less methylation than XY. We suggest that this heterogeneity may reflect the changes in DNA methylation taking place in the germ cell lineage soon after specification.     

Directed neural differentiation of duck embryonic germ cells

Although the avian primordial germ cells (PGCs) have been used to produce transgenic birds, their characteristics largely remain unknown. The isolation, culture, biological characterization, and directed neural differentiation of duck EG cells were assayed in this study. The Results showed that the EG cells were got by isolating embryonic gonad and surrounding tissue from 7-day-old duck embryo. The PGCs co-cultured with their gonadal somatic cells were well grown. After passaging, the EG cells were incubated in medium with cytokines and Mitomycin C on inactivated duck embryonic fibroblasts (DEFs) feeder layers. After several passages, alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) resulted positive, cellular markers detection positive for SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. Karyotype analysis showed the EG cells kept diploid condition and the hereditary feature was stable in accordance with varietal characteristics of duck. These cells grew continuously for 11 passages on DEFs. Under induction of medium with BME, RA, and IBMX, the EG cells lost undifferentiated state, large amount of neural cells appeared with the formation of neural cells networks. Special Nissl body was found by toluidine blue stain after induced for 7 days. Immunofluorescence staining results indicated that differentiated EG cells expressed Nestin, NSE, and GFAP positive. The expression of Nestin, NSE, and GFAP mRNA were positive by RT-PCR. The results revealed that RA can obviously promote the directed differentiation of duck EG cells into neural lineage. The duck EG cells will be useful for the production of transgenic birds, for cell replacement therapy and for studies of germ cell differentiation.     

The developmental fate of green fluorescent mouse embryonic germ cells in chimeric embryos

Primordial germ cells (PGCs),as precursors of mammalian germ lineage,have been gaining more attention as a new resource of pluripotent stem cells,which bring a great possibility to study developmental events of germ cell in vitro and at animal level.EG4 cells derived from 10.5 days post coitum (dpc) PGCs of 129/svJ strain mouse were established and maintained in an undifferentiated state.With an attempt to study the differentiation capability of EG4 cells with a reporter protein:green fluorescence protein,and the possible application of EG4 cells in the research of germ cell development,we have generated several EG4-GFP cell lines expressing enhanced green fluorescence protein (EGFP) and still maintaining typical characteristics of pluripotent stem cells.Then,the differentiation of EG4-GFP cells in vitro as well as their developmental fate in chimeric embryos which were produced by aggregating EG4-GFP cells to 8-cell stage embryos were studied.The results showed that EG4 cells carrying green fluorescence have a potential use in the research of germ cell development and other related studies.     

Avian pluripotent stem cells

Pluripotent embryonic stem cells are undifferentiated cells capable of proliferation and self-renewal and have the capacity to differentiate into all somatic cell types and the germ line. They provide an in vitro model of early embryonic differentiation and are a useful means for targeted manipulation of the genome. Pluripotent stem cells in the chick have been derived from stage X blastoderms and 5.5 day gonadal primordial germ cells (PGCs). Blastoderm-derived embryonic stem cells (ESCs) have the capacity for in vitro differentiation into embryoid bodies and derivatives of the three primary germ layers. When grafted onto the chorioallantoic membrane, the ESCs formed a variety of differentiated cell types and attempted to organize into complex structures. In addition, when injected into the unincubated stage X blastoderm, the ESCs can be found in numerous somatic tissues and the germ line. The potential give rise to somatic and germ line chimeras is highly dependent upon the culture conditions and decreases with passage. Likewise, PGC-derived embryonic germ cells (EGCs) can give rise to simple embryoid bodies and can undergo some differentiation in vitro. Interestingly, chicken EG cells contribute to somatic lineages when injected into the stage X blastoderm, but only germ line chimeras have resulted from EGCs injected into the vasculature of the stage 16 embryo. To date, no lines of transgenic chickens have been generated using ESCs or EGCs. Nevertheless, progress towards the culture of avian pluripotent stem cells has been significant. In the future, the answers to fundamental questions regarding segregation of the avian germ line and the molecular basis of pluripotency should foster the full use of avian pluripotent stem cells.     

小鼠原生殖细胞体外培养及其应用研究

原生殖细胞（ｐｒｉｍｏｒｄｉａｌｇｅｒｍｃｅｌｌ,ＰＧＣ）是胚胎生殖谱系最原始形式的细胞,在体胚胎迁移期ＰＧＣ增殖极为旺盛。体外培养的小鼠迁移期ＰＧＣ在饲养层细胞和三种生长因子（干细胞生长因子、碱性成纤维细胞生长因子及白血病抑制因子）的共同作用下,可发展为长期增殖并维持不分化状态的胚胎性干细胞,即胚胎生殖细胞（ｅｍｂｒｙｏｎｉｃｇｅｒｍｃｅｌｌ,ＥＧ）,具全能性发育潜能。ＥＧ建系成功对于研究生殖细胞发育以及寻找新的转基因动物操作的有效载体具有重要价值。     

Isolation,culture, and characterization of primordial germ cells in Mongolian sheep

This study was performed to culture and preliminarily identify the primordial germ cells (PGCs) isolated from the genital ridge of the Mongolian sheep fetus. The growth characteristics of the sheep PGCs were detected in different culture systems such as culture media, resources, and state and passages of feeder cells. The obtained embryonic germ (EG) cells were identified by morphology, enzymology, and immunofluorescence. The results showed that the sheep EG cell colonies were ridgy, typically nest like, and compact, and had regular edges. Alkaline phosphatase staining reaction was weakly positive. EG cells expressed Kit, Rex-1, Nanog, and Oct-4. Immunofluorescence detection was weakly positive for Oct3/4, whereas positive for SSEA-1, SSEA-3, SSEA-4, TRA-1-61, and TRA-1-80.