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     

猪原始生殖细胞生物学特性的研究

胚胎生殖细胞（embryonic germ cell,EGC）是由胎儿原始生殖细胞（primordial germ cell,PGC）经体外驯化培养获得的一种多潜能干细胞。研究猪PGC生物学特性对于建立猪EGC及了解猪生殖细胞发育机制具有重要意义。该研究以原代培养的猪PGC为对象,探讨了其生长行为特征及其重编程过程中多能性、生殖系标志基因的表达模式。结果显示,26 d胚胎生殖嵴分离的PGC呈碱性磷酸酶阳性,细胞体积及核质比较大;体外培养初期呈现出较强的增殖及迁移能力,培养第5 d细胞增殖达到平台期,此时克隆高表达Oct4、Sox2、Nanog、c-Myc、Klf4和Ifi tm3（P〈0.05）,低表达Blimp1（P〈0.05）,Nanos1和Stella的表达水平与猪胎儿成纤维细胞无差异;猪PGC形成的原代克隆已经具有多向分化潜能。     

Receptor Tyrosine Kinase Signaling and Primordial Germ Cell Development

Primordial germ cells (PGCs) give rise to sperms and eggs. Their development is crucial to species propagation and has to be precisely controlled. Studies in several model organisms have identified many genes involved in the specification and guided migration of PGCs. However, the mechanisms governing the behaviors of this unique type of cells remain to be investigated. Interestingly, PGCs share certain cellular properties with metastasizing cancer cells including proliferation, invasion of other tissues, survival, and migration. Recently we have shown that in Drosophila the receptor tyrosine kinase Torso activates both STAT and Ras during the early phase of PGC development. In later stages, activation of both STAT and Ras, likely by other molecules, is required continuously for PGC migration. The requirement for RTK suggests molecular conservation between flies and mice in PGC development and also suggests that germ cells and cancer cells share certain intracellular signaling strategies.     

Hypersensitivity of Primordial Germ Cells to Compromised Replication-Associated DNA Repair Involves ATM-p53-p21 Signaling

Genome maintenance in germ cells is critical for fertility and the stable propagation of species. While mechanisms of meiotic DNA repair and chromosome behavior are well-characterized, the same is not true for primordial germ cells (PGCs), which arise and propagate during very early stages of mammalian development. Fanconi anemia (FA), a genomic instability syndrome that includes hypogonadism and testicular failure phenotypes, is caused by mutations in genes encoding a complex of proteins involved in repair of DNA lesions associated with DNA replication. The signaling mechanisms underlying hypogonadism and testicular failure in FA patients or mouse models are unknown. We conducted genetic studies to show that hypogonadism of Fancm mutant mice is a result of reduced proliferation, but not apoptosis, of PGCs, resulting in reduced germ cells in neonates of both sexes. Progressive loss of germ cells in adult males also occurs, overlaid with an elevated level of meiotic DNA damage. Genetic studies indicated that ATM-p53-p21 signaling is partially responsible for the germ cell deficiency.     

Primordial Germ Cell Specification from Embryonic Stem Cells

Background

Primordial germ cell (PGC) specification is the first crucial step in germ line development. However, owing to significant challenges regarding the in vivo system, such as the complex cellular environment and potential problems with embryo manipulation, it is desirable to generate embryonic stem (ES) cells that are capable of overcoming these aforementioned limitations in order to provide a potential in vitro model to recapitulate the developmental processes in vivo.

Methodology and Principal Findings

Here, we studied the detailed process of PGC specification from stella-GFP ES cells. We first observed the heterogeneous expression of stella in ES cells. However, neither Stella-positive ES cells nor Stella-negative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors. Second, we derived PGCs from ES cells using two differentiation methods, namely the attachment culture technique and the embryoid body (EB) method. Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method. Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM) that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation.

Conclusions and Significance

The in vitro model we have established can recapitulate the developmental processes in vivo and provides new insights into the mechanism of PGC specification.     

果蝇原生殖细胞特化的分子机制

原生殖细胞在许多有性生殖动物的胚胎发育早期就已特化出来,并进一步分化为生殖细胞以产生新的子代。动物原生殖细胞的特化主要有生殖质决定和诱导两种模式,果蝇原生殖细胞的特化模式属于前者。研究表明,果蝇原生殖细胞特化过程中生殖质组装的关键基因是osk,其调控下游基因转录产物的定位和翻译,如vas和tud。此外,基因转录沉默是原生殖细胞特化过程的一个重要特征,其与生殖质中的成分如基因nos、gcl、pgc的表达产物密切相关。现对果蝇原生殖细胞特化分子机制进行综述。     

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。     

Primordial Germ Cell-Like Cells Differentiated In Vitro from Skin-Derived Stem Cells

Background

We have previously demonstrated that stem cells isolated from fetal porcine skin have the potential to form oocyte-like cells (OLCs) in vitro. However, primordial germ cells (PGCs), which must also be specified during the stem cell differentiation to give rise to these putative oocytes at more advanced stages of culture, were not systematically characterized. The current study tested the hypothesis that a morphologically distinct population of cells derived from skin stem cells prior to OLC formation corresponds to putative PGCs, which differentiate further into more mature gametes.

Methodology/Principal Findings

When induced to differentiate in an appropriate microenvironment, a subpopulation of morphologically distinct cells, some of which are alkaline phosphatase (AP)-positive, also express Oct4, Fragilis, Stella, Dazl, and Vasa, which are markers indicative of germ cell formation. A known differentially methylated region (DMR) within the H19 gene locus, which is demethylated in oocytes after establishment of the maternal imprint, is hypomethylated in PGC-like cells compared to undifferentiated skin-derived stem cells, suggesting that the putative germ cell population undergoes imprint erasure. Additional evidence supporting the germ cell identity of in vitro-generated PGC-like cells is that, when labeled with a Dazl-GFP reporter, these cells further differentiate into GFP-positive OLCs.

Significance

The ability to generate germ cell precursors from somatic stem cells may provide an in vitro model to study some of the unanswered questions surrounding early germ cell formation.     

蒙古绵羊原始生殖细胞用于胚胎干细胞培养的研究

原始生殖细胞是用做分离和克隆胚胎干细胞的一种新的细胞资源。本研究将30-45日龄的蒙古绵羊胚胎生殖嵴及其临近组织用机械剪碎和胰蛋白酶+EDTA消化处理,添加DMEM（低糖）+10%FBS（犊牛血清）、38.0℃、5%CO2和饱和湿度条件下进行培养。其结果：未加任何细胞生长因子的情况下与其胎儿成纤维细胞共培养的方式也能分离得到类胚胎干细胞集落。这些集落细胞经多次克隆传代具有胚胎干细胞的诸多特征,如：具有连续传代的能力,细胞集落有典型鸟巢状结构,AKP染色呈阳性,核型分析结果染色体正常等。这些表明该细胞具有多能性,是绵羊类ES细胞。     

原始生殖细胞体外培养及应用研究

原始生殖细胞是来源于胚胎生殖嵴的一类具有多向分化潜能的干细胞,其形态、细胞表面标志、分化潜能均与来源于囊胚内细胞团的干细胞相似.在饲养细胞层和多种生长因子的共同作用下,可保持原生殖细胞在体外不断增殖而不分化,最终建立EG细胞系.本文就原始生殖细胞体外培养,建立EG细胞系及其应用前景作一综述.     

影响牛胚胎干细胞分离克隆因素的研究

采用与同源胎儿成纤维细胞共同培养及传统饲养层培养方式,以高糖DMEM,添加0.1mM2-巯基乙醇,犊牛血清,细胞因子为培养基,以4-13周龄屠宰牛胎儿为实验材料,探讨影响牛原始生殖细胞分离克隆胚胎干细胞的相关因素,结果发现：当犊牛血清为15%时效果最好;细胞因子添加与否对胚胎干细胞的分离及同源牛胎儿成纤维细胞的贴壁与生长影响并不显著,而在传代过程中中有一定影响;以0.2%胰酶 0.04?TA为细胞消化液效果最佳;以同源胎儿成纤维细胞共培养的方式分离克隆牛胚胎干细胞,本研究观察到效果最好。     

Purification of Primordial Germ Cells from TNAPMouse Embryos Using FACS-gal

Tissue nonspecific alkaline phosphatase (TNAP), the product of theAkp2locus, is expressed in mouse primordial germ cells (PGC) for an extensive period during embryogenesis. Mice with theAkp2^tm1Sormutant allele of TNAP expresslacZ(β-galactosidase; β-gal) under control of theAkp2locus. PGCs were purified fromAkp2^tm1Sorembryos using fluorescence activated cell sorting of β-gal expressing cells (FACS-gal). Analysis of the purified cells by alkaline phosphatase staining and immunocytochemistry with anti-c-kitantibody demonstrated that highly (98%) purified PGCs can be isolated using this method. This technique will facilitate experiments that require highly purified preparations of PGCs including cell culture and gene expression analyses.     

鸡胚血液中原始生殖细胞的分离及其培养的研究

从孵化４８～５５小时鸡胚中抽取血液,每只胚胎可获血液２～６μｌ左右。一步法离心分离原始生殖细胞,可使其浓度由０．１％以下提高到５０％以上。将多余血细胞用微量吸管移走后,加入添加１０％胎牛血清的ＴＣＭ—１９９做为培养基,３７．５℃,５％ＣＯ２,９５％空气,饱和湿度下培养,原始生殖细胞可成活２４小时左右。