Derivation of embryonic germ cells and male gametes from embryonic stem cells

Egg and sperm cells (gametes) of the mouse are derived from a founder population of primordial germ cells that are set aside early in embryogenesis. Primordial germ cells arise from the proximal epiblast, a region of the early mouse embryo that also contributes to the first blood lineages of the embryonic yolk sac. Embryonic stem cells differentiate in vitro into cystic structures called embryoid bodies consisting of tissue lineages typical of the early mouse embryo. Because embryoid bodies sustain blood development, we reasoned that they might also support primordial germ cell formation. Here we isolate primordial germ cells from embryoid bodies, and derive continuously growing lines of embryonic germ cells. Embryonic germ cells show erasure of the methylation markers (imprints) of the Igf2r and H19 genes, a property characteristic of the germ lineage. We show that embryoid bodies support maturation of the primordial germ cells into haploid male gametes, which when injected into oocytes restore the somatic diploid chromosome complement and develop into blastocysts. Our ability to derive germ cells from embryonic stem cells provides an accessible in vitro model system for studies of germline epigenetic modification and mammalian gametogenesis.     

Long-term proliferation of mouse primordial germ cells in culture.

Primordial germ cells (PGCs) are first identifiable as a population of about eight alkaline phosphatase-positive cells in the 7.0 days postcoitum mouse embryo. During the next 6 days of development they proliferate to give rise to the 25,000 cells that will establish the meiotic population. Steel factor is required for PGC survival both in vivo and in vitro and together with leukaemia inhibitory factor stimulates PGC proliferation in vitro. In feeder-dependent culture, PGCs will proliferate for up to 7 days, but their numbers eventually decline and their proliferative capacity is only a fraction of that seen in vivo. Here we report a further factor that stimulates PGC proliferation in vitro, basic fibroblast growth factor (bFGF). Furthermore, bFGF, in the presence of steel factor and leukaemia inhibitory factor, stimulates long-term proliferation of PGCs, leading to the derivation of large colonies of cells. These embryonic germ cells resemble embryonic stem cells, pluripotent cells derived from preimplantation embryos, or feeder-dependent embryonal carcinoma cells, pluripotent stem cells of PGC-derived tumours (teratomas and teratocarcinomas). To our knowledge, these results provide the first system for long-term culture of PGCs.     

Soma-germline interactions coordinate homeostasis and growth in the Drosophila gonad

The ability of organs such as the liver or the lymphoid system to maintain their original size or regain it after injury is well documented. However, little is known about how these organs sense that equilibrium is breached, and how they cease changing when homeostasis is reached. Similarly, it remains unclear how, during normal development, different cell types within an organ coordinate their growth. Here we show that during gonad development in the fruitfly Drosophila melanogaster the proliferation of primordial germ cells (PGCs) and survival of the somatic intermingled cells (ICs) that contact them are coordinated by means of a feedback mechanism composed of a positive signal and a negative signal. PGCs express the EGF receptor (EGFR) ligand Spitz, which is required for IC survival. In turn, ICs inhibit PGC proliferation. Thus, homeostasis and coordination of growth between soma and germ line in the larval ovary is achieved by using a sensor of PGC numbers (EGFR-mediated survival of ICs) coupled to a correction mechanism inhibiting PGC proliferation. This feedback loop ensures that sufficient numbers of PGCs exist to fill all the stem-cell niches that form at the end of larval development. We propose that similar feedback mechanisms might be generally used for coordinated growth, regeneration and homeostasis.     

静宁鸡原生殖细胞的分离及形态学观察

原生殖细胞(PGCs)是配子的始祖细胞,它可发育演变生成卵细胞或精子细胞.上世纪90年代发展了体外培养PGCs技术,现在PGCs已能通过体外培养形成多潜能干细胞(ES),对于研究体外分化机制和细胞治疗具有重要意义.文章通过对静宁鸡种蛋进行36~82 h孵化,并对其血涂片经PAS染色后,在显微镜下观察其原生殖细胞(PGCs)的形态结构,以便为鸡的胚胎干细胞分离培养奠定基础.实验结果:原生殖细胞呈圆形或卵圆形,明显较其他细胞大2~4倍,PGCs的细胞核颜色较深,偏向一侧,后期常聚成一团.     

Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector

Embryonic stem cells isolated directly from mouse embryos can be cultured for long periods in vitro and subsequently repopulate the germ line in chimaeric mice. During the culture period these embryonic cells are accessible for experimental genetic manipulation. Here we report the use of retroviral vectors to introduce exogenous DNA sequences into a stem-cell line and show that these modified cells contribute extensively to the somatic and germ-cell lineages in chimaeric mice. Compared with current methods for manipulation of the mouse genome, this approach has the advantage that powerful somatic-cell genetic techniques can be used to modify and to select cells with germ-line potential, allowing the derivation of transgenic strains with pre-determined genetic changes. We have by this means inserted many proviral vector sequences that provide new chromosomal molecular markers for linkage studies in the mouse and that also may cause insertional mutations.     

Effect of Steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture.

Despite the importance of germ cells to the survival of species, surprisingly little is known about their embryological origin, proliferation, migration and entry into mitotic arrest or meiosis. Mutations in the murine Dominant White Spotting (W) and Steel genes, which respectively encode the c-kit tyrosine kinase receptor and the c-kit ligand (or Steel factor), impair the development of primordial germ cells (PGCs) in vivo, as well as haematopoietic stem cells and neural crest-derived melanoblasts. Here we use a monoclonal antibody against c-kit tyrosine kinase receptor and recombinant Steel factor to study the c-kit receptor-ligand system in cultured PGCs. In addition, we show that leukaemia inhibitory factor (also known as differentiation inhibitory activity), a factor secreted by STO fibroblasts, can stimulate proliferation of primordial germ cells in vitro.     

半滑舌鳎早期胚胎性腺原基分化的组织学

采用连续组织切片对半滑舌鳎胚胎及仔鱼进行了观察研究,首次描述了半滑舌鳎胚胎发育过程中原始生殖细胞(primordial germ cells,PGCs)出现的部位及迁移特征,以及卵黄合胞体,鳔与性腺原基的发育分化.结果发现,PGCs出现于神经胚期的靠近卵黄囊的囊胚层.PGCs的特征为体积比周围细胞大,核大透亮,随后在肌肉期的脊索壁上出现.孵化前期的PGCs迁移到肠原基附近,肠系膜旁可见尚在移动的PGCs和10日龄的仔鱼中在肾管旁出现性腺原基,以后PGCs数量逐渐增多参与性腺的形成.本研究为半滑舌鳎的发育生物学以及养殖生产实践提供参考.     

鸟类原生殖细胞的实验观察

本文系统地报导了鸟类原生殖细胞(PGC_s)的实验观察。初步提出了关于使用简易方法制备鸟类PGC_s标本的一系列技术(包括取胚、取胚血、制作胚血涂片、活胚标本、整胚装片和胚组织切片等)。实验观察表明,鸟类PGC_s可以在光镜下借助普通细胞学和组织化学相结合的方法进行鉴别。     

Comparative pluripotency analysis of mouse embryonic stem cells derived from wild-type and infertile hermaphrodite somatic cell nuclear transfer blastocysts

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer （SCNT）, holds great promise for treating many human diseases using regenerative medicine. Teratoma formation and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells （hESCs） have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmis- sion, which would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines lack full multi- or pluripotency has not yet been evaluated. In this study, we established 16 mouse ESC lines, including 3 genetically defective nuclear transfer- ESC （ntESC） lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted via the germ line, in contrast with normal ntESCs. Our results in- dicate that teratoma formation assays with hESCs might be an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards are required to assess the safety of hESCs for therapeutic cloning.     

Follicles were reconstituted from dissociated mouse fetal ovarian cellsin vitro

Early folliculogenesis involved in the interaction of germ cells and somatic cells is a complicated physiological event. Female germ cells are committed to differentiate into oocytes and finish complete development in the functional units of follicles. Thus there will be great significance in basal research and practices to evaluate the possibility of ovarian cells to reconstitute into follicles in vitro. In the present research, 12-16 dpc (days post coitum) mouse fetal ovarian cells were respectively isolated using collagenase digestion and cultured in droplets in vitro. The results revealed that the fetal ovarian cells of 12-16 dpc appeared to form multiple cell aggregates and tissue-like pieces in vitro. However, 12-13 dpc ovarian cells failed to form the follicles. 14-15 dpc ovarian cells were competent to form a few follicle-like complexes. Furthermore many small typical follicles were reconstituted from 16 dpc ovarian cells in vitro. The results showed for the first time that mouse embryonic ovarian cells were able to form the follicles in vitro. It was a gradual progression for the female germ cells to achieve the ability to induce somatic cells differentiation and reconstitu-tion into follicles, which may directly lead to the success in reorganization and transplantation of genetically modified ovary in vitro.     

Generation of pluripotent stem cells from adult human testis

Human primordial germ cells and mouse neonatal and adult germline stem cells are pluripotent and show similar properties to embryonic stem cells. Here we report the successful establishment of human adult germline stem cells derived from spermatogonial cells of adult human testis. Cellular and molecular characterization of these cells revealed many similarities to human embryonic stem cells, and the germline stem cells produced teratomas after transplantation into immunodeficient mice. The human adult germline stem cells differentiated into various types of somatic cells of all three germ layers when grown under conditions used to induce the differentiation of human embryonic stem cells. We conclude that the generation of human adult germline stem cells from testicular biopsies may provide simple and non-controversial access to individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells.     

Requirement for mast cell growth factor for primordial germ cell survival in culture.

Mast-cell growth factor (MGF) is encoded by the murine steel (Sl) locus and is a ligand for the tyrosine kinase receptor protein encoded by the proto-oncogene c-kit at the murine dominant white spotting (W) locus. Mutations at both these loci affect mast cells, primordial germ cells (PGCs), haemopoietic stem cells and melanocytes. In many Sl and W mutants, the rapid proliferation of PGC that normally occurs between day 7 and 13.5 of embryonic development fails to occur. As c-kit is expressed in PGCs while MGF is expressed in the surrounding mesenchyme, MGF might promote the proliferation of PGCs. Here we report that MGF is essential for PGC survival in culture, but does not stimulate PGC proliferation. Moreover, whereas both the transmembrane and soluble proteolytic cleavage forms of MGF stimulate mast-cell proliferation, soluble MGF has a relatively limited ability to support survival of PGCs in culture, thus explaining the sterility in mice carrying the steel-dickie (Sld) mutation, which encodes only a soluble form of MGF, and providing a functional role for a transmembrane growth factor.     

实验用五指山小型猪胚胎生殖细胞的分离培养

目的对从猪的原始生殖细胞分离获得胚胎干细胞的方法进行初步研究。方法胎儿取自怀孕26～28d的实验用五指山母猪。分离胎儿生殖嵴获得PGCs,接种于STO饲养层上,以DMEM+2mmol/L谷氨酰胺+0.1mmol/L非必需氨基酸+0.1mmol/Lβ-巯基乙醇+100IU/mL青霉素+100μg/mL链霉素+15%FCS作为培养基,对细胞进行培养传代。结果培养的细胞传至4～5代,形态多样,经AKP染色、SSEA-1免疫荧光染色、Oct-4免疫荧光标记等方法鉴定为阳性,具有胚胎干细胞的特征。     

保护剂浓度对第19期鸡胚原始生殖细胞存活率的影响

采用Ficoll密度梯度离心 ,提取第 19期 (孵化 72h)性腺中的原始生殖细胞 (PGCs) ,用不同浓度的冷冻保护液进行冷冻保存。复苏后的PGCs用台盼蓝染色检测其存活率 ,并进行体外培养。结果发现 :在同一浓度下 ,不同的冷冻保护液之间存在显著 (P <0 0 5 )或极显著 (P <0 0 1)差异。在同一种冷冻保护液下 ,不同的浓度之间存在显著(P <0 0 5 )或极显著 (P <0 0 1)差异 ,具体表现为冷冻保护液浓度越大 ,复苏后细胞的存活率越低。PGCs复苏后于体外培养 ,可增殖形成细胞克隆 ,并可传代培养     

Chromatin dynamics during epigenetic reprogramming in the mouse germ line

A unique feature of the germ cell lineage is the generation of totipotency. A critical event in this context is DNA demethylation and the erasure of parental imprints in mouse primordial germ cells (PGCs) on embryonic day 11.5 (E11.5) after they enter into the developing gonads. Little is yet known about the mechanism involved, except that it is apparently an active process. We have examined the associated changes in the chromatin to gain further insights into this reprogramming event. Here we show that the chromatin changes occur in two steps. The first changes in nascent PGCs at E8.5 establish a distinctive chromatin signature that is reminiscent of pluripotency. Next, when PGCs are residing in the gonads, major changes occur in nuclear architecture accompanied by an extensive erasure of several histone modifications and exchange of histone variants. Furthermore, the histone chaperones HIRA and NAP-1 (NAP111), which are implicated in histone exchange, accumulate in PGC nuclei undergoing reprogramming. We therefore suggest that the mechanism of histone replacement is critical for these chromatin rearrangements to occur. The marked chromatin changes are intimately linked with genome-wide DNA demethylation. On the basis of the timing of the observed events, we propose that if DNA demethylation entails a DNA repair-based mechanism, the evident histone replacement would represent a repair-induced response event rather than being a prerequisite.