In Vitro Germ Cell Differentiation from Cynomolgus Monkey Embryonic Stem Cells

Background

Mouse embryonic stem (ES) cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro.

Methods and Findings

To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis). VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB) formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA) 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene.

Conclusion

VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the identification and characterization of germ cells derived from ES cells are possible by using reported germ cell markers in vivo, including SSEA1, OCT-4, and VASA, in vitro as well as in vivo. These findings are thus considered to help elucidate the germ cell developmental process in primates.     

Interspecies chimera between primate embryonic stem cells and mouse embryos: monkey ESCs engraft into mouse embryos, but not post-implantation fetuses

Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspring has yet to be demonstrated in human or nonhuman primates (NHPs). Here, rhesus and baboons ESCs were investigated in interspecific mouse chimera generated by aggregation or blastocyst injection. Aggregation chimera produced mouse blastocysts with GFP-nhpESCs at the inner cell mass (ICM), and embryo transfers (ETs) generated dimly-fluorescencing abnormal fetuses. Direct injection of GFP-nhpESCs into blastocysts produced normal non-GFP-fluorescencing fetuses. Injected chimera showed >70% loss of GFP-nhpESCs after 21 h culture. Outgrowths of all chimeric blastocysts established distinct but separate mouse- and NHP-ESC colonies. Extensive endogenous autofluorescence compromised anti-GFP detection and PCR analysis did not detect nhpESCs in fetuses. NhpESCs localize to the ICM in chimera and generate pregnancies. Because primate ESCs do not engraft post-implantation, and also because endogenous autofluorescence results in misleading positive signals, interspecific chimera assays for pluripotency with primate stem cells is unreliable with the currently available ESCs. Testing primate ESCs reprogrammed into even more na?ve states in these inter-specific chimera assays will be an important future endeavor.     

A unique combination of male germ cell miRNAs coordinates gonocyte differentiation

The last 100 years have seen a concerning decline in male reproductive health associated with decreased sperm production, sperm function and male fertility. Concomitantly, the incidence of defects in reproductive development, such as undescended testes, hypospadias and testicular cancer has increased. Indeed testicular cancer is now recognised as the most common malignancy in young men. Such cancers develop from the pre-invasive lesion Carcinoma in Situ (CIS), a dysfunctional precursor germ cell or gonocyte which has failed to successfully differentiate into a spermatogonium. It is therefore essential to understand the cellular transition from gonocytes to spermatogonia, in order to gain a better understanding of the aetiology of testicular germ cell tumours. MicroRNA (miRNA) are important regulators of gene expression in differentiation and development and thus highly likely to play a role in the differentiation of gonocytes. In this study we have examined the miRNA profiles of highly enriched populations of gonocytes and spermatogonia, using microarray technology. We identified seven differentially expressed miRNAs between gonocytes and spermatogonia (down-regulated: miR-293, 291a-5p, 290-5p and 294*, up-regulated: miR-136, 743a and 463*). Target prediction software identified many potential targets of several differentially expressed miRNA implicated in germ cell development, including members of the PTEN, and Wnt signalling pathways. These targets converge on the key downstream cell cycle regulator Cyclin D1, indicating that a unique combination of male germ cell miRNAs coordinate the differentiation and maintenance of pluripotency in germ cells.     

Differentiation of murine premigratory primordial germ cells in culture.

In the mouse embryo, primordial germ cells first appear in the extraembryonic mesoderm and divide rapidly while migrating to the fetal gonad. Shortly after their arrival in the gonad, germ cells sexually differentiate as proliferation ceases. Previous studies have established that primordial germ cells proliferate and migrate in feeder layer culture. To explore cellular regulation of fetal germ cell development, we have used germ cell nuclear antigen 1 (GCNA1), a marker normally expressed only in postmigratory germ cells, to investigate the developmental potency of both pre- and postmigratory cells in this culture system. We found that explanted premigratory germ cells will initiate expression of this marker and are, therefore, capable of undertaking some aspects of gonocyte differentiation without intimate exposure to the fetal gonad. We have also tested whether postmigratory gonocytes are stable in culture. As detected by either alkaline phosphatase or GCNA1, we did not detect long-term survival of either prospermatogonia or oogonia under conditions that support the survival, proliferation, and differentiation of earlier premigratory cells. These observations are consistent with an autonomous cellular mechanism governing the initial stages of gonocyte differentiation, and suggest that differentiation towards gonocytes is accompanied by a change in requirements for cell survival.     

胚胎干细胞诱导分化为雄性生殖细胞的研究进展

胚胎干细胞（embryonic stem cells,ES细胞）具有自我更新及无限分化潜能,理论上可以分化为生殖细胞。目前,在人及鼠中已有体外诱导ES细胞分化为成熟精子的报道。系统阐述影响ES细胞分化为雄性生殖细胞的内源性及外源性因素,并结合国内外最新研究进展总结其诱导分化方法,展望应用前景,期望为从事相关研究的学者提供参考。     

Generation of multipotential mesendodermal progenitors from mouse embryonic stem cells via sustained Wnt pathway activation

Pluripotent embryonic stem cells (ESCs) are capable of differentiating into cell types belonging to all three germ layers within the body, which makes them an interesting and intense field of research. Inefficient specific differentiation and contamination with unwanted cell types are the major issues in the use of ESCs in regenerative medicine. Lineage-specific progenitors generated from ESCs could be utilized to circumvent the issue. We demonstrate here that sustained activation of the Wnt pathway (using Wnt3A or an inhibitor of glycogen synthase kinase 3beta) in multiple mouse and human ESCs results in meso/endoderm-specific differentiation. Using monolayer culture conditions, we have generated multipotential "mesendodermal progenitor clones" (MPC) from mouse ESCs by sustained Wnt pathway activation. MPCs express increased levels of meso/endodermal and mesendodermal markers and exhibit a stable phenotype in culture over a year. The MPCs have enhanced potential to differentiate along endothelial, cardiac, vascular smooth muscle, and skeletal lineages than undifferentiated ESCs. In conclusion, we demonstrate that the Wnt pathway activation can be utilized to generate lineage-specific progenitors from ESCs, which can be further differentiated into desired organ-specific cells.     

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.     

In vitro generation of germ cells from murine embryonic stem cells

The demonstration of germ cell and haploid gamete development from embryonic stem cells (ESCs) in vitro has engendered a unique set of possibilities for the study of germ cell development and the associated epigenetic phenomenon. The process of embryoid body (EB) differentiation, like teratoma formation, signifies a spontaneous differentiation of ESCs into cells of all three germ layers, and it is from these differentiating aggregates of cells that putative primordial germ cells (PGCs) and more mature gametes can be identified and isolated. The differentiation system presented here requires the differentiation of murine ESCs into EBs and the subsequent isolation of PGCs as well as haploid male gametes from EBs at various stages of differentiation. It serves as a platform for studying the poorly understood process of germ cell allocation, imprint erasure and gamete formation, with 4-6 weeks being required to isolate PGCs as well as haploid cells.     

RSPO1/β-catenin signaling pathway regulates oogonia differentiation and entry into meiosis in the mouse fetal ovary

Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog gene and factors synthesized by the somatic Sertoli cells promote gonocyte differentiation. In the female, ovarian differentiation requires activation of the WNT/β-catenin signaling pathway in the somatic cells by the secreted protein RSPO1. Using mouse models, we now show that Rspo1 also activates the WNT/β-catenin signaling pathway in germ cells. In XX Rspo1(-/-) gonads, germ cell proliferation, expression of the early meiotic marker Stra8, and entry into meiosis are all impaired. In these gonads, impaired entry into meiosis and germ cell sex reversal occur prior to detectable Sertoli cell differentiation, suggesting that β-catenin signaling acts within the germ cells to promote oogonial differentiation and entry into meiosis. Our results demonstrate that RSPO1/β-catenin signaling is involved in meiosis in fetal germ cells and contributes to the cellular decision of germ cells to differentiate into oocyte or sperm.     

Induction of primordial germ cells from mouse induced pluripotent stem cells derived from adult hepatocytes

Pluripotent stem cells can be established by various methods, but they share several cytological properties, including germ cell differentiation in vitro, independently of their origin. Although mouse induced pluripotent stem (iPS) cells can produce functional gametes in vivo, it is still unclear whether or not they have the ability to produce presumptive germ cells in vitro. Here, we show that mouse iPS cells derived from adult hepatocytes were able to differentiate into presumptive germ cells marked by mouse vasa homolog (Mvh) expression in feeder‐free or suspension cultures. Embryoid body (EB) formation from iPS cells also induced the formation of round‐shaped cells resembling immature oocytes. Mvh⁺ cells formed clumps by co‐aggregation with differentiation‐supporting cells, and increased expression of germ cell markers was detected in these cell aggregates. Differentiation culture of presumptive germ cells from iPS cells could provide a conventional system for facilitating our understanding of the mechanisms underlying direct reprogramming and germline competency. Mol. Reprod. Dev. 77: 802–811, 2010. © 2010 Wiley‐Liss, Inc.     

Bidirectional induction toward paraxial mesodermal derivatives from mouse ES cells in chemically defined medium

Embryonic stem cells (ESCs) are a renewable cell source of tissue for regenerative therapies. The addition of bone morphogenetic protein 4 (BMP4) to serum-free ESC cultures can induce primitive streak-like mesodermal cells. In differentiated mouse ESCs, platelet-derived growth factor receptor-α (PDGFR-α) and E-cadherin (ECD) are useful markers to distinguish between paraxial mesodermal progenitor cells and undifferentiated and endodermal cells, respectively. Here, we demonstrate methods for BMP4-mediated induction of paraxial mesodermal progenitors using PDGFR-α and ECD as markers for purification and characterization. Serum-free monolayers of ESCs cultured with BMP4 could efficiently promote paraxial mesodermal differentiation akin to embryonic mesodermal development. BMP4 treatment alone induced paraxial mesodermal progenitors that could differentiate into osteochondrogenic cells in vitro and in vivo. Furthermore, early removal of BMP4 followed by lithium chloride (LiCl) promoted the differentiation to myogenic progenitor cells. These myogenic progenitors were able to differentiate further in vitro into mature skeletal muscle cells. Thus, we successfully induced the efficient bidirectional differentiation of mouse ESCs toward osteochondrogenic and myogenic cell types using chemically defined conditions.     

干细胞命运决定的分子调控网络

来源于囊胚期胚胎内细胞团的胚胎干细胞具有独特的生物学特性,包括无限自我更新的能力以及分化为内胚层、中胚层和外胚层各种细胞的潜能.阐明胚胎干细胞全能性维持以及向各种特定细胞分化的分子机制,不仅有助于我们了解胚胎发育过程,而且将促进胚胎干细胞尽早应用于疾病治疗.本文主要就干细胞的一种命运决定过程,维持胚胎干细胞全能性或失去全能性开始分化,结合最新的研究进展讨论该过程中的分子调控网络,包括信号转导通路、表达调控网络以及表观遗传调控.     

Primate spermatogenesis: new insights into comparative testicular organisation, spermatogenic efficiency and endocrine control

Owing to the close phylogenetic relationship of Platyrrhini (New World monkeys) and Catarrhini (Old World monkeys) to man, nonhuman primates are often used as models for the study of male reproductive physiology and endocrinology. This review aims at providing new data and insights into comparative primate spermatogenesis, dealing specifically with quantitative aspects of germinal epithelial organisation and germ cell production, and with the roles of gonadotrophic hormones in this process. Typically, the seminiferous epithelium is composed of specific germ cell associations (spermatogenic stages). In rodents, prosimians and most Catarrhini, tubular cross sections contain a single spermatogenic stage whereas in Platyrrhini, great apes and man multi-stage tubules are present. Since Platyrrhini represent a more basal type of primate, this spermatogenic feature must have developed convergently. The primate multi-stage tubular arrangement was previously believed to be associated with low spermatogenic efficiency. However, recent studies using new methodological approaches and comparing primate species from all taxa have revealed that multistage organisation is compatible with highly efficient spermatogenesis. In fact, meta-analysis demonstrated that the efficiency of spermatogenesis in several nonhuman primate species is comparable to that of rodents which are considered as species with highly efficient germ cell production. The duration of the spermatogenic process was not related to organisation or efficiency of spermatogenesis. Sertoli cell work load was species-specific but had no impact on germ cell numbers and on the efficiency of spermatogenesis. The gonadotrophic hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH) are the primary regulators of primate testicular function. Recent studies revealed that in New World monkeys chorionic gonadotrophin (CG)--the primate pregnancy hormone--regulates testosterone production instead of LH. Receptor studies demonstrated a dual action of the closely related hormones LH and CG in primates. It is hypothesised that following the divergence of the Platyrrhini lineage from Catarrhini, the LH/CG system evolved independently with ancestral functions of the LH/CG system retained in the neotropical taxa. In summary, key spermatogenic features are preserved across all primate taxa whereas male reproductive endocrinology features appear substantially different in the neotropical primates compared to other primate lineages.     

胚胎干细胞向造血干/祖细胞定向诱导分化的研究进展

胚胎干细胞(embryonic stem cell,ES细胞)是指由胚胎内细胞团(inner cell mass,ICM)细胞经体外抑制培养而筛选得到的细胞,具有无限增殖潜能,在体外可以向造血细胞分化,有可能为造血干细胞移植和血细胞输注开辟新的来源．此外,ES细胞向造血干/祖细胞的定向诱导分化也为阐明哺乳动物造血发育的细胞和分子机制提供了良好的体外模型．对ES细胞向造血干/祖细胞定向分化的研究进展进行了综述．     

Cloning of non-human primates: the road "less traveled by"

Early studies on cloning of non-human primates by nuclear transfer utilized embryonic blastomeres from preimplantation embryos which resulted in the reproducible birth of live offspring. Soon after, the focus shifted to employing somatic cells as a source of donor nuclei (somatic cell nuclear transfer, SCNT). However, initial efforts were plagued with inefficient nuclear reprogramming and poor embryonic development when standard SCNT methods were utilized. Implementation of several key SCNT modifications was critical to overcome these problems. In particular, a non-invasive method of visualizing the metaphase chromosomes during enucleation was developed to preserve the reprogramming capacity of monkey oocytes. These modifications dramatically improved the efficiency of SCNT, yielding high blastocyst development in vitro. To date, SCNT has been successfully used to derive pluripotent embryonic stem cells (ESCs) from adult monkey skin fibroblasts. These remarkable advances have the potential for development of human autologous ESCs and cures for many human diseases. Reproductive cloning of nonhuman primates by SCNT has not been achieved yet. We have been able to establish several pregnancies with SCNT embryos which, so far, did not progress to term. In this review, we summarize the approaches, obstacles and accomplishments of SCNT in a non-human primate model.     

胚胎干细胞及诱导多能干细胞向心肌细胞分化和调控的研究进展

胚胎干细胞（embryonic stem cells,ESCs）具有自我更新、无限增殖和多向分化的特性,包括分化成心脏组织的多种类型细胞。经体细胞重编程产生的诱导多能干细胞（induced pluripotent stem cells,iPS）也被证明有类似胚胎干细胞的特性。但这些多能干细胞向心肌细胞自发分化的效率非常低,因此,如何有效地诱导这些多能干细胞向心肌细胞的定向分化对深入认识心肌发生发育的关键调控机制和实现其在药物发现和再生医学,如心肌梗塞、心力衰竭的细胞治疗以及心肌组织工程中的应用均具有非常重要的意义。该文重点综述了近年来胚胎干细胞及诱导多能干细胞向心肌细胞分化和调控的研究进展,并探讨了这一研究领域亟待解决的关键问题和这些多能干细胞的应用前景。     

FGF9 promotes survival of germ cells in the fetal testis

In addition to its role in somatic cell development in the testis, our data have revealed a role for Fgf9 in XY germ cell survival. In Fgf9-null mice, germ cells in the XY gonad decline in numbers after 11.5 days post coitum (dpc), while germ cell numbers in XX gonads are unaffected. We present evidence that germ cells resident in the XY gonad become dependent on FGF9 signaling between 10.5 dpc and 11.5 dpc, and that FGF9 directly promotes XY gonocyte survival after 11.5 dpc, independently from Sertoli cell differentiation. Furthermore, XY Fgf9-null gonads undergo true male-to-female sex reversal as they initiate but fail to maintain the male pathway and subsequently express markers of ovarian differentiation (Fst and Bmp2). By 14.5 dpc, these gonads contain germ cells that enter meiosis synchronously with ovarian gonocytes. FGF9 is necessary for 11.5 dpc XY gonocyte survival and is the earliest reported factor with a sex-specific role in regulating germ cell survival.