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.     

Derivation of embryonic germ cells from post migratory primordial germ cells, and methylation analysis of their imprinted genes by bisulfite genomic sequencing

The embryonic germ cell (EGCs) of mice is a kind of pluripotent stem cell that can be generated from pre- and post-migratory primordial germ cells (PGCs). Most previous studies on DNA methylation of EGCs were restricted to 12.5 days post coitum (dpc). This study was designed to establish and characterize murine EGC lines from migrated PGCs as late as 13.5 dpc and to estimate the degrees of methylation of their imprinted genes as well as of the non-imprinted locus, Oct4, using an accurate and quantitative method of measurement. We established five independent EGC lines from post migratory PGCs of 11.5-13.5 dpc from C57BL/6xDBA/2 F1 hybrid mouse fetuses. All the EGCs exhibited the typical features of pluripotent cells including hypomethylation of the Oct4 regulatory region. We examined the methylation status of three imprinted genes; Igf2, Igf2r and H19 in the five EGC lines using bisulfite genomic sequencing analysis. Igf2r was almost unmethylated in all the EGC lines irrespective of the their sex and stage of isolation; Igf2 and H19 were more methylated than Igf2r, especially in male EGCs. Moreover, EGCs derived at 13.5 dpc exhibited higher levels of DNA methylation than those from earlier stages. These results suggest that in vitro derived EGCs acquire different epigenotypes from their parental in vivo migratory PGCs, and that sex-specific de novo methylation occurs in the Igf2 and H19 genes of EGCs.     

Global gene expression profiling reveals similarities and differences among mouse pluripotent stem cells of different origins and strains

Pluripotent stem cell lines with similar phenotypes can be derived from both blastocysts (embryonic stem cells, ESC) and primordial germ cells (embryonic germ cells, EGC). Here, we present a compendium DNA microarray analysis of multiple mouse ESCs and EGCs from different genetic backgrounds (strains 129 and C57BL/6) cultured under standard conditions and in differentiation-promoting conditions by the withdrawal of Leukemia Inhibitory Factor (LIF) or treatment with retinoic acid (RA). All pluripotent cell lines showed similar gene expression patterns, which separated them clearly from other tissue stem cells with lower developmental potency. Differences between pluripotent lines derived from different sources (ESC vs. EGC) were smaller than differences between lines derived from different mouse strains (129 vs. C57BL/6). Even in the differentiation-promoting conditions, these pluripotent cells showed the same general trends of gene expression changes regardless of their origin and genetic background. These data indicate that ESCs and EGCs are indistinguishable based on global gene expression patterns alone. On the other hand, a detailed comparison between a group of ESC lines and a group of EGC lines identified 20 signature genes whose average expression levels were consistently higher in ESC lines, and 84 signature genes whose average expression levels were consistently higher in EGC lines, irrespective of mouse strains. Similar analysis identified 250 signature genes whose average expression levels were consistently higher in a group of 129 cell lines, and 337 signature genes whose average expression levels were consistently higher in a group of C57BL/6 cell lines. Although none of the genes was exclusively expressed in either ESCs versus EGCs or 129 versus C57BL/6, in combination these signature genes provide a reliable separation and identification of each cell type. Differentiation-promoting conditions also revealed some minor differences between the cell lines. For example, in the presence of RA, EGCs showed a lower expression of muscle- and cardiac-related genes and a higher expression of gonad-related genes than ESCs. Taken together, the results provide a rich source of information about the similarities and differences between ESCs and EGCs as well as 129 lines and C57BL/6 lines. Such information will be crucial to our understanding of pluripotent stem cells. The results also underscore the importance of studying multiple cell lines from different strains when making comparisons based on gene expression analysis.     

Induction of pluripotency in primordial germ cells

Primordial germ cells (PGCs) are the founder cells of all gametes. PGCs differentiate from pluripotent epiblasts cells by mesodermal induction signals during gastrulation. Although PGCs are unipotent cells that eventually differentiate into only sperm or oocytes, they dedifferentitate to pluripotent stem cells known as embryonic germ cells (EGCs) in vitro and give rise to testicular teratomas in vivo, which indicates a "metastable" differentiation state of PGCs. We have shown that an appropriate level of phosphoinositide-3 kinase (PI3K)/Akt signaling, balanced by positive and negative regulators, ensures the establishment of the male germ lineage by preventing its dedifferentiation. Specifically, hyper-activation of the signal leads to testicular teratomas and enhances EGC derivation efficiency. In addition, PI3K/Akt signaling promotes PGC dedifferentiation via inhibition of the tumor suppressor p53, a downstream molecule of the PI3K/Akt signal. On the other hand, Akt activation during mesodermal differentiation of embryonic stem cells (ESCs) generates PGC-like pluripotent cells, a process presumably induced through equilibrium between mesodermal differentiation signals and dedifferentiation-inducing activity of Akt. The transfer of these cells to ESC culture conditions results in reversion to an ESC-like state. The interconversion between ESC and PGC-like cells helps us to understand the metastability of PGCs. The regulatory mechanisms of PGC dedifferentiation are discussed in comparison with those involved in the dedifferentiation of testicular stem cells, ESC pluripotency, and somatic nuclear reprogramming.     

Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.     

The early human germ cell lineage does not express SOX2 during in vivo development or upon in vitro culture

NANOG, POU5F1, and SOX2 are required by the inner cell mass of the blastocyst and act cooperatively to maintain pluripotency in both mouse and human embryonic stem cells. Inadequacy of any one of them causes loss of the undifferentiated state. Mouse primordial germ cells (PGCs), from which pluripotent embryonic germ cells (EGCs) are derived, also express POU5F1, NANOG, and SOX2. Thus, a similar expression profile has been predicted for human PGCs. Here we show by RT-PCR, immunoblotting, and immunohistochemistry that human PGCs express POU5F1 and NANOG but not SOX2, with no evidence of redundancy within the group B family of human SOX genes. Although lacking SOX2, proliferative human germ cells can still be identified in situ during early development and are capable of culture in vitro. Surprisingly, with the exception of FGF4, many stem cell-restricted SOX2 target genes remained detected within the human SOX2-negative germ cell lineage. These studies demonstrate an unexpected difference in gene expression between human and mouse. The human PGC is the first primary cell type described to express POU5F1 and NANOG but not SOX2. The data also provide a new reference point for studies attempting to turn human stem cells into gametes by normal developmental pathways for the treatment of infertility.     

应用诱导多能干细胞治疗的进展与挑战

诱导多能干细胞(Induced pluripotent stem cells,i PSCs)是利用特定的转录因子诱导体细胞获得的,像胚胎干细胞一样,可以进行无限的自我更新,并具有分化成三个胚层的能力。iPSC有可能提供无限的自体细胞治疗,目前研究已经证实,不同种类疾病的患者提供的成体细胞诱导后可产生种类繁多的iPSC,这项技术给目前无有效治疗手段的多类疾病带来了治疗的希望,并有可能避免利用胚胎干细胞(embryonic stem cells,ESCs)治疗面临的伦理问题和免疫排斥反应。本文回顾iPSC技术优化过程,着重关注应用i PSC建立细胞模型、进行细胞治疗的进展,并探讨iPSC在基础研究及临床应用中遇到的挑战。     

The germ cell determinant Blimp1 is not required for derivation of pluripotent stem cells

Blimp1 (Prdm1), the key determinant of primordial germ cells (PGCs), plays a combinatorial role with Prdm14 during PGC specification from postimplantation epiblast cells. They together initiate epigenetic reprogramming in early germ cells toward an underlying pluripotent state, which is equivalent to embryonic stem cells (ESCs). Whereas Prdm14 alone can?promote reprogramming and is important for the propagation of the pluripotent state, it is not known whether Blimp1 is similarly involved. By using a genetic approach, we demonstrate that Blimp1 is?dispensable for the derivation and maintenance of ESCs and postimplantation epiblast stem cells (epiSCs). Notably, Blimp1 is also dispensable for reprogramming epiSCs to ESCs. Thus, although Blimp1 is obligatory for PGC specification, it is not required for the reversion of epiSCs to ESCs and for their maintenance thereafter. This study suggests that reprogramming, including that of somatic cells to ESCs, may not entail an obligatory route through a Blimp1-positive PGC-like state.     

Turning germ cells into stem cells

Primordial germ cells (PGCs), the embryonic precursors of the gametes of the adult animal, can give rise to two types of pluripotent stem cells. In vivo, PGCs can give rise to embryonal carcinoma cells, the pluripotent stem cells of testicular tumors. Cultured PGCs exposed to a specific cocktail of growth factors give rise to embryonic germ cells, pluripotent stem cells that can contribute to all the lineages of chimeric embryos including the germline. The conversion of PGCs into pluripotent stem cells is a remarkably similar process to nuclear reprogramming in which a somatic nucleus is reprogrammed in the egg cytoplasm. Understanding the genetics of embryonal carcinoma cell formation and the growth factor signaling pathways controlling embryonic germ cell derivation could tell us much about the molecular controls on developmental potency in mammals.     

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。     

Impaired meiotic competence in putative primordial germ cells produced from mouse embryonic stem cells

There are still several unanswered questions and problems about the recently claimed possibility of producing functional germ cells in vitro from pluripotent embryonic stem cells (ESCs). In the present paper, we compared by single-cell analysis the capability of putative primordial germ cells (PGCs), produced in vitro from ESCs, and that of endogenous PGCs isolated from embryos, to enter and progress through meiotic prophase I. Using a protocol previously reported to be suitable to produce female germ cells from mouse ESC monolayers, we first identified putative PGCs by analysing the expression pattern of several markers such as SSEA1, APase, OCT4, NANOG, MVH and SCP3 of pre- and post migratory PGCs. Next, after isolation of such cells from culture, we tested their meiotic capability. The evaluation at 2-5 days of culture of the number of cells showing meiotic nuclear SCP3 staining in cytospreads showed that it remained nearly constant in the putative PGCs, whereas it increased markedly in endogenous PGCs. Moreover, we observed that in putative PGCs, the nuclear distribution or expression of SCP3 and other meiotic markers such as DMC1, gH2AX and SCP1 were always highly abnormal in comparison to that observed in endogenous cultured PGCs. We conclude that although the formation of cells showing characteristics of PGCs can occur efficiently from ESCs in vitro, these cells possess impaired capability to enter and progress through meiotic prophase I.     

Human primordial germ cells and embryonic germ cells, and their use in cell therapy

Human embryonic germ (hEG) cells derive from the transformation of primordial germ cells (PGCs) under appropriate culture conditions with embryonic fibroblast feeder cells. Although the pluripotent and proliferative capacity of hEG cells is thought to be equivalent to that of human embryonic stem (hES) cells, the difficulties of isolating and maintaining hEG cell lines in vitro have restricted their availability for experimental use. Despite this, some of the factors involved in PGC development, their transformation into embryonic germ cells and the differentiation of embryonic germ cells to specific cell phenotypes have been explored. The potential use of hEG cells in cell therapy applications will, however, depend on a more thorough understanding of how to derive and maintain these cells in vitro.     

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.     

Characterization, isolation and culture of primordial germ cells in domestic animals: recent progress and insights from the ovine species

Primordial germ cell (PGC) allocation, characterization, lineage restriction, and differentiation have been extensively studied in the mouse. Murine PGC can be easily identified using markers as alkaline phosphatase content or the expression of pluripotent markers such as Pou5f1, Nanog, Sox2, Kit, SSEA1, and SSEA4. These tools allowed us to clarify certain aspects of the complex interactions of somatic and germinal cells in the establishment of the germ cell lineage, its segregation from the neighbouring somatic tissue, and the guidance mechanisms during migration that direct most of the germ cells into the genital ridges. Few data are available from other domestic animals and here we reported our preliminary studies on the isolation, characterization, and in vitro culture of sheep PGCs. Sheep PGCs can be identified with the markers previously used in mouse, but, in some cases, these markers are not coherently expressed in the same cell depending on the grade of differentiation and on technical problems related to commercial antibodies used. Pluripotency of PGCs in culture (EGCs) from domestic animals also needs further evaluation even though the derivation of embryonic pluripotent cell lines from large mammals may be an advantage as they are more physiologically similar to the human and perhaps more relevant for clinical translation studies. Comprehensive epigenetic reprogramming of the genome in early germ cells, and derived EGCs including extensive erasure of epigenetic modifications, may be relevant for gaining insight into events that lead to reprogramming and establishment of totipotency. EGCs can differentiate in vitro in a various range of tissues, form embryonic bodies, but in many cases failed to generate tumours when transplanted into immunodeficient mice and are not able to generate germline chimeric animals after their transfer. Such incomplete information clearly indicates the urge to improve the studies on derivation of stem cells in farm animals and shows the need for a multidisciplinary investigation in order to create farm animal models to set up suitable ethical and technical systems for cell regenerative therapies in humans.