Pluripotent stem cells: Maintenance of genetic and epigenetic stability and prospects of cell technologies

Permanent lines of pluripotent stem cells can be obtained from humans and monkeys using different techniques and from different sources—inner cell mass of the blastocyst, primary germ cells, parthenogenetic oocytes, and mature spermatogonia—as well as by transgenic modification of various adult somatic cells. Despite different origin, all pluripotent lines demonstrate considerable similarity of the major biological properties: active self-renewal and differentiation into various somatic and germ cells in vitro and in vivo, similar gene expression profiles, and similar cell cycle structure. Ten years of intense studies on the stability of different human and monkey embryonic stem cells demonstrated that, irrespective of their origin, long-term in vitro cultures lead to the accumulation of chromosomal and gene mutations as well as epigenetic changes that can cause oncogenic transformation of cells. This review summarizes the research data on the genetic and epigenetic stability of different lines of pluripotent stem cells after long-term in vitro culture. These data were used to analyze possible factors of the genome and epigenome instability in pluripotent lines. The prospects of using pluripotent stem cells of different origin in cell therapy and pharmacological studies were considered.     

生殖细胞形成的调控机制及体外培养体系的研究进展

生殖细胞是多细胞生物体遗传物质传递的载体,在发育生物学、临床医学及畜牧业生产等领域中具有广阔的应用前景。原始生殖细胞作为胚胎体内最早出现的生殖细胞,在发育过程中受多种信号因子的诱导,发生特化、迁移、分化及减数分裂,最终形成单倍体的配子,此过程在遗传学和表观遗传学方面受到严格的调控。另外,多能性干细胞向生殖细胞的分化以及生殖细胞的体外培养方面在最近均取得了较大的进展。该文将主要围绕原始生殖细胞,综述最近几年来关于生殖细胞形成中的转录调控及体外培养体系的进展。     

Kinetics of reprogramming in cell fusion hybrids

Somatic cells can obtain pluripotency by fusion with pluripotent stem cells. The resulting fusion hybrids display pluripotential characteristics, such as inactivation of tissue-specific genes, differentiation potential to all three germ layers, and a specific epigenetic state corresponding to the pluripotent cells. However, the fusion hybrid cells are not identical to the pluripotent fusion partner cells although they are similar to the pluripotent cells. Recently, we showed that fusion-induced reprogramming was not a solely unidirectional process. In this review, we address how much somatic cells "remember and lose" their original characteristics after fusion with pluripotent cells.     

原始生殖细胞发生的机制

生殖细胞的发生是发育和遗传的基础。在几乎所有哺乳动物中,原始生殖细胞（primordial germ cell,PGC）均由近端上胚层体细胞在周边细胞特定的信号诱导下特化而成。目前的研究已经发现一些与生殖细胞特化有关的信号分子和关键转录调控元件,以及特化后生殖细胞获得的与体细胞不同的生物特性。生殖细胞的特化是一个结合了体细胞发育程序的抑制、细胞多能性程序的启动和全基因组表观遗传重编程三个方面的动态的复杂过程。多能性干细胞（胚胎干细胞或诱导型多能干细胞）具有发育全能性,能分化为机体任何一种细胞类型,包括生殖细胞。利用多能性干细胞体外分化形成生殖细胞有助于深入系统地研究配子发生的调控机制,为干细胞在不育症治疗方面的应用带来新希望。     

Environmental epigenetic modifications and reprogramming-recalcitrant genes

The term “environmental epigenetic modifications” refers to alterations in phenotype triggered by environmental stimuli via epigenetic mechanisms. Epidemiologic and animal model studies show that a subset of such environmental epigenetic marks may affect susceptibility to chronic diseases. A growing body of evidence regarding incompleteness of reprogramming indicates that the potential retention of pathogenic environmental epigenetics in human induced pluripotent stem cells (iPSCs) should be seriously considered. Given this possibility, the optimization of methods for the generation of human induc pluripotent stem cells may require the identification of epigenetically appropriate somatic cell sources. Similarly, techniques for controlling epigenetic modification by environmental factors may also play a critical role in the development of epigenetically stable sources of pluripotent stem cells.     

Autonomous regulation of sex-specific developmental programming in mouse fetal germ cells

In mice, unique events regulating epigenetic programming (e.g., genomic imprinting) and replication state (mitosis versus meiosis) occur during fetal germ cell development. To determine whether these processes are autonomously programmed in fetal germ cells or are dependent upon ongoing instructive interactions with surrounding gonadal somatic cells, we isolated male and female germ cells at 13.5 days postcoitum (dpc) and maintained them in culture for 6 days, either alone or in the presence of feeder cells or gonadal somatic cells. We examined allele-specific DNA methylation in the imprinted H19 and Snrpn genes, and we also determined whether these cells remained mitotic or entered meiosis. Our results show that isolated male germ cells are able to establish a characteristic "paternal" methylation pattern at imprinted genes in the absence of any support from somatic cells. On the other hand, cultured female germ cells maintain a hypomethylated status at these loci, characteristic of the normal "maternal" methylation pattern in endogenous female germ cells before birth. Further, the surviving female germ cells entered first meiotic prophase and reached the pachytene stage, whereas male germ cells entered mitotic arrest. These results indicate that mechanisms controlling both epigenetic programming and replication state are autonomously regulated in fetal germ cells that have been exposed to the genital ridge prior to 13.5 dpc.     

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.     

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.     

Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells.

Genomic reprogramming of primordial germ cells (PGCs), which includes genome-wide demethylation, prevents aberrant epigenetic modifications from being transmitted to subsequent generations. This process also ensures that homologous chromosomes first acquire an identical epigenetic status before an appropriate switch in the imprintable loci in the female and male germ lines. Embryonic germ (EG) cells have a similar epigenotype to PGCs from which they are derived. We used EG cells to investigate the mechanism of epigenetic modifications in the germ line by analysing the effects on a somatic nucleus in the EG-thymic lymphocyte hybrid cells. There were striking changes in methylation of the somatic nucleus, resulting in demethylation of several imprinted and non-imprinted genes. These epigenetic modifications were heritable and affected gene expression as judged by re-activation of the silent maternal allele of Peg1/Mest imprinted gene in the somatic nucleus. This remarkable change in the epigenotype of the somatic nucleus is consistent with the observed pluripotency of the EG-somatic hybrid cells as they differentiated into a variety of tissues in chimeric embryos. The epigenetic modifications observed in EG-somatic cell hybrids in vitro are comparable to the reprogramming events that occur during germ cell development.     

A germ cell-specific gene, Prmt5, works in somatic cell reprogramming

Germ cells possess the unique ability to acquire totipotency during development in vivo as well as give rise to pluripotent stem cells under the appropriate conditions in vitro. Recent studies in which somatic cells were experimentally converted into pluripotent stem cells revealed that genes expressed in primordial germ cells (PGCs), such as Oct3/4, Sox2, and Lin28, are involved in this reprogramming. These findings suggest that PGCs may be useful for identifying factors that successfully and efficiently reprogram somatic cells into toti- and/or pluripotent stem cells. Here, we show that Blimp-1, Prdm14, and Prmt5, each of which is crucial for PGC development, have the potential to reprogram somatic cells into pluripotent stem cells. Among them, Prmt5 exhibited remarkable reprogramming of mouse embryonic fibroblasts into which Prmt5, Klf4, and Oct3/4 were introduced. The resulting cells exhibited pluripotent gene expression, teratoma formation, and germline transmission in chimeric mice, all of which were indistinguishable from those induced with embryonic stem cells. These data indicate that some of the factors that play essential roles in germ cell development are also active in somatic cell reprogramming.     

Centromeric DNA hypomethylation as an epigenetic signature discriminates between germ and somatic cell lineages

Germ cells have unique features strikingly distinguishable from somatic cells. The functional divergence between these two cell lineages has been postulated to result from epigenetic mechanisms. Here we show that the chromosomal centric and pericentric (C/P) regions in male and female germline cells are specifically DNA-hypomethylated, despite the hypermethylation status in somatic cells. In multipotent germline stem cells, the C/P region was initially hypomethylated and then shifted to the hypermethylation status during differentiation into somatic lineage in vitro. Moreover, the somatic-type hypermethylation pattern was maintained in the somatic cell-derived nuclear transfer embryos throughout preimplantation development. These results imply that the identity of germ cell lineage may be warranted by the hypomethylation status of the C/P region as an epigenetic signature.     

Epigenetic regulation of germ cell differentiation

Germ cells and somatic cells have the identical genome. However, unlike the mortal fate of somatic cells, germ cells have the unique ability to differentiate into gametes that retain totipotency and produce an entire organism upon fertilization. The processes by which germ cells differentiate into gametes, and those by which gametes become embryos, involve dramatic cellular differentiation accompanied by drastic changes in gene expression, which are tightly regulated by genetic circuitries as well as epigenetic mechanisms. Epigenetic regulation refers to heritable changes in gene expression that are not due to changes in primary DNA sequence. The past decade has witnessed an ever-increasing understanding of epigenetic regulation in many different cell types/tissues during embryonic development and adult homeostasis. In this review, we focus on recent discoveries of epigenetic regulation of germ cell differentiation in various metazoan model organisms, including worms, flies, and mammals.