Differentiation Of Human Germ Cells Research Articles

Sorting and Manipulation of Human PGC-LC Using PDPN and Hanging Drop Cultures.

The generation of oocytes from induced pluripotent stem cells (iPSCs) was proven efficient with mouse cells. However, no human iPSCs have yet been reported to generate cells able to complete oogenesis. Additionally, efficient sorting of human Primordial Germ Cell-like Cells (hPGC-LCs) without genomic integration of fluorescent reporter for their downstream manipulation is still lacking. Here, we aimed to develop a model that allows human germ cell differentiation in vitro in order to study the developing human germline. The hPGC-LCs specified from two iPS cell lines were sorted and manipulated using the PDPN surface marker without genetic modification. hPGC-LCs obtained remain arrested at early stages of maturation and no further differentiation nor meiotic onset occurred when these were cultured with human or mouse fetal ovarian somatic cells. However, when cultured independently of somatic ovarian cells, using BMP4 and the hanging drop-transferred EBs system, early hPGC-LCs further differentiate efficiently and express late PGC (DDX4) and meiotic gene markers, although no SYCP3 protein was detected. Altogether, we characterized a tool to sort hPGC-LCs and an efficient in vitro differentiation system to obtain pre-meiotic germ cell-like cells without using a gonadal niche.

A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

Dysregulation of genetic pathways during human germ cell development leads to infertility. Here, we analyzed bona fide human primordial germ cells (hPGCs) to probe the developmental genetics of human germ cell specification and differentiation. We examined distribution of OCT4 occupancy in hPGCs relative to human embryonic stem cells (hESCs). We demonstrate that development, from pluripotent stem cells to germ cells, is driven by switching partners with OCT4 from SOX2 to PAX5 and PRDM1. Gain- and loss-of-function studies revealed that PAX5 encodes a critical regulator of hPGC development. Moreover, analysis of epistasis indicates that PAX5 acts upstream of OCT4 and PRDM1. The PAX5-OCT4-PRDM1 proteins form a core transcriptional network that activates germline and represses somatic programs during human germ cell differentiation. These findings illustrate the power of combined genome editing, cell differentiation and engraftment for probing human developmental genetics that have historically been difficult to study.

Fate of induced pluripotent stem cells following transplantation to murine seminiferous tubules

Studies of human germ cell development are limited in large part by inaccessibility of germ cells during development. Moreover, although several studies have reported differentiation of mouse and human germ cells from pluripotent stem cells (PSCs) in vitro, differentiation of human germ cells from PSCs in vivo has not been reported. Here, we tested whether mRNA reprogramming in combination with xeno-transplantation may provide a viable system to probe the genetics of human germ cell development via use of induced pluripotent stem cells (iPSCs). For this purpose, we derived integration-free iPSCs via mRNA-based reprogramming with OCT3/4, SOX2, KLF4 and cMYC alone (OSKM) or in combination with the germ cell-specific mRNA, VASA (OSKMV). All iPSC lines met classic criteria of pluripotency. Moreover, global gene expression profiling did not distinguish large differences between undifferentiated OSKM and OSKMV iPSCs; however, some differences were observed in expression of pluripotency factors and germ cell-specific genes, and in epigenetic profiles and in vitro differentiation studies. In contrast, transplantation of undifferentiated iPSCs directly into the seminiferous tubules of germ cell-depleted immunodeficient mice revealed divergent fates of iPSCs produced with different factors. Transplantation resulted in morphologically and immunohistochemically recognizable germ cells in vivo, particularly in the case of OSKMV cells. Significantly, OSKMV cells also did not form tumors while OSKM cells that remained outside the seminiferous tubule proliferated extensively and formed tumors. Results indicate that mRNA reprogramming in combination with transplantation may contribute to tools for genetic analysis of human germ cell development.

Human germ cell differentiation from pluripotent embryonic stem cells and induced pluripotent stem cells.

Although 10-15 % of couples are infertile, little is known of the diverse, underlying pathologies in men and women with poor germ cell production; furthermore, for those with few or no high-quality germ cells, there are few options available for treatment. Thus, over the last decade, concerted efforts have been aimed at developing a biological system to probe the fundamentals of human egg and sperm production via pluripotent stem cell cells with the hopes of informing clinical decisions and ultimately providing alternative methods for therapy which may include developing a source of germ cells ultimately for reproductive purposes.

Generation of Human Induced Pluripotent Stem Cells Using Epigenetic Regulators Reveals a Germ Cell-Like Identity in Partially Reprogrammed Colonies

Previous studies have shown that induced pluripotent stem cells (iPSCs) can be derived from fibroblasts by ectopic expression of four transcription factors, OCT4, SOX2, KLF4 and c-MYC using various methods. More recent studies have focused on identifying alternative approaches and factors that can be used to increase reprogramming efficiency of fibroblasts to pluripotency. Here, we use nucleofection, morpholino technologies and novel epigenetic factors, which were chosen based on their expression profile in human embryos, fibroblasts and undifferentiated/differentiated human embryonic stem cells (hESCs) and conventionally generated iPSCs, to reprogram human fibroblasts into iPSCs. By over expressing DNMT3B, AURKB, PRMT5 and/or silencing SETD7 in human fibroblasts with and without NANOG, hTERT and/or SV40 overexpression, we observed the formation of colonies resembling iPSCs that were positive for certain pluripotency markers, but exhibited minimal proliferation. More importantly, we also demonstrate that these partially-reprogrammed colonies express high levels of early to mid germ cell-specific genes regardless of the transfection approach, which suggests conversion to a germ cell-like identity is associated with early reprogramming. These findings may provide an additional means to evaluate human germ cell differentiation in vitro, particularly in the context of pluripotent stem cell-derived germ cell development, and contribute to our understanding of the epigenetic requirements of the reprogramming process.

Status of human germ cell differentiation from pluripotent stem cells

Historically, the quality of life of infertile couples has been greatly diminished by the loss of opportunity to conceive. However, beginning with the advent of IVF in the late 1970s, novel clinical interventions have greatly changed the outlook for those with severe forms of infertility. Yet, in cases in which the quality and quantity of germ cells are most compromised, there are few options. In the present paper, the current status of germ cell development from stem cells is reviewed in light of potential utility for basic science and clinical applications.

NANOS3 function in human germ cell development.

Human infertility is common and frequently linked to poor germ cell development. Yet, human germ cell development is poorly understood, at least in part due to the inaccessibility of germ cells to study especially during fetal development. Here, we explored the function of a highly conserved family of genes, the NANOS genes, in the differentiation of human germ cells from human embryonic stem cells. We observed that NANOS-1, -2 and -3 mRNAs and proteins were expressed in human gonads. We also noted that NANOS3 was expressed in germ cells throughout spermatogenesis and oogenesis and thus, focused further efforts on this family member. NANOS3 expression was highest in human germ cell nuclei where the protein co-localized with chromosomal DNA during mitosis/meiosis. Reduced expression of NANOS3 (via morpholinos or short hairpin RNA) resulted in a reduction in germ cell numbers and decreased expression of germ cell-intrinsic genes required for the maintenance of pluripotency and meiotic initiation and progression. These data provide the first direct experimental evidence that NANOS3 functions in human germ cell development; indeed, NANOS3 is now one of just two genes that has been directly shown to function in germ cell development across diverse species from flies, worms, frogs and mice to humans [the other is BOULE, a member of the Deleted in Azoospermia (DAZ) gene family]. Findings may contribute to our understanding of the basic biology of human germ cell development and may provide clinical insights regarding infertility.

Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells

Historically, our understanding of molecular genetic aspects of human germ cell development has been limited, at least in part due to inaccessibility of early stages of human development to experimentation. However, the derivation of pluripotent stem cells may provide the necessary human genetic system to study germ cell development. In this study, we compared the potential of human induced pluripotent stem cells (iPSCs), derived from adult and fetal somatic cells to form primordial and meiotic germ cells, relative to human embryonic stem cells. We found that ∼5% of human iPSCs differentiated to primordial germ cells (PGCs) following induction with bone morphogenetic proteins. Furthermore, we observed that PGCs expressed green fluorescent protein from a germ cell-specific reporter and were enriched for the expression of endogenous germ cell-specific proteins and mRNAs. In response to the overexpression of intrinsic regulators, we also observed that iPSCs formed meiotic cells with extensive synaptonemal complexes and post-meiotic haploid cells with a similar pattern of ACROSIN staining as observed in human spermatids. These results indicate that human iPSCs derived from reprogramming of adult somatic cells can form germline cells. This system may provide a useful model for molecular genetic studies of human germline formation and pathology and a novel platform for clinical studies and potential therapeutical applications.

Germ cell differentiation in the marmoset (Callithrix jacchus) during fetal and neonatal life closely parallels that in the human

BACKGROUNDTesticular germ cell tumours (TGCT) are thought to originate from fetal germ cells that fail to differentiate normally, but no animal model for these events has been described. We evaluated the marmoset (Callithrix jacchus) as a model by comparing perinatal germ cell differentiation with that in humans.METHODSImmunohistochemical profiling was used to investigate germ cell differentiation (OCT4, NANOG, AP-2γ, MAGE-A4, VASA, NANOS-1) and proliferation (Ki67) in fetal and neonatal marmoset testes in comparison with the human and, to a lesser extent, the rat.RESULTSIn marmosets and humans, differentiation of gonocytes into spermatogonia is associated with the gradual loss of pluripotency markers such as OCT4 and NANOG, and the expression of germ cell-specific proteins such as VASA. This differentiation occurs asynchronously within individual cords during fetal and early postnatal life. This contrasts with rapid and synchronous germ cell differentiation within and between cords in the rat. Similarly, germ cell proliferation in the marmoset and human occurs throughout perinatal life, in contrast to rats in which proliferation ceases during this period.CONCLUSIONSThe marmoset provides a good model for normal human germ cell differentiation and proliferation. The perinatal marmoset may be a useful model in which to establish factors that lead to failure of normal germ cell differentiation and the origins of TGCT.

Bone Morphogenetic Proteins Induce Germ Cell Differentiation from Human Embryonic Stem Cells

The growth factors bone morphogenetic protein-4 (BMP4), BMP7, and BMP8b are required for specification of primordial germ cells (PGCs) in mice. Disruption of the genes that encode these factors leads to a severe reduction in number, or the complete absence, of PGCs. In addition, several studies have demonstrated that human BMP4 can promote PGC differentiation from mouse embryonic stem (ES) cells and in organ cultures. Here, we sought to determine whether recombinant human BMPs could induce differentiation of germ cells from human (h) ES cells. We found that addition of recombinant human BMP4 increased the expression of the germ cell-specific markers VASA and SYCP3 during differentiation of hES cells to embryoid bodies (EBs). In addition, BMP7 and BMP8b showed additive effects on germ cell induction when added together with BMP4. Finally, we observed that addition of BMPs to differentiating ES cells also increased the percentage of cells that stained positively for VASA. We note that the effects of recombinant BMPs were modest but reproducible and suggest that addition of BMPs to differentiation media increases differentiation of human germ cells from hES cells.