Culture Of Primordial Germ Cells Research Articles

First study on repeatable culture of primordial germ cells from various embryonic regions with giant feeder cells in Japanese quail (Coturnix japonica)

Japanese quail (JQ, Coturnix japonica) is a farmed animal with a high economic value and has been used extensively as an avian model for research. Germline chimera production based on cryopreserved primordial germ cells (PGCs) is possible for conservation management of quail breeds as successful isolation has been reported of PGCs from their blood and gonads. However, the repeatable cultivation protocol has not been elucidated yet, which has hindered technological development. The current study characterized cultivation of pregonadal PGCs isolated from embryonic parts; embryonic blood (cPGCs), whole embryonic tissues (tPGCs), parts of tail buds (tbPGCs), and a mixture of blood and tail bud tissues (ctbPGCs). The results showed that the cultivation system required the presence of specific embryonic cells to act as a feeder for JQ-PGCs and that such a system facilitated more successful cultivation, as shown by the percentages of isolation and cultivation in tbPGCs (100%, 100%, respectively), tPGCs (60%, 55%, respectively), and ctbPGCs (60%, 30%, respectively), but not in cPGCs (0%) cultured on a mitomycin-treated JQ feeder cell-line. Once the co-culture system had been established, the PGCs could be propagated for at least 5 months. These PGCs expressed germ cell-specific markers (DAZL and CVH) and could colonize embryonic gonads. Conclusively, the isolation of pregonadal PGCs and their long-term cultivation in vitro requires a unique embryonic cell, giant cell feeder, that is indispensable for the proliferation of PGCs. Characterization of cell signaling sustaining a mutual interaction between the PGCs and the specific feeder cells will elucidate a superior environment for in vitro cultivation, as well as support the minimal transfer of used xenobiotics in chimera production.

The constitutively active pSMAD2/3 relatively improves the proliferation of chicken primordial germ cells

AbstractIn many multicellular organisms, mature gametes originate from primordial germ cells (PGCs). Improvements in the culture of PGCs are important not only for developmental biology research, but also for preserving endangered species, and for genome editing and transgenic animal technologies. SMAD2/3 appear to be powerful regulators of gene expression; however, their potential positive impact on the regulation of PGC proliferation has not been taken into consideration. Here, the effect of TGF‐β signaling as the upstream activator of SMAD2/3 transcription factors was evaluated on chicken PGCs' proliferation. For this, chicken PGCs at stages 26−28 Hamburger−Hamilton were obtained from the embryonic gonadal regions and cultured on different feeders or feeder‐free substrates. The results showed that TGF‐β signaling agonists (IDE1 and Activin‐A) improved PGC proliferation to some extent while treatment with SB431542, the antagonist of TGF‐β, disrupted PGCs' proliferation. However, the transfection of PGCs with constitutively active SMAD2/3 (SMAD2/3CA) resulted in improved PGC proliferation for more than 5 weeks. The results confirmed the interactions between overexpressed SMAD2/3CA and pluripotency‐associated genes NANOG, OCT4, and SOX2. According to the results, the application of SMAD2/3CA could represent a step toward achieving an efficient expansion of avian PGCs.

In vitro culture of reptile PGCS to preserve endangered species.

Primordial germ cells (PGCs), are the source of gametes in vertebrates. There are similarities in the development of PGCs of reptiles with avian and mammalian species PGCs development. PGCs culture has been performed for avian and mammalian species but there is no report for reptilian PGCs culture. In vitro culture of PGCs is needed to produce transgenic animals, preservation of endangered animals and for studies on cell behaviour and research on fertility. Reptiles are traded as exotic pets and a source of food and they are valuable for their skin and they are useful as model for medical research. Transgenic reptile has been suggested to be useful for pet industry and medical research. In this research different aspects of PGCs development was compared in three main classes of vertebrates including mammalian, avian and reptilian species. It is proposed that a discussion on similarities between reptilian PGCs development with avian and mammalian species helps to find clues for studies of reptilian PGCs development details and finding an efficient protocol for in vitro culture of reptilian PG.

Genome editing in chickens

Chicken (Gallus gallus) is a major protein source and an important model organism in the avian species. Although genome editing has enabled genetic modifications of various organisms and has a significant impact on academia and industry, its application to chickens has been comparatively delayed owing to difficulties in handling their one-cell fertilized eggs. Thus, researchers have attempted to produce genome-edited chickens using primordial germ cells (PGCs), which are precursor cells of sperm or eggs. Currently, genome-edited chickens can be produced with the development of avian biotechnologies, PGCs culture methods, and germline chimerism systems, in particular. In this review, we describe the current status of genome editing in chickens, including avian biotechnologies, with a primary focus on the achievements of Japanese researchers. In addition, we discuss the remaining issues and make suggestions for future research.

Selection of culture media for chicken PGC long‐term cultivation

Primordial germ cells (PGCs) are precursors of eggs and spermatozoa and serve as an effective means for the transgenic chickens production, including using CRISPR / Cas9 technology. The aim study is selection of PGCs culture media suitable for long‐term cultivation. All experiments on chickens were conducted with ethical approval of the Russian Research Institute of Farm Animal Genetics and Breeding ‐ Branch of the L.K. Ernst Federal Research Center for Animal Husbandry (Protocol Number: 2020‐3). For the work, eggs of the Pushkin breed chickens (n = 21) were selected from the Bioresource collection RRIFAGB (Pushkin, St. Petersburg). PGCs were selected from the embryos dorsal aorta for 3‐4 days of incubation (stage Hamburger‐Hamilton 14) using a microinjector (Narishige IM‐11‐2, Japan). Cells were cultured for 21 days at 37 ° C with 5% CO2. Three variants of the culture medium were used. The first basic medium was Opti‐MEM (Reduced Serum Medium, GlutaMAX Supplement) (Gibco, Thermo Fisher) with the addition of sodium pyruvate 1M, nucleosides 100X ‐ 2.5 ml, Chicken Serum (Gibco, Thermo Fisher) ‐ 2%, 2‐mercaptoethanol (NF, VWR) ‐ 3.9 μl. On day 21, the concentration of cells in the first culture reached 1,2х103, and some of the wells with cells were overgrown. The second base medium was similar to the first base medium, but with the addition of antimycotic antibiotic (Thermo Fisher) up to 1X. The medium with the use of the antibiotic‐antimycotic was less susceptible to overgrowth, on day 21, the concentration of cells in the culture reached 3.5x104. The third basic medium was KnockOut DMEM / F‐12, without L‐glutamine (Gibco, ThermoFisher) with the addition sodium pyruvate 1M, nucleosides 100X ‐ 2.5 ml, Human Activin A Recombinant Protein (Gibco, Thermo Fisher) ‐ 25 ng / μl, Human FGF‐basic (FGF‐2 / bFGF) Recombinant Protein (Gibco, Thermo Fisher) ‐ 10 ng / μl, Chicken Serum (Gibco, Thermo Fisher) ‐ 2%, 2‐mercaptoethanol (NF, VWR) ‐ 3.9 μl), antimycotic antibiotic (Thermo Fisher) up to 1X. The third medium with the addition of Activin A and FGF‐2 / bFGF supported the cell culture better, overgrowth was practically not observed, cells were actively proliferating, оn day 21, the concentration of cells in the culture reached 2.7x105. The PGCs presence was confirmed by expression high level (p≤0.01) specific marker genes PGCs such as CXCR4 (chemokine receptor CXC type 4) and PIWIL1 (Piwi‐like protein 1) in cultured cells. In a control tissue small intestine sample of an adult chicken (330 days old), the above genes expression was completely absent.

Effect of Glutamine Stability on the Long-term Culture and Line Establishment of Chicken Primordial Germ Cells

Primordial germ cells (PGCs) are precursors for mature gametes, which transmit genetic information to the next generation. Due to the importance of PGCs in many fields including developmental biology, genome editing, transgenesis, and conservation of avian genetic resources, a lot of research has focused on the cultivation of PGCs. Despite considerable progress in the establishment of specified culture media for the expansion of PGCs, a well-defined PGC culture medium has not yet been developed. This might be due to the complexity of the nutritional requirements of PGCs in the culture. Besides the nutritional needs including vitamins, amino acids, salts, carbohydrates, and growth factors, a certain source of energy must be provided to sustain growth and viability. Glutamine is a major energy source for cultured cells, which is commonly added in cell culture media at higher concentrations than other amino acids. However, glutamine is very labile and rapidly degrades in solutions such as culture media. This generates ammonia as a by-product, which is toxic to the cultured cells and can affect cell viability and protein glycosylation. Therefore, the stability of glutamine in culture conditions is another concern for the long-termed culture of PGCs. Here, we study the effect of glutamine stability on PGC culture using glutamine and GlutaMax (a commercial stabilized dipeptide form of glutamine). We found that the addition of GlutaMax in the medium promotes PGC proliferation. This effect might be exerted by minimizing toxic ammonia buildup that results in maximizing cell performance, and also increasing media stability.

Mouse Primordial Germ Cells: In Vitro Culture and Conversion to Pluripotent Stem Cell Lines.

Primordial germ cells (PGCs) are the embryonic precursors of the gametes. Despite decades of research, in vitro culture of PGCs remains a major challenge and has previously relied on undefined components such as serum and feeders. Notably, PGCs cultured for extended periods do not maintain their lineage identity but instead undergo conversion to form pluripotent stem cell lines called embryonic germ (EG) cells in response to LIF/STAT3 signaling. Here we report both established and new methodologies to derive EG cells, in a range of different conditions. We show that basic fibroblast growth factor is not required for EG cell conversion. We detail the steps taken in our laboratory to systematically remove complex components and establish a fully defined protocol that allows efficient conversion of isolated PGCs to pluripotent EG cells. In addition, we demonstrate that PGCs can adhere and proliferate in culture without the support of feeder cells or serum. This may well suggest novel approaches to establishing short-term culture of PGCs in defined conditions.

Germline engineering of the chicken genome using CRISPR/Cas9 by in vivo transfection of PGCs

Development of simple and readily adoptable methods to mediate germline engineering of the chicken genome will have many applications in research, agriculture and industrial biotechnology. We report germline targeting of the endogenous chicken Interferon Alpha and Beta Receptor Subunit 1 (IFNAR1) gene by in vivo transgenic expression of the high-fidelity Cas9 (Cas9-HF1) and guide RNAs (gRNAs) in chickens. First, we developed a Tol2 transposon vector carrying Cas9-HF1, IFNAR1-gRNAs (IF-gRNAs) and green fluorescent protein (GFP) transgenes (pTgRCG) and validated in chicken fibroblast DF1 cells. Next, the pTgRCG plasmid was directly injected into the dorsal aorta of embryonic day (ED) 2.5 chicken embryos targeting the circulating primordial germ cells (PGCs). The resulting chimera roosters generated a fully transgenic generation 1 (G1) hen with constitutive expression of Cas9-HF1 and IF-gRNAs (G1_Tol2-Cas9/IF-gRNA). We detected a spectrum of indels at gRNA-targeted loci in the G1_Tol2-Cas9/IF-gRNA hen and the indels were stably inherited by the G2 progeny. Breeding of the G1_Tol2-Cas9/IF-gRNA hen resulted in up to 10% transgene-free heterozygote IFNAR1 mutants, following null-segregation of the Tol2 insert. The method described here will provide new opportunities for genome editing in chicken and other avian species that lack PGC culture.

Expression and possible roles of extracellular signal-related kinases 1-2 (ERK1-2) in mouse primordial germ cell development.

In the present work, we described the expression and activity of extracellular signal-related kinases 1-2 (ERK1-2) in mouse primordial germ cells (PGCs) from 8.5–14.5 days post coitum (dpc) and investigated whether these kinases play a role in regulating the various processes of PGC development. Using immunofluorescence and immunoblotting to detect the active phosphorylated form of ERK1-2 (p-ERK1-2), we found that the kinases were present in most proliferating 8.5–10.5 dpc PGCs, low in 11.5 dpc PGCs, and progressively increasing between 12.5–14.5 dpc both in female and male PGCs. In vitro culture experiments showed that inhibiting activation of ERK1-2 with the MEK-specific inhibitor U0126 significantly reduced the growth of 8.5 dpc PGCs in culture but had little effect on 11.5–12.5 dpc PGCs. Moreover, we found that the inhibitor did not affect the adhesion of 11.5 dpc PGCs, but it significantly reduced their motility features onto a cell monolayer. Further, while the ability of female PGCs to begin meiosis was not significantly affected by U0126, their progression through meiotic prophase I was slowed down. Notably, the activity of ERK1-2 was necessary for maintaining the correct expression of oocyte-specific genes crucial for germ cells survival and the formation of primordial follicles.

Reviving rare chicken breeds using genetically engineered sterility in surrogate host birds

In macrolecithal species, cryopreservation of the oocyte and zygote is not possible due to the large size and quantity of lipid deposited within the egg. For birds, this signifies that cryopreserving and regenerating a species from frozen cellular material are currently technically unfeasible. Diploid primordial germ cells (PGCs) are a potential means to freeze down the entire genome and reconstitute an avian species from frozen material. Here, we examine the use of genetically engineered (GE) sterile female layer chicken as surrogate hosts for the transplantation of cryopreserved avian PGCs from rare heritage breeds of chicken. We first amplified PGC numbers in culture before cryopreservation and subsequent transplantation into host GE embryos. We found that all hatched offspring from the chimera GE hens were derived from the donor rare heritage breed broiler PGCs, and using cryopreserved semen, we were able to produce pure offspring. Measurement of the mutation rate of PGCs in culture revealed that 2.7 × 10-10 de novo single-nucleotide variants (SNVs) were generated per cell division, which is comparable with other stem cell lineages. We also found that endogenous avian leukosis virus (ALV) retroviral insertions were not mobilized during in vitro propagation. Taken together, these results show that mutation rates are no higher than normal stem cells, essential if we are to conserve avian breeds. Thus, GE sterile avian surrogate hosts provide a viable platform to conserve and regenerate avian species using cryopreserved PGCs.

Chicken Mesenchymal Stem Cells as Feeder Cells Facilitate the Cultivation of Primordial Germ Cells from Circulating Blood and Gonadal Ridge

Long-term maintenance of chicken primordial germ cells (PGCs) in vitro has tremendous potential for transgenic chicken production. Feeder cells are essential for the establishment and culture of chicken PGCs in vitro. Buffalo rat liver (BRL) cells are the most commonly used feeder cells for PGCs culture; however, this feeder layers from other animal species usually cause immunogenic contaminations, compromising the potential of PGCs in applications. Therefore, we tested chicken source mensenchymal stem cell (MSCs) derived from bone marrow as feeder cells to further improve PGC culture conditions. MSCs derived from chicken bone marrow have a powerful capacity to proliferate and secrete cytokines. We found chicken primordial germ cells derived from circulating blood (cPGCs) and gonads (gPGCs) can be maintained and proliferated with MSCs feeder layer cells. PGCs co-cultured on MSCs feeder retained their pluripotency, expressed PGCs specific genes and stemness markers, and maintained undifferentiated state. Our study indicated that the xeno-free MSCs-feeders culture system is a good candidate for growth and expansion of PGCs as the stepping stone for transgenic chicken research.

Production of Transgenic Chickens Using Cultured Primordial Germ Cells and Gonocytes.

The unique characteristics of the avian embryo, with its large opaque yolk, have necessitated the development of different approaches to transgenesis from those that have been successful in mammalian species. Genetic modification of birds was greatly advanced by the ability to grow long-term cultures of primordial germ cells (PGCs). These cells are obtained from embryos, established in culture, and can be propagated without losing the ability to contribute to the germline when reintroduced into a host animal. PGCs can be genetically modified in culture using traditional transfection and selection techniques, including gene targeting and site-specific nuclease approaches. Here, we describe our methods for deriving cell lines, long-term culture, genetic modification, production of germline chimeras and obtaining fully transgenic birds with the desired genetic modifications.

Long-term in vitro culture and preliminary establishment of chicken primordial germ cell lines.

Primordial germ cells (PGCs) are precursors of functional gametes and can be used as efficient transgenic tools and carriers in bioreactors. Few methods for long-term culture of PGCs are available. In this study, we tested various culture conditions for PGCs, and used the optimum culture system to culture chicken gonad PGCs for about three hundred days. Long-term-cultured PGCs were detected and characterized by karyotype analysis, immunocytochemical staining of SSEA-1, c-kit, Sox2, cDAZL, and quantitative RT-PCR for specific genes like Tert, DAZL, POUV, and NANOG. Cultured PGCs labeled with PKH26 were reinjected into Stage X recipient embryos and into the dorsal aorta of Stage 14–17 embryos to assay their ability of migration into the germinal crescent and gonads, respectively. In conclusion, the most suitable culture system for PGCs is as follows: feeder layer cells treated with 20 μg/mL mitomycin C for 2 hours, and with 50% conditioned medium added to the factor culture medium. PGCs cultured in this system retain their pluripotency and the unique ability of migration without transformation, indicating the successful preliminary establishment of chicken primordial germ cell lines and these PGCs can be considered for use as carriers in transgenic bioreactors.

Distinct requirements for energy metabolism in mouse primordial germ cells and their reprogramming to embryonic germ cells

Primordial germ cells (PGCs), undifferentiated embryonic germ cells, are the only cells that have the ability to become gametes and to reacquire totipotency upon fertilization. It is generally understood that the development of PGCs proceeds through the expression of germ cell-specific transcription factors and characteristic epigenomic changes. However, little is known about the properties of PGCs at the metabolite and protein levels, which are directly responsible for the control of cell function. Here, we report the distinct energy metabolism of PGCs compared with that of embryonic stem cells. Specifically, we observed remarkably enhanced oxidative phosphorylation (OXPHOS) and decreased glycolysis in embryonic day 13.5 (E13.5) PGCs, a pattern that was gradually established during PGC differentiation. We also demonstrate that glycolysis and OXPHOS are important for the control of PGC reprogramming and specification of pluripotent stem cells (PSCs) into PGCs in culture. Our findings about the unique metabolic property of PGCs provide insights into our understanding of the importance of distinct facets of energy metabolism for switching PGC and PSC status.

Differentiation of Mouse Primordial Germ Cells into Functional Oocytes In Vitro

Various complex molecular events in oogenesis cannot be observed in vivo. As a bioengineering technique for female reproductive tissues, in vitro culture systems for female germ cells have been used to analyze oogenesis and preserve germ cells for over 20 years. Recently, we have established a new methodological approach for the culture of primordial germ cells (PGCs) and successfully obtained offspring. Our PGC culture system will be useful to clarify unresolved mechanisms of fertility and sterility from the beginning of mammalian oogenesis, before meiosis. This review summarizes the history of culture methods for mammalian germ cells, our current in vitro system, and future prospects for the culture of germ cells.

Dose-dependent functions of fibroblast growth factor 9 regulate the fate of murine XY primordial germ cells.

Male differentiation of primordial germ cells (PGCs) is initiated by the inhibition of entry into meiosis and exposure to male-inducing factor(s), which are regulated by somatic elements of the developing gonad. Fibroblast growth factor 9 (FGF9) produced by pre-Sertoli cells is essential for male gonadal differentiation and also contributes to survival and male differentiation of XY PGCs. However, it is not clear how FGF9 regulates PGC fate. Using a PGC culture system, we identified dose-dependent, fate-determining functions of FGF9 in XY PGCs. Treatment with low levels of FGF9 (0.2 ng/ml) increased expression of male-specific Dnmt3L and Nanos2 in XY PGCs. Conversely, treatment with high levels of FGF9 (25 ng/ml) suppressed male-specific gene expression and stimulated proliferation of XY PGCs. Western blotting showed that low FGF9 treatment enhanced p38 MAPK (mitogen-activated protein kinase) phosphorylation in the same cells. In contrast, high FGF9 treatment significantly stimulated the ERK (extracellular signal-regulated kinase)1/2 signaling pathway in XY PGCs. We investigated the relationship between the ERK1/2 signaling pathway stimulated by high FGF9 and regulation of PGC proliferation. An ERK1/2 inhibitor (U0126) suppressed the PGC proliferation that would otherwise be stimulated by high FGF9 treatment, and increased Nanos2 expression in XY PGCs. Conversely, a p38 MAPK inhibitor (SB202190) significantly suppressed Nanos2 expression that would otherwise be stimulated by low FGF9 in XY PGCs. Taken together, our results suggest that stage-specific expression of FGF9 in XY gonads regulates the balance between proliferation and differentiation of XY PGCs in a dose-dependent manner.

Proliferation in culture of primordial germ cells derived from embryonic stem cell: induction by retinoic acid.

An in vitro system that supports primordial germ cells (PGCs) survival and proliferation is useful for enhancement of these cells and efficient transplantation in infertility disorders. One approach is cultivation of PGCs under proper conditions that allow self-renewal and proliferation of PGCs. For this purpose, we compared the effects of different concentrations of retinoic acid (RA), and the effect of PGCs co-culture (Co-C) with SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cells on the proliferation of embryonic stem cells (ESCs)-derived PGCs. One-day-old embryoid body (EB) was cultured for 4 days in simple culture system in the presence of 5 ng/ml bone morphogenetic protein-4 (BMP4) (SCB group) for PGC induction. For PGC enrichment, ESCs-derived germ cells were cultured for 7 days in the presence of different doses (0–5 μM) of RA, both in the simple and STO Co-C systems. At the end of the culture period, viability and proliferation rates were assessed and expression of mouse vasa homologue (Mvh), α6 integrin, β1 integrin, stimulated by retinoic acid 8 (Stra8) and piwi (Drosophila)-like 2 (Piwil2) was evaluated using quantitative PCR. Also, the inductive effects were investigated immunocytochemically with Mvh and cadherin1 (CDH1) on the selected groups. Immunocytochemistry/PCR results showed higher expression of Mvh, the PGC-specific marker, in 3 μM RA concentrations on the top of the STO feeder layer. Meanwhile, assessment of the Stra8 mRNA and CDH1 protein, the specific makers for spermatogonia, showed no significant differences between groups. Based on the results, it seems that in the presence of 3 μM RA on top of the STO feeder layer cells, the majority of the cells transdifferentiated into germ cells were PGCs.

Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE).

Generating transgenic and gene edited mammals involves in vitro manipulation of oocytes or single cell embryos. Due to the comparative inaccessibility of avian oocytes and single cell embryos, novel protocols have been developed to produce transgenic and gene edited birds. While these protocols are relatively efficient, they involve two generation intervals before reaching complete somatic and germline expressing transgenic or gene edited birds. Most of this work has been done with chickens, and many protocols require in vitro culturing of primordial germ cells (PGCs). However, for many other bird species no methodology for long term culture of PGCs exists. Developing methodologies to produce germline transgenic or gene edited birds in the first generation would save significant amounts of time and resource. Furthermore, developing protocols that can be readily adapted to a wide variety of avian species would open up new research opportunities. Here we report a method using sperm as a delivery mechanism for gene editing vectors which we call sperm transfection assisted gene editing (STAGE). We have successfully used this method to generate GFP knockout embryos and chickens, as well as generate embryos with mutations in the doublesex and mab-3 related transcription factor 1 (DMRT1) gene using the CRISPR/Cas9 system. The efficiency of the method varies from as low as 0% to as high as 26% with multiple factors such as CRISPR guide efficiency and mRNA stability likely impacting the outcome. This straightforward methodology could simplify gene editing in many bird species including those for which no methodology currently exists.

Improvement of Chicken Primordial Germ Cell Maintenance In Vitro by Blockade of the Aryl Hydrocarbon Receptor Endogenous Activity.

Primordial germ cells (PGCs) are the undifferentiated progenitors of gametes. Germline competent PGCs can be developed as a cell-based system for genetic modification in chickens, which provides a valuable tool for transgenic technology with both research and industrial applications. This implies manipulation of PGCs, which, in recent years, encouraged a lot of research focused on the study of PGCs and the way of improving their culture. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that besides mediating toxic responses to environmental contaminants plays pivotal physiological roles in various biological processes. Since a novel compound that acts as an antagonist of this receptor has been reported to promote expansion of hematopoietic stem cells, we conducted the present study with the aim of determining whether addition of an established AHR antagonist to the standard culture medium used nowadays for in vitro chicken PGCs culture improves ex vivo expansion. We have found that addition of α-naphthoflavone in culture medium promotes the amplification of undifferentiated cells and that this effect is exerted by the blockade of AHR action. Our results constitute the first report of the successful use of a readily available AHR antagonist to improve avian PGCs expansion, and they further extend the knowledge of the effects of AHR modulation in undifferentiated cells.

Human Primordial Germ Cell Specification--Breakthrough In Culture and Hopes for Therapeutic Utilization

Germ cells are the precursors to the gametes that carry genetic and epigenetic information between human generations and generate a new individual. Because germ cells are specified early during embryogenesis, at the time of embryo implantation, they are inaccessible for research. Our understanding of their biology has therefore developed slowly since their identification over one hundred years ago. As a result of research into the properties of human and mouse embryonic stem cells and primordial germ cells, scientists have now succeeded in efficiently generating human primordial germ cells in culture by embryonic stem cell and induced pluripotent stem cell culture. In this review we will discuss the state of our knowledge of human primordial germ cells and how research into the pluripotent properties of human and mouse embryonic germ cells has led to this breakthrough. In addition we will discuss the possible utilization of a cell culture system of human primordial germ cells for research into and treatment of germ cell related abnormalities.