[Frontiers in Bioscience 15, 46-56, January 1, 2010]
In vitro derivation of germ cells from embryonic stem cells
Kinarm Ko, Karin Huebner, Jeanine Mueller-Keuker, Hans R. Schoeler
Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Muenster, 48149, Germany
TABLE OF CONTENTS
1. ABSTRACT
Embryonic stem cells (ESCs) have the potential to differentiate into cells of all tissue types, as demonstrated by their capability to contribute to the development of all germ layers in chimeras and to form teratomas. Of interest, however, is the lack of evidence of gametogenic gonadal tissues within teratomas, such as testicular tubules or follicles. Although this may reflect the fact that in vivo germ cell development and subsequent gametogenesis are complex biological processes, the generation of putative primordial germ cells (PGCs) has been reported during the differentiation of mouse and human ESCs in in vitro systems. In addition, mouse ESCs can even lead to the development of putative gametes, but the efficiency of this process needs to be improved and the functionality of the gametes remains to be proven. Interest in the generation of fully functional gametes from ESCs lies in an experimental model fundamental to the study of reproductive biology, a potential unlimited source of oocytes as a reprogramming bioreactor, and perhaps even in therapeutic applications to overcome human reproductive problems.
2. IN VIVO DEVELOPMENT OF GERM CELLS
Germ cells are a unique population of cells in mammals, transferring their genetic information from one generation to the next, and generating a fully functional organism by processes induced by their fertilization (1, 2). The production of gametes, - i.e., germ cells in their most differentiated state - is therefore one of the most important biological events in the propagation of a species. The earliest biological event in germ cell development is the formation of PGCs - the embryonic precursors of gametes - which begins around the time of gastrulation (1). In the mouse PGCs remain in a rather undifferentiated stage until the beginning of sexual differentiation, at 12.5 day post coitum (dpc). PGCs differentiate into prospermatogonia and oogonia at a late stage during the development of male and female mammalian fetuses, respectively. After birth, prospermatogonia further differentiate into spermatogonial stem cells (SSCs) (3), which are capable of undergoing self-renewal and serve as a reservoir of sperm precursor cells. Female germ cell differentiation, on the other hand, is characterized by the development of oogonia into primary oocytes prior to birth, arrest of meiotic progression until puberty, and maturation of secondary oocytes into mature oocytes thereafter (4).
2.1. Development of primordial germ cells
The development of PGCs in vivo can be divided into three distinct biological processes: specification of PGCs in the proximal epiblast, proliferation of PGCs, and migration of PGCs into the fetal genital ridge (5). Each of these processes involves region- and stage-specific signaling pathways mediated either by the interaction between PGCs and somatic cells, or between PGCs themselves.
The specification of PGCs is regulated by complex molecular mechanisms in which multiple molecular signals occur between PGC precursor cells and neighboring somatic cells, as well as between the precursor cells themselves. Regional specification of all germ layers, including the germline in the epiblast, occurs shortly before the beginning of gastrulation. The proximal region of the epiblast, which responds to signals from extraembryonic ectoderm, contains a population of cells committed to develop into PGCs (6). Early events in the specification of PGCs are regulated by signaling molecules and transcription factors. In the mouse, at about 5.5 dpc, bone morphogenetic protein (BMP) 4 and BMP8b, both members of the TGF beta superfamily, are released from extraembryonic ectoderm and exert their functions in the epiblast (7-9). In response to these signals, a subpopulation of cells in the proximal epiblast region at 6.0 dpc express mothers against decapentaplegic (SMAD) 1 and SMAD5 or simply SMADs, and are committed to develop into PGCs (10, 11). About six cells of the epiblast at 6.25 dpc that express fragilis/Ifitm3 in response to BMPs will subsequently express B-lymphocyte-induced maturation protein-1 (Blimp1)/Prdm1. These Blimp1-expressing cells will acquire germ cell competence and separate from the somatic cells (6). Blimp1 has been identified as a key signaling molecule involved in the inhibition of PGC differentiation into the mesodermal cell lineage, which expresses Hoxb1 among other mesodermal marker genes (12). Consistent signals in the proximal region of the epiblast are required for the maintenance of predetermined PGCs and for further recruitment of PGCs until 6.5 dpc. About 40 Stella-positive cells, which comprise the founder PGC population, settle at the posterior end of the primitive streak at 7.25 dpc (6, 12, 13). PGCs become motile at about 7.5 dpc, start migrating into the hindgut by 8.5 dpc, and localize therein at 9.0 dpc. At 9.5 dpc, they leave the hindgut and begin migrating through the dorsal mesentery (14-17). Migratory PGCs enter the developing genital ridges at about 10.5 dpc (16, 17) and complete their migration by 12.5 dpc.
2.2. Factors required for survival and proliferation of PGCs after specification
Subsequent to the specification of PGCs, germ cells need to maintain germ cell competence and proliferate without differentiating into somatic cell lineages. It has been reported that the POU domain transcription factor Oct4 is required for the maintenance of germ cell competence as well as for the survival of PGCs (14). During specification of mouse PGCs, expression of Oct4 is restricted to the founder PGC population at 7.25 dpc. In female fetal germ cells, Oct4 expression is maintained until sexual differentiation at 13.5 dpc and then starts to decline (18). In male fetal germ cells, Oct4 expression is maintained even after sexual differentiation (18). Later, Oct4 expression in the adult testis is restricted to SSCs (19). It is worth mentioning that Oct4 is a key factor not only for the pluripotency and self-renewal property of ESCs (20), but also for the survival of germ cells (14, 21). It is also interesting to note that ESCs and PGCs share common gene expression profiles; representative pluripotency-related genes, such as Oct4, Sox2, and Nanog, are all expressed in both ESCs and PGCs (22, 23). However, unlike ESCs, PGCs are unipotent, differentiating only as gametes. Still, stemness is a property common to both ESCs and PGCs.
In contrast to female gonads, male gonads contain germline stem cells (GSCs)- also referred to as SSCs - which persist after birth. These are postnatal male germ cell precursors that self-renew and undergo spermatogenesis to develop into male gametes (24). It should be mentioned that there are other known somatic stem cells that exhibit stemness. However, only SSCs express Oct4, one of the ESC-specific marker genes (25). Adult female germ cell derivatives also express Oct4, although they are not actively proliferating (14, 26). Analysis of oocyte growth has revealed the upregulation of Oct4 expression de novo during the later stages of prophase I of meiotic division in female germ cells (27). These data suggest that downregulation of Oct4 in male and female germ cells might trigger meiotic commitment (27). The upregulation of Oct4 expression in oocytes at the completion of meiotic prophase I further suggests a specific involvement of this transcription factor in oocyte growth and in the acquisition of meiotic competence (27).
Activation of and downstream signaling from the c-kit tyrosine kinase receptor are essential for the survival and proliferation of PGCs during PGC migration (28). Defects in either the c-kit receptor or its ligand, e.g. stem cell factor/steel factor (SCF), can cause depletion of germ cells (29). The importance of c-kit signaling in PGC survival is also observed in in vitro cultures (30). When PGCs are cultured in medium without SCF, most of the cells do not survive, which reflects the scenario in vivo. But when PGCs are cultured in the presence of SCF, survival is prolonged and the cells proliferate. c-Kit mediated signaling is also important for growth and differentiation of both male and female postnatal germ cells (31-33). A number of studies in the mouse model have demonstrated an important role of SCF in proliferation and in prevention of apoptosis of differentiated spermatogonia, as well as in either initiation or maintenance of meiosis (33-35). In the female, c-kit signaling between ovarian somatic cells and oocytes is associated with follicular growth and involved in the meiotic process (31).
2.3. Sexual differentiation of PGCs
In the mouse, PGCs proliferate during migration but stop dividing at about 13.5 dpc. Forty PGC founder cells give rise to approximately 25,000 cells in the fetal gonad at 13.5 dpc (1). PGCs are indistinguishable in both male and female gonads until 13.5 dpc. In male gonads, PGCs arrest in the G0/G1 stage of the mitotic cell cycle and develop into prospermatogonia (36), which differentiate to spermatogonia that, in turn, resume cell proliferation shortly after birth and begin the process of spermatogenesis. In female gonads, PGCs develop into oogonia that enter prophase I of meiosis, arrest in the diplotene stage at late gestation to complete meiosis after puberty and to develop into oocytes (4). It has been suggested that the different fate of PGCs in male and female mammalian development is regulated by retinoic acid (RA) (37, 38). In female fetal gonads, RA induces the expression of Stimulated by Retinoic Acid Gene 8 (Stra8), leading to the initiation of meiosis. In male fetal gonads, however, RA undergoes degradation by Cyp26b1, preventing PGCs from entering meiosis (37). It is interesting to note that expression of Stra8 is found in premeiotic spermatogenic cells in postnatal testes, during puberty, concomitant with a reduction in Cyb26b1 expression (39). Thus, RA may still function as a meiotic inducer molecule responsible for the initiation of the meiotic process, albeit at different time points during the sexual development of both sexes (18, 37, 39).
2.4. Generation of gametes from fetal and postnatal germ cells
The generation of gametes from fetal germ cells, either PGCs or sexually differentiated germ cells, has been reported by several studies in mouse. In fact, gametogenesis was observed when fetal gonads were transplanted underneath the kidney capsule or under the skin (40-42). These studies described the development of transplanted fetal male gonads into putative adult testicular tissue containing round spermatids within the testicular tubules, suggesting that fetal PGCs can differentiate into fertility-competent male germ cells. Upon transplantation of fetal male germ cells isolated from late-gestation embryos into testicular tubules of W/W mice, fully mature sperm could be obtained that were capable of fertilizing oocytes, subsequently giving rise to live offspring (43). Of interest is the fact that even PGCs at 10.5 dpc can still differentiate into fully mature sperm by testicular transplantation (43). Furthermore, SSCs, which are postnatal male germ cells, can develop into GSCs in in vitro culture (44, 45). These GSCs established in vitro have the capability to undergo self-renewal without differentiation and restore spermatogenesis upon transplantation into germ cell-depleted testis. One study demonstrated that competent oocytes could differentiate from fetal female germ cells by in vitro culture of 12.5-dpc fetal female gonads (46). Consistent with that study, our unpublished data showed that in vitro culture of fetal female gonads induced oogenesis, but would not bring about complete maturation of the oocytes. By contrast, a recent study reported the differentiation of fetal female germ cells into fully matured oocytes by in vivo culture of fetal gonads. Fully mature oocytes were obtained from 3-week in vivo cultured fetal gonads after transplantation underneath the kidney capsules (42, 47, 48). Fully mature oocytes were also obtained from primordial follicles by in vitro maturation culture (48), gaining developmental competence and giving rise to live pups subsequent to fertilization.
Overall, these results suggest that fetal or postnatal germ cells can differentiate into functional gametes by either in vitro or in vivo culture systems, suggesting that the derivation of PGCs or germ cells of later developmental stages from ESCs, such as SSCs in adult male gonads and primordial follicles in adult female gonads, may be the first step in the generation of gametes from ESCs.
3. IN VITRO DIFFERENTIATION OF ESCS INTO GERM CELLS
Several reports have recently demonstrated the generation of germ cells of different stages from ESCs. Depending on the in vitro differentiation system, these ESC-derived PGCs could further differentiate into gametes by either in vitro or in vivo culture. Furthermore, in vitro-derived postnatal germ cells, such as SSCs and primary follicular cells, could also successfully differentiate into male gametes and oocytes, respectively (49, 50).
3.1. Formation of germ cell precursors from ESCs
In vivo development of germ cells involves complex biological processes, which require specific environmental cues to support induction of germ cell precursors and maturation of the precursors into gametes. It is therefore remarkable that it is possible to obtain germ cells from cultured ESCs in an in vitro environment. Although it is still unclear whether such complex processes can take place in a Petri dish and whether they, in fact, mimic the in vivo scenario, it seems likely that distinct steps in germ cell development, required for the production of specific germ cell precursors, do take place. In vitro-derived germ cell precursors can be largely defined as several specific cells, these being PGCs, prospermatogonia, oogonia, and SSCs, depending on the timeline of germ cell development and sex determinants. PGCs are the earliest germ cell precursors in both male and female mammals. Several groups reported the formation of PGCs from ESCs in vitro (50-52). Our laboratory was the first to report the generation of putative PGCs of various stages in a monolayer ESC differentiation culture system. Identification of these cell populations was based on detection of expression of the germ cell-specific Oct4-GFP (gcOct4-GFP) transgene, along with that of stage-specific marker genes (c-kit and Vasa) (50). After 3 days of differentiation, we first detected early-stage PGCs (gcOct4-GFP+/c-kit+) and later, putative migratory PGCs (GFP+/c-kit+/Vasa-) and post-migratory PGCs (GFP-/c-kit+/Vasa+). Our results demonstrate the generation of PGCs from ESCs in a monolayer culture induced by a seemingly spontaneous differentiation process. We also observed that Vasa-positive cells detached from the monolayer and formed aggregates in the supernatant. These aggregates resembled late-stage oogonia, the precursors of oocytes, suggesting that these putative PGCs could further differentiate into presumptive female germ cell precursors.
Toyooka et al. used transgenic ESCs that contained the Lac-Z gene under the control of the Vasa promoter and reported the generation of Vasa-positive putative PGCs from embryoid bodies (EBs). These PGCs were capable of undergoing germ cell maturation (51). Formation of PGCs in this EB culture system appeared to be faster than in monolayer differentiation cultures. Vasa-positive putative PGCs could be detected after only one day of EB differentiation. The formation of Vasa-positive cells could be facilitated by coaggregation of EBs with BMP4-producing cells. While direct treatment of the EB differentiation culture with BMP4 appeared to have no effect, close contact with BMP4-producing cells appeared to have a positive effect on the formation of Vasa-positive cells. By contrast, a recent study using human ESCs showed that direct treatment of differentiating human EB cultures with BMP4 led to a dose-dependent, positive effect on Vasa expression (53). The addition of BMP4 also led to an increase in SYCP3 expression, although the effect was modest. This study clearly shows that PGC differentiation from human ESCs is promoted by the addition of BMP4 to EB cultures, in contrast to PGC differentiation from murine ESCs. This difference may be due to the fact that human ESCs are more closely related to mouse epiblast cells than to mouse ESCs, as it has recently been suggested that human ESCs indeed have properties in common with mouse epiblast-derived stem cells (54, 55). As described previously, PGC specification occurs in cells of the epiblast, which respond to BMP4 signaling. Nevertheless, both studies demonstrate the generation of putative PGCs from ESCs, a process promoted by BMP4. Utilizing a different approach, Geijsen et al. reported the detection of putative PGCs that express SSEA1 in differentiating EB cultures, and claimed that PGC formation in EBs was promoted by treatment with RA (52). The effect of RA on proliferation or survival of PGCs isolated from embryos has been demonstrated in several studies (56). It is therefore possible that putative PGCs, which may have spontaneously differentiated into EBs, proliferated in response to RA, rather than RA having triggered the formation of PGCs. To characterize the generated cell population, SSEA1-positive cells were isolated and expanded under culture conditions used to derive embryonic germ (EG) cells. DNA methylation analysis in imprinted genes revealed that the expanded cells had lost their methylation pattern, suggesting that EB-derived SSEA1-positive cells were indeed PGCs.
More recently, Nayernia et al. reported the derivation of male germ cell precursors of a postnatal stage from ESCs (49). In their study, RA was used for the initial differentiation of ESCs into Stra8-GFP-positive cells, which were further cultured in RA-free culture medium to establish putative SSCs. Our own findings revealed that in ESCs treated with RA, Stra8 expression was elevated within 24 hours when analyzed by reverse transcriptase PCR (unpublished data). In the aforementioned study, the initial elevation of Stra8 by continuous treatment with RA for 10 days may have been favorable for the differentiation of ESCs into SSCs (49). These so-called SSCs could then be maintained as cell lines. However, the imprinting patterns of these putative SSCs were different from the paternal imprinting patterns in SSCs derived from the adult testis: H19 is fully methylated, while the Igf2r gene is unmethylated by analysis of bisulfite sequencing (unpublished data). In fact, the imprinted genes in the ESC-derived SSCs did not show proper paternal imprinting patterns in the H19, Igf2r, and Snrpn loci before treatment of RA (49). Nonetheless, the putative SSCs showed gene expression profiles similar to in vivo SSCs. Of interest is the induction of meiosis by RA treatment, along with changes in imprinting patterns, as demonstrated by the somatically imprinted H19 gene and the paternally imprinted Igf2r and Snrpn genes (49).
3.2. Strategies for the derivation of gametes from ESC-derived germ cell precursors
In vivo germ cell precursors readily mature into functional gametes (42, 43, 47). Therefore, the generation and further enrichment of germ cell precursors in vitro can be critical and beneficial for the in vitro derivation of mature gametes. Several strategies have already been used to generate gametes from germ cell precursors of various stages.
In our monolayer differentiation system, aggregates containing Vasa-positive cells, which comprised putative oogonia, were further differentiated under maturation conditions (Figure 1A) (50). After 2 weeks in culture, follicle-like structures had formed, which expressed key genes for female estrogen biosynthesis. After 26 days in culture, oocyte-like cells were released from the follicle-like structures, which contained an outer layer resembling the zona pellucida. Occasionally, blastocyst-like structures developed from oocyte-like cells, possibly due to parthenogenetic activation. It has been reported that parthenogenesis could be induced from in vivo preantral follicle-derived oocytes (57). Our results provide indirect evidence for the functionality of oocyte-like cells derived from ESCs, for they have the capability to undergo spontaneous parthenogenesis.
To induce maturation of ESC-derived PGCs into male gametes, Toyooka et al. first obtained aggregates of these putative PGCs with male fetal gonadal somatic cells, and subsequently cultured them in vivo under the testicular capsule (Figure 1B) (51). Seminiferous tubules formed within these aggregates and produced sperm-like cells. Even though that study did not provide direct evidence for the functionality of the putative sperm, these results demonstrate that fetal gonads can be artificially generated and that ESC-derived PGCs and their wild-type somatic cell counterparts can induce spermatogenesis.
It has been demonstrated that maturation of ESC-derived PGCs can be induced solely by in vitro differentiation culture (Figure 1B) (52). Geijsen et al. reported the in vitro derivation of haploid male germ cells from ESCs. EBs at day 11 of differentiation were found to express genes associated with spermatogenesis and gonadal somatic cells. Putative haploid male germ cells could be isolated by staining EBs with a FE-J1 surface marker characteristic of male meiotic germ cells. The male germ cells were further tested for functionality by intracytoplasmic injection into oocytes. Although a significant number of injected oocytes developed into blastocysts, live offspring and, thus, ultimate proof of complete spermatogenesis in vitro, could not be obtained. Nayernia et al. reported remarkable results on the generation of functional male gametes from ESCs using two germ cell marker genes: Stra8 (premeiotic germ cell marker)-GFP and protamine 1 (Prm1: postmeiotic germ cell marker)-DsRed (49). Stra8-GFP was used to establish SSCs from an ESC monolayer culture in the presence of RA and Prm1-DsRed served as a marker for sperm after further maturation of the established SSCs, again, in the presence of RA. Treatment with RA appears to lead to different outcomes depending on the germ cell stage. RA is known to promote PGC survival; however, Nayernial et al. (49) described induction of meiosis by RA in SSCs resulting in the generation of male haploid gametes that successfully fertilized oocytes, which subsequently gave rise to live offspring. RA has recently been identified as an effector molecule in meiotic initiation in female fetal gonads and, possibly, in male adult gonads as well (37, 38, 58). The study by Nayernia et al. is the first to demonstrate the generation of functional gametes from ESCs. In addition, those outstanding results suggest that meiotic processes can be completed in SSCs isolated from the testis or in SSCs established in culture by treatment with RA.
3.3. Direct differentiation of gametes from ESCs
The culturing of EBs with conditioned medium prepared from male adult gonadal tissues has been shown to directly result in the generation of putative female gametes. Of interest is the formation of follicle-like cells at the borders of EBs observed after 6-7 days in co-culture (Figure 1B) (59). Signals from testicular tissues, which are similar to those in ovaries, may, in fact, facilitate the differentiation of ESCs into female rather than male gonadal cells (59). Growth factors found in the testes are also present in ovarian cells and during oogenesis. Since one-day differentiated EBs are known to contain putative PGCs, albeit a small percentage, it is possible that these preexisting germ cells readily respond to gametogenic cues in the conditioned medium (59). The establishment of gametogenic cues in regular differentiation cultures appears to require a relatively long time in culture (50, 52). Therefore, conditioned media appear to be more effective and faster than monolayer and EB culture systems described above at supporting the generation of gametes directly from ESCs. Other groups have also reported the beneficial effects of unknown factors from adult gonadal tissues. The co-culture of EBs with ovarian granulosa cells was found to effectively induce the formation of oocyte-like cells within 10 days in differentiation culture (Figure 1B) (60). The identification of key molecules from adult gonadal tissues is likely to improve the generation of germ cells from ESCs and their subsequent maturation.
3.4. In vitro microenvironment of ESC and germ cell cultures
Given the fact that in vivo germ cell development and subsequent germ cell maturation are complex biological processes that require precise cues from the germ cells themselves and from surrounding somatic cells, the achievement of in vitro germ cell differentiation from both, mouse and human ESCs seems remarkable (49-52, 61). It is still unclear how the somatic microenvironment and a range of germ cell determinants that support germ cell formation, such as certain growth factors and hormone stimulus, are created in ESC differentiation cultures. Two culture systems, monolayer and EB culture, are routinely used for germ cell differentiation, both of which are also commonly used for the generation of cell types of other lineages (49-52, 59, 60). The monolayer culture system provides a microenvironment suitable for the differentiation of both male and female germ cells. Our laboratory showed that female germ cells could be generated by the differentiation of mouse ESCs grown in monolayer (50). After 12 days in culture, we observed follicle-like structures. We also detected estradiol in the culture medium, which we assumed was released by the follicles. These findings are suggestive of a microenvironment comprising female germ cells and their supporting gonadal somatic cells. Nayernia et al. were able to obtain male germ cells from ESCs in a monolayer culture, which, in contrast to our system, depended on treatment with RA (49). Within 10 days of RA treatment, putative SSC precursors were generated from ESCs and were further expanded and established as SSC lines.
Several other groups have used EB culture systems to generate germ cell precursors or to directly produce gametes. Germ cell formation in an EB culture system appears to occur faster than in a monolayer culture system. PGCs can be detected as early as day 1 in differentiating EBs (51). Although a small number of PGCs differentiate spontaneously, BMPs and RA were found to facilitate PGC formation in EBs (51, 52). When EBs were cultured in conditioned medium prepared from adult gonadal cells or when they were co-cultured with adult gonadal cells, putative female gametes were found to form in the EBs (59, 60). These results suggest that the EB culture system also supports the generation of both male and female germ cells, depending on the stimulus to germ cell formation added to the system. Therefore, the crucial cues in directing ESC differentiation toward either the male or female germ cell lineages appear to rely on the determinants present in the culture systems. The microenvironment, created in both the monolayer and the EB culture systems, can support the formation of germ cell precursors that subsequently respond to additional determinants to further drive differentiation toward either the male or female gamete lineages.
3.5. Dedifferentiation of germ cells into ESC-like cells
It has been reported that germ cells can undergo spontaneous conversion into pluripotent cells. However, more than a decade has passed since it was first shown that mouse PGCs could be converted into embryonic germ (EG) cells, these being equivalent to ESCs with regard to pluripotency (Figure 2) (62, 63). More recently, ESC-like cells were also generated after the derivation of SSCs from neonatal or adult testicular cells (Figure 2) (64, 65). The dedifferentiation of unipotent cells into pluripotent, ESC-like cells is an interesting phenomenon in germ cell biology. A descriptive explanation for this conversion is that germ cells and ESCs both exhibit common gene profiles and have self-renewing capacities both in vivo and in vitro. The signaling pathways underlying these shared characteristics involved in the transformation of germ cells into ESC-like cells are still unknown.
Spontaneous cross-transformation between germ cells and ESCs has not been described to occur in any other somatic cells, unless specific factors that transform germ cells into ESC-like cells are artificially introduced (66). The fetal germ cell precursors PGCs can be converted into EG cells, which are pluripotent. Recent studies have reported that ESC-like cells could be obtained from either neonatal or adult testis (64, 65, 67). As no immediate evidence for direct dedifferentiation was provided, it is more likely that these pluripotent cells originated from SSCs residing in the testis. The latter hypothesis could be proven by demonstrating that established in vitro SSC lines can be directly transformed into pluripotent ESC-like cells.
4. ALTERNATIVE SOURCES FOR THE GENERATION OF GERM CELLS
Several recent studies have shown the potential of cells from alternative sources to be harnessed for the generation of putative germ cells (68-71). Dyce et al. demonstrated that stem cells isolated from porcine fetal skin were capable of differentiating into oocyte-like cells (68). When the skin stem cells were cultured in the presence of porcine follicular fluid, expression of germ cell-specific genes, such as Oct4, GDF9b, Dazl, and Vasa, was detected in a subpopulation of cultured cells, which later formed cell aggregates (68). These aggregates underwent further differentiation in oocyte-growth medium containing gonadotropin, forming oocyte-cumulus-like complexes and releasing putative oocyte-like cells. The results of this study suggest that adult skin stem cells, which may represent a suitable cell source for the generation of gametes, may have the capacity to develop into oocytes in vitro.
Recent studies have also described the use of other alternative sources for the generation of germ cells in vitro. Nayernia et al. demonstrated the ability of bone marrow stem (BMS) cells to differentiate into putative male germ cells (69, 70). The differentiation of mouse BMS cells into male germ cells was induced by RA treatment (69). About 3% of the BMS cells differentiated into male germ cells, which displayed gene expression profiles characteristic of germ cells. Some of the BMS cells that had been transplanted into seminiferous tubules of germ cell-depleted testes colonized the tubules and developed into spermatogenic cells. More recently, the same group demonstrated that human BMS cells were even capable of differentiating into male germ cells. Although direct evidence for the functionality of BMS cell-derived human male germ cells was not presented, expression of early germ cell markers, such as Oct4, Fragilis, Stella, and Vasa, as well as male germ cell markers, such as Dazl, TSPY, Piwil2, and Stra8, was clearly shown (70). While these are preliminary findings, they do offer some direction on the utility of BMS cells as an alternate source of cells for the generation of germ cells.
Another recent study has demonstrated that pancreatic stem cells (PSCs) derived from adult rat pancreas are capable of differentiating into putative oocyte-like cells (71). PSCs spontaneously formed three-dimensional aggregates in suspension culture, which then released oocyte-like cells. Of interest is the expression of oocyte marker genes, such as Oct4, SSEA-1, SCP3, DMC1, Vasa, CD9, and GDF9, exhibited by these oocyte-like cells. PSCs, like BMS cells, have a multipotent phenotype and can thus differentiate in vitro into various cell types. The alternative cell sources for the derivation of germ cells described above are of a multipotent phenotype. While pluripotent ESCs can contribute to the formation of germ cells in chimeras, multipotent cells cannot. The description of in vitro germ cell formation from multipotent cells can therefore possibly be attributed to a subpopulation of multipotent cells that have undergone spontaneous dedifferentiation in vitro toward the pluripotent stage, from which germ cells may arise.
5. CONCLUSION AND PERSPECTIVES
To improve the process of generating germ cells from ESCs, it is crucial to carefully and efficiently monitor the processes of in vitro germ cell formation and, subsequently, to isolate germ cells from these cultures. As in any other differentiation protocol, identifying a specific cell population and isolating the desired cell population is a challenge. At present, this is accomplished by morphological examination and analysis of germ cell markers. Germ cells of different stages can be detected by the use of different germ cell markers. In order to derive and identify germ cells from ESCs, the use of efficient determinants in cultures must be improved, and markers with greater specificity need to be developed.
The study of human development is difficult since early and critical steps in germ cell development occur after implantation, after which point manipulation of embryos is rendered impossible due to ethical considerations. The in vitro generation of germ cell precursors and mature gametes from ESCs can provide models to study germ cell development and to develop reproductive technologies. Recently, an intriguing study reported the generation of ESCs from primates by means of somatic cell nuclear transfer (SCNT) technology, demonstrating the potential of SCNT for human therapeutic applications (72). However, the large number of oocytes used in that study and the ethical considerations concerning the use of human oocytes make this methodology unsuitable for human applications. Artificial oocytes from ESCs may at least function as a "bioreactor" to reprogram somatic nuclei (Figure 2). This prospect holds tremendous potential impact on therapeutic cloning in the generation of patient-specific pluripotent cells to be applied in regenerative medicine. So far, one report using a mouse model demonstrated that male gametes derived from ESCs were functional in that they were capable of giving rise to live pups, thus showing the potential utility of ESCs in the generation of functional gametes (49). Although several groups, including our own, have shown that female gametes can be generated from mouse ESCs, the functionality of these female gametes has not yet been demonstrated. In addition, it remains to be determined whether such oocyte-like cells can be obtained from human ESCs and whether they will be capable of reprogramming a somatic nucleus. Therefore, the current protocols need to be improved in order to obtain functional oocytes, at least for the purpose of SCNT.
The importance of investigating the relationship between germ cells and ESCs is emphasized by recent reports demonstrating that adult germ cells can be utilized to generate pluripotent cells (65, 66). Distinct from other somatic cells, germ cells appear to have the unique property of undergoing transformation into pluripotent cells, either spontaneously or by parthenogenetic activation. This attribute suggests that there are unique signaling mechanisms that mediate the transformation of germ cells and ESCs into each other. Once these mechanisms are uncovered, and our understanding of basic germ cell properties is enhanced, we can fully explore the potential usage of germ cells in the generation of pluripotent cells, and work toward the technological advancements necessary to differentiate functional gametes from ESCs.
6. ACKNOWLEDGEMENTS
This work was supported by the Max Planck Society.
7. REFERENCES
1. A. McLaren: Primordial germ cells in the mouse. Dev Biol 262, 1-15 (2003)
doi:10.1016/S0012-1606(03)00214-8
http://dx.doi.org/10.1016/S0012-1606(03)00214-8
2. P. J. Donovan and M. P. de Miguel: Turning germ cells into stem cells. Curr Opin Genet Dev 13, 463-71 (2003)
doi:10.1016/j.gde.2003.08.010
http://dx.doi.org/10.1016/j.gde.2003.08.010
3. K. E. Orwig, B. Y. Ryu, M. R. Avarbock and R. L. Brinster: Male germ-line stem cell potential is predicted by morphology of cells in neonatal rat testes. Proc Natl Acad Sci U S A 99, 11706-11 (2002)
doi:10.1073/pnas.182412099
http://dx.doi.org/10.1073/pnas.182412099
4. M. E. Pepling: From primordial germ cell to primordial follicle: mammalian female germ cell development. Genesis 44, 622-32 (2006)
doi:10.1002/dvg.20258
http://dx.doi.org/10.1002/dvg.20258
5. L. L. Tres, C. Rosselot and A. L. Kierszenbaum: Primordial germ cells: what does it take to be alive? Mol Reprod Dev 68, 1-4 (2004)
doi:10.1002/mrd.20056
http://dx.doi.org/10.1002/mrd.20056
6. M. Saitou, S. C. Barton and M. A. Surani: A molecular programme for the specification of germ cell fate in mice. Nature 418, 293-300 (2002)
doi:10.1038/nature00927
http://dx.doi.org/10.1038/nature00927
7. K. A. Lawson, N. R. Dunn, B. A. Roelen, L. M. Zeinstra, A. M. Davis, C. V. Wright, J. P. Korving and B. L. Hogan: Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev 13, 424-36 (1999)
doi:10.1101/gad.13.4.424
http://dx.doi.org/10.1101/gad.13.4.424
8. Y. Ying, X. Qi and G. Q. Zhao: Induction of primordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signaling pathways. Proc Natl Acad Sci U S A 98, 7858-62 (2001)
doi:10.1073/pnas.151242798
http://dx.doi.org/10.1073/pnas.151242798
9. Y. Ying and G. Q. Zhao: Cooperation of endoderm-derived BMP2 and extraembryonic ectoderm-derived BMP4 in primordial germ cell generation in the mouse. Dev Biol 232, 484-92 (2001)
doi:10.1006/dbio.2001.0173
http://dx.doi.org/10.1006/dbio.2001.0173
10. C. H. Heldin, K. Miyazono and P. ten Dijke: TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 390, 465-71 (1997)
doi:10.1038/37284
http://dx.doi.org/10.1038/37284
11. J. Massague and D. Wotton: Transcriptional control by the TGF-beta/Smad signaling system. Embo J 19, 1745-54 (2000)
doi:10.1093/emboj/19.8.1745
http://dx.doi.org/10.1093/emboj/19.8.1745
12. Y. Ohinata, B. Payer, D. O'Carroll, K. Ancelin, Y. Ono, M. Sano, S. C. Barton, T. Obukhanych, M. Nussenzweig, A. Tarakhovsky, M. Saitou and M. A. Surani: Blimp1 is a critical determinant of the germ cell lineage in mice. Nature 436, 207-13 (2005)
doi:10.1038/nature03813
http://dx.doi.org/10.1038/nature03813
13. A. McLaren and K. A. Lawson: How is the mouse germ-cell lineage established? Differentiation 73, 435-7 (2005)
doi:10.1111/j.1432-0436.2005.00049.x
http://dx.doi.org/10.1111/j.1432-0436.2005.00049.x
14. J. Kehler, E. Tolkunova, B. Koschorz, M. Pesce, L. Gentile, M. Boiani, H. Lomeli, A. Nagy, K. J. McLaughlin, H. R. Scholer and A. Tomilin: Oct4 is required for primordial germ cell survival. EMBO Rep 5, 1078-83 (2004)
doi:10.1038/sj.embor.7400279
http://dx.doi.org/10.1038/sj.embor.7400279
15. K. A. Molyneaux, J. Stallock, K. Schaible and C. Wylie: Time-lapse analysis of living mouse germ cell migration. Dev Biol 240, 488-98 (2001)
doi:10.1006/dbio.2001.0436
http://dx.doi.org/10.1006/dbio.2001.0436
16. J. M. Clark and E. M. Eddy: Fine structural observations on the origin and associations of primordial germ cells of the mouse. Dev Biol 47, 136-55 (1975)
doi:10.1016/0012-1606(75)90269-9
http://dx.doi.org/10.1016/0012-1606(75)90269-9
17. P. J. Donovan, D. Stott, L. A. Cairns, J. Heasman and C. C. Wylie: Migratory and postmigratory mouse primordial germ cells behave differently in culture. Cell 44, 831-8 (1986)
doi:10.1016/0092-8674(86)90005-X
http://dx.doi.org/10.1016/0092-8674(86)90005-X
18. D. B. Menke, J. Koubova and D. C. Page: Sexual differentiation of germ cells in XX mouse gonads occurs in an anterior-to-posterior wave. Dev Biol 262, 303-12 (2003)
doi:10.1016/S0012-1606(03)00391-9
http://dx.doi.org/10.1016/S0012-1606(03)00391-9
19. H. R. Scholer, S. Ruppert, N. Suzuki, K. Chowdhury and P. Gruss: New type of POU domain in germ line-specific protein Oct-4. Nature 344, 435-9 (1990)
doi:10.1038/344435a0
http://dx.doi.org/10.1038/344435a0
20. H. Niwa, J. Miyazaki and A. G. Smith: Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24, 372-6 (2000)
doi:10.1038/74199
http://dx.doi.org/10.1038/74199
21. C. T. Dann, A. L. Alvarado, L. A. Molyneux, B. S. Denard, D. L. Garbers and M. H. Porteus: Spermatogonial stem cell self-renewal requires OCT4, a factor downregulated during retinoic acid-induced differentiation. Stem Cells 26, 2928-37 (2008)
doi:10.1634/stemcells.2008-0134
http://dx.doi.org/10.1634/stemcells.2008-0134
22. P. Western, J. Maldonado-Saldivia, J. van den Bergen, P. Hajkova, M. Saitou, S. Barton and M. A. Surani: Analysis of Esg1 expression in pluripotent cells and the germline reveals similarities with Oct4 and Sox2 and differences between human pluripotent cell lines. Stem Cells 23, 1436-42 (2005)
doi:10.1634/stemcells.2005-0146
http://dx.doi.org/10.1634/stemcells.2005-0146
23. S. Yamaguchi, H. Kimura, M. Tada, N. Nakatsuji and T. Tada: Nanog expression in mouse germ cell development. Gene Expr Patterns 5, 639-46 (2005)
doi:10.1016/j.modgep.2005.03.001
http://dx.doi.org/10.1016/j.modgep.2005.03.001
24. D. G. de Rooij and L. D. Russell: All you wanted to know about spermatogonia but were afraid to ask. J Androl 21, 776-98 (2000)
25. J. M. Oatley, M. R. Avarbock, A. I. Telaranta, D. T. Fearon and R. L. Brinster: Identifying genes important for spermatogonial stem cell self-renewal and survival. Proc Natl Acad Sci U S A 103, 9524-9 (2006)
doi:10.1073/pnas.0603332103
http://dx.doi.org/10.1073/pnas.0603332103
26. A. Rajkovic, S. A. Pangas, D. Ballow, N. Suzumori and M. M. Matzuk: NOBOX deficiency disrupts early folliculogenesis and oocyte-specific gene expression. Science 305, 1157-9 (2004)
doi:10.1126/science.1099755
http://dx.doi.org/10.1126/science.1099755
27. M. Pesce, X. Wang, D. J. Wolgemuth and H. Scholer: Differential expression of the Oct-4 transcription factor during mouse germ cell differentiation. Mech Dev 71, 89-98 (1998)
doi:10.1016/S0925-4773(98)00002-1
http://dx.doi.org/10.1016/S0925-4773(98)00002-1
28. M. P. De Miguel, L. Cheng, E. C. Holland, M. J. Federspiel and P. J. Donovan: Dissection of the c-Kit signaling pathway in mouse primordial germ cells by retroviral-mediated gene transfer. Proc Natl Acad Sci U S A 99, 10458-63 (2002)
doi:10.1073/pnas.122249399
http://dx.doi.org/10.1073/pnas.122249399
29. C. Runyan, K. Schaible, K. Molyneaux, Z. Wang, L. Levin and C. Wylie: Steel factor controls midline cell death of primordial germ cells and is essential for their normal proliferation and migration. Development 133, 4861-9 (2006)
doi:10.1242/dev.02688
http://dx.doi.org/10.1242/dev.02688
30. M. Pesce, M. G. Farrace, M. Piacentini, S. Dolci and M. De Felici: Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis). Development 118, 1089-94 (1993)
31. B. Motro and A. Bernstein: Dynamic changes in ovarian c-kit and Steel expression during the estrous reproductive cycle. Dev Dyn 197, 69-79 (1993)
32. K. J. Hutt, E. A. McLaughlin and M. K. Holland: KIT/KIT ligand in mammalian oogenesis and folliculogenesis: roles in rabbit and murine ovarian follicle activation and oocyte growth. Biol Reprod 75, 421-33 (2006)
doi:10.1095/biolreprod.106.051516
http://dx.doi.org/10.1095/biolreprod.106.051516
33. K. Yoshinaga, S. Nishikawa, M. Ogawa, S. Hayashi, T. Kunisada and T. Fujimoto: Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function. Development 113, 689-99 (1991)
34. M. A. Bedell and A. Mahakali Zama: Genetic analysis of Kit ligand functions during mouse spermatogenesis. J Androl 25, 188-99 (2004)
35. B. H. Schrans-Stassen, H. J. van de Kant, D. G. de Rooij and A. M. van Pelt: Differential expression of c-kit in mouse undifferentiated and differentiating type A spermatogonia. Endocrinology 140, 5894-900 (1999)
doi:10.1210/en.140.12.5894
http://dx.doi.org/10.1210/en.140.12.5894
36. K. Ohbo, S. Yoshida, M. Ohmura, O. Ohneda, T. Ogawa, H. Tsuchiya, T. Kuwana, J. Kehler, K. Abe, H. R. Scholer and T. Suda: Identification and characterization of stem cells in prepubertal spermatogenesis in mice small star, filled. Dev Biol 258, 209-25 (2003)
doi:10.1016/S0012-1606(03)00111-8
http://dx.doi.org/10.1016/S0012-1606(03)00111-8
37. J. Koubova, D. B. Menke, Q. Zhou, B. Capel, M. D. Griswold and D. C. Page: Retinoic acid regulates sex-specific timing of meiotic initiation in mice. Proc Natl Acad Sci U S A 103, 2474-9 (2006)
doi:10.1073/pnas.0510813103
http://dx.doi.org/10.1073/pnas.0510813103
38. J. Bowles and P. Koopman: Retinoic acid, meiosis and germ cell fate in mammals. Development 134, 3401-11 (2007)
doi:10.1242/dev.001107
http://dx.doi.org/10.1242/dev.001107
39. D. H. Volle, R. Duggavathi, B. C. Magnier, S. M. Houten, C. L. Cummins, J. M. Lobaccaro, G. Verhoeven, K. Schoonjans and J. Auwerx: The small heterodimer partner is a gonadal gatekeeper of sexual maturation in male mice. Genes Dev 21, 303-15 (2007)
doi:10.1101/gad.409307
http://dx.doi.org/10.1101/gad.409307
40. H. Ohta and T. Wakayama: Full-term development of offspring using round spermatids produced ectopically from fetal male germ cells. J Reprod Dev 50, 429-37 (2004)
doi:10.1262/jrd.50.429
http://dx.doi.org/10.1262/jrd.50.429
41. S. Schlatt, A. Honaramooz, M. Boiani, H. R. Scholer and I. Dobrinski: Progeny from sperm obtained after ectopic grafting of neonatal mouse testes. Biol Reprod 68, 2331-5 (2003)
doi:10.1095/biolreprod.102.014894
http://dx.doi.org/10.1095/biolreprod.102.014894
42. M. Snow, S. L. Cox, G. Jenkin, A. Trounson and J. Shaw: Generation of live young from xenografted mouse ovaries. Science 297, 2227 (2002)
doi:10.1126/science.1073693
http://dx.doi.org/10.1126/science.1073693
43. S. Chuma, M. Kanatsu-Shinohara, K. Inoue, N. Ogonuki, H. Miki, S. Toyokuni, M. Hosokawa, N. Nakatsuji, A. Ogura and T. Shinohara: Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis. Development 132, 117-22 (2005)
doi:10.1242/dev.01555
http://dx.doi.org/10.1242/dev.01555
44. M. Nagano, M. R. Avarbock, E. B. Leonida, C. J. Brinster and R. L. Brinster: Culture of mouse spermatogonial stem cells. Tissue Cell 30, 389-97 (1998)
doi:10.1016/S0040-8166(98)80053-0
http://dx.doi.org/10.1016/S0040-8166(98)80053-0
45. M. Kanatsu-Shinohara, N. Ogonuki, K. Inoue, H. Miki, A. Ogura, S. Toyokuni and T. Shinohara: Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 69, 612-6 (2003)
doi:10.1095/biolreprod.103.017012
http://dx.doi.org/10.1095/biolreprod.103.017012
46. Y. Obata, T. Kono and I. Hatada: Gene silencing: maturation of mouse fetal germ cells in vitro. Nature 418, 497 (2002)
doi:10.1038/418497a
http://dx.doi.org/10.1038/418497a
47. T. Qing, H. Liu, W. Wei, X. Ye, W. Shen, D. Zhang, Z. Song, W. Yang, M. Ding and H. Deng: Mature oocytes derived from purified mouse fetal germ cells. Hum Reprod 23, 54-61 (2007)
doi:10.1093/humrep/dem334
http://dx.doi.org/10.1093/humrep/dem334
48. J. Liu, J. Van der Elst, R. Van den Broecke and M. Dhont: Live offspring by in vitro fertilization of oocytes from cryopreserved primordial mouse follicles after sequential in vivo transplantation and in vitro maturation. Biol Reprod 64, 171-8 (2001)
doi:10.1095/biolreprod64.1.171
http://dx.doi.org/10.1095/biolreprod64.1.171
49. K. Nayernia, J. Nolte, H. W. Michelmann, J. H. Lee, K. Rathsack, N. Drusenheimer, A. Dev, G. Wulf, I. E. Ehrmann, D. J. Elliott, V. Okpanyi, U. Zechner, T. Haaf, A. Meinhardt and W. Engel: In vitro-differentiated embryonic stem cells give rise to male gametes that can generate offspring mice. Dev Cell 11, 125-32 (2006)
doi:10.1016/j.devcel.2006.05.010
http://dx.doi.org/10.1016/j.devcel.2006.05.010
50. K. Hubner, G. Fuhrmann, L. K. Christenson, J. Kehler, R. Reinbold, R. De La Fuente, J. Wood, J. F. Strauss, 3rd, M. Boiani and H. R. Scholer: Derivation of oocytes from mouse embryonic stem cells. Science 300, 1251-6 (2003)
doi:10.1126/science.1083452
http://dx.doi.org/10.1126/science.1083452
51. Y. Toyooka, N. Tsunekawa, R. Akasu and T. Noce: Embryonic stem cells can form germ cells in vitro. Proc Natl Acad Sci U S A 100, 11457-62 (2003)
doi:10.1073/pnas.1932826100
http://dx.doi.org/10.1073/pnas.1932826100
52. N. Geijsen, M. Horoschak, K. Kim, J. Gribnau, K. Eggan and G. Q. Daley: Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427, 148-54 (2004)
doi:10.1038/nature02247
http://dx.doi.org/10.1038/nature02247
53. K. Kee, J. M. Gonsalves, A. T. Clark and R. A. Pera: Bone morphogenetic proteins induce germ cell differentiation from human embryonic stem cells. Stem Cells Dev 15, 831-7 (2006)
doi:10.1089/scd.2006.15.831
http://dx.doi.org/10.1089/scd.2006.15.831
54. I. G. Brons, L. E. Smithers, M. W. Trotter, P. Rugg-Gunn, B. Sun, S. M. Chuva de Sousa Lopes, S. K. Howlett, A. Clarkson, L. Ahrlund-Richter, R. A. Pedersen and L. Vallier: Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191-5 (2007)
doi:10.1038/nature05950
http://dx.doi.org/10.1038/nature05950
55. P. J. Tesar, J. G. Chenoweth, F. A. Brook, T. J. Davies, E. P. Evans, D. L. Mack, R. L. Gardner and R. D. McKay: New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196-9 (2007)
doi:10.1038/nature05972
http://dx.doi.org/10.1038/nature05972
56. U. Koshimizu, M. Watanabe and N. Nakatsuji: Retinoic acid is a potent growth activator of mouse primordial germ cells in vitro. Dev Biol 168, 683-5 (1995)
doi:10.1006/dbio.1995.1113
http://dx.doi.org/10.1006/dbio.1995.1113
57. S. T. Lee, M. H. Choi, E. J. Lee, S. P. Gong, M. Jang, S. H. Park, H. Jee, D. Y. Kim, J. Y. Han and J. M. Lim: Establishment of autologous embryonic stem cells derived from preantral follicle culture and oocyte parthenogenesis. Fertil Steril 90, 1910-20 (2008)
doi:10.1016/j.fertnstert.2007.01.099
http://dx.doi.org/10.1016/j.fertnstert.2007.01.099
58. Q. Zhou, Y. Li, R. Nie, P. Friel, D. Mitchell, R. M. Evanoff, D. Pouchnik, B. Banasik, J. R. McCarrey, C. Small and M. D. Griswold: Expression of Stimulated by Retinoic Acid Gene 8 (Stra8) and Maturation of Murine Gonocytes and Spermatogonia Induced by Retinoic Acid In Vitro. Biol Reprod 79, 35-42 (2008)
doi:10.1095/biolreprod.107.066795
http://dx.doi.org/10.1095/biolreprod.107.066795
59. O. Lacham-Kaplan, H. Chy and A. Trounson: Testicular cell conditioned medium supports differentiation of embryonic stem (ES) cells into ovarian structures containing oocytes. Stem Cells 24, 266-73 (2006)
doi:10.1634/stemcells.2005-0204
http://dx.doi.org/10.1634/stemcells.2005-0204
60. T. Qing, Y. Shi, H. Qin, X. Ye, W. Wei, H. Liu, M. Ding and H. Deng: Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells. Differentiation 75, 902-11 (2007)
61. K. Tilgner, S. P. Atkinson, A. Golebiewska, M. Stojkovic, M. Lako and L. Armstrong: Isolation of primordial germ cells from differentiating human embryonic stem cells. Stem Cells 26, 3075-85 (2008)
doi:10.1634/stemcells.2008-0289
http://dx.doi.org/10.1634/stemcells.2008-0289
62. J. L. Resnick, L. S. Bixler, L. Cheng and P. J. Donovan: Long-term proliferation of mouse primordial germ cells in culture. Nature 359, 550-1 (1992)
doi:10.1038/359550a0
http://dx.doi.org/10.1038/359550a0
63. Y. Matsui, K. Zsebo and B. L. Hogan: Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70, 841-7 (1992)
doi:10.1016/0092-8674(92)90317-6
http://dx.doi.org/10.1016/0092-8674(92)90317-6
64. M. Kanatsu-Shinohara, K. Inoue, J. Lee, M. Yoshimoto, N. Ogonuki, H. Miki, S. Baba, T. Kato, Y. Kazuki, S. Toyokuni, M. Toyoshima, O. Niwa, M. Oshimura, T. Heike, T. Nakahata, F. Ishino, A. Ogura and T. Shinohara: Generation of pluripotent stem cells from neonatal mouse testis. Cell 119, 1001-12 (2004)
doi:10.1016/j.cell.2004.11.011
http://dx.doi.org/10.1016/j.cell.2004.11.011
65. K. Guan, K. Nayernia, L. S. Maier, S. Wagner, R. Dressel, J. H. Lee, J. Nolte, F. Wolf, M. Li, W. Engel and G. Hasenfuss: Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 440, 1199-203 (2006)
doi:10.1038/nature04697
http://dx.doi.org/10.1038/nature04697
66. K. Takahashi and S. Yamanaka: Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-76 (2006)
doi:10.1016/j.cell.2006.07.024
http://dx.doi.org/10.1016/j.cell.2006.07.024
67. M. Seandel, D. James, S. V. Shmelkov, I. Falciatori, J. Kim, S. Chavala, D. S. Scherr, F. Zhang, R. Torres, N. W. Gale, G. D. Yancopoulos, A. Murphy, D. M. Valenzuela, R. M. Hobbs, P. P. Pandolfi and S. Rafii: Generation of functional multipotent adult stem cells from GPR125+ germline progenitors. Nature 449, 346-50 (2007)
doi:10.1038/nature06129
http://dx.doi.org/10.1038/nature06129
68. P. W. Dyce, L. Wen and J. Li: In vitro germline potential of stem cells derived from fetal porcine skin. Nat Cell Biol 8, 384-90 (2006)
doi:10.1038/ncb1388
http://dx.doi.org/10.1038/ncb1388
69. K. Nayernia, J. H. Lee, N. Drusenheimer, J. Nolte, G. Wulf, R. Dressel, J. Gromoll and W. Engel: Derivation of male germ cells from bone marrow stem cells. Lab Invest 86, 654-63 (2006)
doi:10.1038/labinvest.3700429
http://dx.doi.org/10.1038/labinvest.3700429
70. N. Drusenheimer, G. Wulf, J. Nolte, J. H. Lee, A. Dev, R. Dressel, J. Gromoll, J. Schmidtke, W. Engel and K. Nayernia: Putative human male germ cells from bone marrow stem cells. Soc Reprod Fertil Suppl 63, 69-76 (2007)
71. S. Danner, J. Kajahn, C. Geismann, E. Klink and C. Kruse: Derivation of oocyte-like cells from a clonal pancreatic stem cell line. Mol Hum Reprod 13, 11-20 (2007)
doi:10.1093/molehr/gal096
http://dx.doi.org/10.1093/molehr/gal096
72. J. A. Byrne, D. A. Pedersen, L. L. Clepper, M. Nelson, W. G. Sanger, S. Gokhale, D. P. Wolf and S. M. Mitalipov: Producing primate embryonic stem cells by somatic cell nuclear transfer. Nature 450, 497-502 (2007)
doi:10.1038/nature06357
http://dx.doi.org/10.1038/nature06357
Abbreviations: ESCs: embryonic stem cells, PGCs: primordial germ cells, SSCs: spermatogonial stem cells, Blimp1: B-lymphocyte-induced maturation protein-1, BMP: bone morphogenetic protein, RA: retinoic acid, BMS: bone marrow stem, SCNT: somatic cell nuclear transfer
Key Words: Embryonic Stem Cells, Primordial Germ Cells, In Vitro Gametogenesis, Sperm, Oocytes, Review
Send correspondence to: Hans R. Schoeler, Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Muenster, 48149, Germany, Tel: 49-251-70365-300, Fax: 49-251-70365-399, E-mail:office@mpi-muenster.mpg.de