2.1. In Vitro Derivation of PGC-Like Cells (PGCLCs) from Pluripotent Stem Cells
In 2009, Ohinata et al. established the basic conditions for directed PGC specification in mouse pluripotent epiblast cells [
44]. Pregastrula stage epiblasts were treated with BMP4, BMP8b, SCF, EGF, and LIF to induce the expression of Blimp1 and Prdm14. These epiblast-derived PGC-like cells (epiPGCs) expressing
Blimp1-mVenus and
Stella-ECFP showed genetic and epigenetic characteristics consistent with in vivo PGCs. When these epiPGCs were transplanted into neonatal testes, they developed into mature spermatozoa, which could fertilize oocytes and give rise to healthy offspring [
44]. This research showed the basis of signal interactions essential for PGC specification from pluripotent cells and proved that the epiblast is the direct precursor of PGCs. Ohinata’s study prompted researchers to use PSCs to derive PGC-like cells (PGCLCs) in vitro, since PSCs are the in vitro derivatives of the peri-implantation epiblast.
Initial studies on in vitro germ cell specification from PSCs have mainly focused on the derivation of PGCs through embryoid body (EB) formation from ESCs or EGCs [
1,
45,
46]. Culture in the absence of LIF and other growth factors associated with self-renewal [
1] and culture with BMP4 [
47] or retinoic acid [
48] could induce the differentiation of PSCs into PGC-like cells (PGCLCs). Toyooka et al. isolated PGCLCs expressing Mvh (DEAD box helicase 4) during PGC differentiation and cocultured them with BMP4-producing cells for 1 d, followed by aggregation with gonadal cells isolated from E12.5 to 15.5 mice [
46]. Transplantation of these aggregates into the testis capsule resulted in the formation of testicular tubules, wherein ESC-derived mature sperm were formed. Geijsen et al. also suggested that differentiation through EB formation resulted in meiotic male germ cells that could fertilize oocytes and develop the blastocyst stage [
48]. However, since these germ cell derivation protocols were random differentiation strategies using EB formation, the PGC specification was very heterogeneous and inefficient (approximately 0.5–3.6% per EB), raising the need for a directed induction method for PGC specification. To increase the PGC specification from PSCs, PSCs should be maintained in a homogenous population that is germline competent. Ying et al. suggested that germline competent naïve PSCs could be maintained under culturing with MAPK and GSK inhibitors (referred to as 2i) and LIF, called the ground state [
49]. The ground state of pluripotency is equivalent to that of the E4.5 epiblast of preimplantation blastocysts in gene expression patterns. Thus, the 2iL condition in mice enables the induction of germline competent ESCs/iPSCs from all mouse strains tested [
49,
50,
51].
Hayashi et al. first proposed the stepwise differentiation of mouse PSCs into PGCLCs in 2011 [
3]. They showed that mouse PSCs in ground state pluripotency were differentiated into epiblast-like cells (EpiLCs), an intermediate precursor for PGC formation, in the presence of activin A and bFGF for 2 days [
3]. After culturing under conditions similar to those in Ohinata’s experiment (derivation of epiblast to PGCLCs), EpiLCs were subsequently differentiated into PGCLCs [
51]. Global transcriptional profiles revealed that specification of PGCLCs from EpiLCs recapitulated the in vivo specification of epiblast to PGCs, suggesting that EpiLCs were germline competent precursors corresponding to pre-gastrulating epiblasts at around E5.75, and PGCLCs had properties similar to those of E9.5 PGCs [
3]. Transplanting these male or female PGCLCs into germ cell-deficient mice results in the formation of a gonad-like structure containing gamete-like cells. In males, PGCLCs were further differentiated in an in vivo environment, such as seminiferous tubules of W/Wv mice lacking endogenous sperm, in which PGCLCs could resume spermatogenesis [
3,
52]. PGCLC-derived sperm developed in host testes could give rise to fertile offspring after intracytoplasmic injection (ICSI) into wild-type oocytes and transplantation into surrogate mothers [
3]. Similar to the in vitro derivation of sperm, female ovary-like structures and oocyte-like cells could be derived from EpiLC-derived PGCLCs, which were differentiated from mouse ESCs and iPSCs [
4]. Female PGCLCs also develop into oocytes, contributing to fertile offspring after in vitro maturation and fertilization with normal sperm [
4]. These in vitro (EpiLC and PGCLC formation) and in vivo (transplantation of PGCLCs into host gonads) conjugated methods serve as bridges for the complete in vitro gametogenesis technology and provide important information for signaling cascades during germ cell fate transition.
Human PSCs can differentiate into human PGCLCs (hPGCLCs) spontaneously [
53,
54] or under defined culture conditions [
55,
56]. Gkountela et al. differentiated hESCs via serum-induced random differentiation in EBs for 9 days and sorted TRA-1-81
+/cKIT
+ hPGCLCs (0.3% per sorted living population), which also expressed
BLIMP1 and
OCT4 with or without
NANOS3 expression [
53]. Kee et al. used another germ cell marker, VASA-GFP reporter, for hPGCLC isolation from differentiating hESCs [
54]. VASA-GFP
+ cells expressed other early germ cell markers,
DAZL,
BLIMP1, and
STELLA. They also suggested that overexpression of BOULE alone or together with DAZL could enhance hPGCLC specification and meiotic progression, suggesting the importance of BOULE and DAZL for germ cell progression in humans [
54]. For hPGCLC enrichment, Yu et al. treated the EBs formed from hESCs and iPSCs with activin A, BMP4, and retinoic acid, by which PGCLCs expressing DDX4, folliculogenesis-related markers, and postnatal oocyte-specific markers were derived [
57]. Aggregates of PGCLCs with human granulosa cells gave rise to ovarian follicle-like structures in xenografted mice [
57].
Irie et al. suggested a defined culture condition for the specification of hPGCLCs from hESCs and hiPSCs. To efficiently derive hPGCLCs, they used distinct states of PSCs that were preconditioned in a medium containing four inhibitors (4i), including CHIR99021 (GSK-3 inhibitor), PD0325901 (MEK inhibitor), SB203580 (p38 MAPK inhibitor), and SP600125 (JNK inhibitor), sustaining cells in near ground state pluripotency and aiding in easy differentiation into germ cell lineage [
55]. To induce hPGCLCs, hPSCs in 4i medium were cultured in bFGF/TGFβ-containing medium for 2 days, followed by a suspension culture in the presence of BMP2 (or BMP4), LIF, stem cell factor (SCF), and epidermal growth factor (EGF). The derived hPGCLCs were similar to human PGCs (hPGCs) in terms of gene expression and epigenetic patterns. They showed that SOX17, which is known as a transcription factor for endoderm specification, might be important for hPGCLC specification and maintenance since SOX17 began to be expressed as early as day 1 after suspension culture and was associated with BLIMP1, both of which are highly expressed in hPGCs; BLIMP alone repressed somatic differentiation and enhanced germ cell specification together with SOX17 [
55]. Although the preconditioned hPSC state was sufficient for producing hPGCs, Sasaki et al. pointed out that hPSCs kept in 4i medium were not genuinely at the ground state, as cells did not show consistent upregulation in naïve pluripotency markers [
56]. Instead, Sasaki et al. proposed the induction of PGCs from primed hPSCs by introducing new cell types, incipient mesoderm-like cells (iMeLCs), as intermediate cells in the process of inducing hPGCLCs from hiPSCs [
56]. Double transgenic hiPSCs bearing
BLIMP1-
tdTomato and
TFAP2C-
EGFP were used as reporters for human fetal germ cells [
58,
59]. As a first step for germ cell differentiation, hiPSCs were stimulated with activin A and CHIR99021, which produced iMeLC-expressing genes for pluripotency and the mesoderm (
T,
EOMES,
SP5, and
MIXL1). After suspension culturing with BMP4, SCF, EGF, and LIF for 8 days, these iMeLCs were converted into hPGCLCs (
BLIMP1-
2A-
tdTomato+ and
TFAP2C-
2A-
EGFP+) expressing early human germ cell markers, such as OCT4, NANOG, BLIMP1, TFAP2C, and SOX17. GO term analysis after RNA sequencing revealed that potential regulators of hPGCLC specification from iMeLCs include
BLIMP1 and
SOX17, which were also suggested by Irie et al. [
55,
56].
hPGCLC derivation using hESCs was facilitated by the stimulation of activin A [
60] or by adding vitamin C to the induction medium [
61]. Activin A is also a well-known inducer of fetal and postnatal oogenesis, indicating that it is involved in germ cell development [
62]. Epigenetic changes induced by vitamin C through enhancing the expression of ten-eleven translocation (TET) may positively affect germ cell differentiation [
61].
2.2. In Vitro Derivation of Spermatogonia and Oogonia from Pluripotent Stem Cells
PSC-derived PGCLCs were injected into neonatal testes to generate mature sperm from PSCs. However, PGCLCs are not suitable cell types for transplantation into the testis, since the niche for PGCs is not the postnatal testis, but the prenatal proximal epiblast, hindgut, and genital ridge. After birth, the earliest germ cell types in the testis are spermatogonia that reside in the basement membrane of the seminiferous epithelium. These spermatogonia are male germ line stem cells, so-called spermatogonial stem cells (SSCs), which continuously self-renew and differentiate into spermatocytes and haploid gametes by passing between two Sertoli cells [
39]. Currently, long-term in vitro culture of SSCs is possible, although PGCs cannot be cultured in vitro [
63,
64]. Undifferentiated SSCs established in an in vitro culture system expressed
Vasa,
Pax7,
Plzf,
Gfra1,
Etv5, and
Bcl6b [
65]. In contrast, female germline stem cells, or oogonia, transiently exist (between E12.5 and E13.5) and undergo meiosis during embryonic development [
66]. Thus far, an in vitro culture of oogonia is not possible.
Nayernia et al. first attempted to generate in vitro-derived SSCs using
Stra8-
EGFP and
Prm1-
DsRed double transgenic mouse ESCs [
67]. They suggested that Stra8-EGFP
+ cells were ESC-derived SSCs and that Prm1-DsRed
+ could differentiate into haploid germ cells. However, Stra8-EGFP
+ cells express PGC-specific genes; therefore, this differentiation protocol may yield heterogeneous germ cell populations, including SSCs. In addition, the ESC-derived spermatids they suggested are not fully characterized; therefore, it is not clear whether the cells are true spermatids. Ishikura et al. generated well-characterized in vitro-derived SSCs differentiated from PSCs using reconstituted testes formed by aggregating ESC-derived PGCLCs with E12.5 testicular somatic cells [
68]. Aggregates of PGCLCs and testicular somatic cells formed seminiferous tubule-like structures, in which PLZF-positive SSC-like cells were detected within 21 days of culture [
68]. SSC-like cells showed similar gene expression profiles and DNA methylomes to those of in vivo SSCs. In addition, after transplantation of these SSC-like cells into adult W/W
v mouse testes instead of neonatal testes, they developed into spermatids and spermatozoa, which could form embryos after an injection into oocytes and developed to term after transfer of the embryos to the surrogate mother.
In human, Sasaki and colleagues et al. generated prospermatogonia-like cells from hiPSCs through long-term culturing of the xenogeneic aggregates of hiPSC-derived human germ cells with mouse testicular somatic cells [
69]. hPGCLCs were obtained from xenogeneic aggregates after 14 days of culture on an air–liquid interface (ALI) system where xenogeneic aggregates were cultured on the surface of the porous filter. In the ALI culture system, hiPSC-derived hPGCLCs were differentiated into multiplying (M)-prospermatogonia on day 77 and finally differentiated into primary transitional (T1)-prospermatogonia at day 120. Although they did not test the functionality of the hiPSC-derived hPGCLCs, single-cell RNA sequencing analysis revealed that the gene expression patterns of in vivo M- and T1-prospermatogonia were very similar to those of in vivo M- and T1-prospermatogonia, respectively. As hiPSC-derived T1-prospermatogonia expressed a fraction of genes associated with “spermatogenesis” and “spermatogenic failure” these cells can be used as an in vitro platform for research on the mechanism of human spermatogenesis and male infertility [
69].
Recently, human female germline stem cells (oogonia) were generated in vitro from hiPSCs (
BLIMP1-
tdTomato and
TFAP2C-
EGFP double transgenic) using Sasaki’s approach, by which hiPSC-derived hPGCLCs were generated via iMeLCs [
56,
70]. BLIMP1-tdTomato
+/TFAP2C-EGFP
+ hPGCLCs were sorted by fluorescence-activated cell sorting (FACS) and cultured for further specification in xenogeneic ovary structures formed by aggregation of hiPSC-derived hPGCLCs with mouse ovarian somatic cells, as in a prospermatogonia-like cell-inducing protocol [
69]. At day 7 after culture, aggregates began to form cyst-like structures and TFAP2C-EGFP
+ cells developed into oogonia-like cells, which expressed DAZL and DDX4 and were delineated by mouse granulosa cells at culture day 77. On culture day 120, BLIMP1-tdTomato
+/TFAP2C-EGFP
+ cells appeared to differentiate into BLIMP1-tdTomato
+/TFAP2C-EGFP
- cells, which correspond to fetal germ cells expressing genes for meiosis initiation (
STRA8) except for meiotic recombination (
SPO1l,
PRDM9,
DMC1, or
SYCP1). Transcriptome assays revealed that hiPSC-derived oogonia-like cells (TFAP2C-EGFP
+) were similar to those of week 7 and 9 oogonia and gonocytes of human embryos [
70,
71].
These reports suggest that male and female germline stem cells can be established from PSCs in vitro within reconstituted testes and ovary structures, where embryonic gonadal somatic cells were aggregated with PSC-derived PGCLCs. Of note, in vitro oogonia may provide a useful tool for elucidating the mechanism of female germline stem cell development and female infertility, which has not been studied well due to ethical issues and limitations in using human-origin materials.
2.4. In Vitro Oogenesis from Pluripotent Stem Cells
Female germ cells were formed within ovarian follicles, which are an in vivo niche for the maturation of female gametes. One follicle contains one meiotically arrested oocyte surrounded by multilayered granulosa cells at puberty [
73]. During follicular maturation in vivo, primary oocytes complete meiosis I, begin meiosis II, and arrest at the metaphase of meiosis II (MII). Primordial follicles can mature in vitro by stimulating oocyte growth and meiotic competency by EGF [
74] and cAMP [
75,
76], respectively. In vitro oogenesis could also be recapitulated in PGCs obtained from E12.5 gonads by stimulation of follicle formation using α-MEM-based medium supplemented with fetal bovine serum (FBS) and an estrogen-receptor antagonist (ICI 187, 780) [
77]. These studies prompted an alternative method to derive female gametes from PSCs, where germline specification is not started.
The first published report showing germ cell differentiation from PSCs in vitro was on female gamete derivation from mouse ESCs [
1]. Hübner et al. generated oocyte-like structures, which were randomly formed through EB formation, followed by two-dimensional culture. These non-directed differentiation protocols, or random differentiation, allowed even male ES cells to differentiate into oocyte-like structures. Similarly, Lacham-Kaplan et al. obtained presumptive ovary-like structures from male ESC-derived EBs using the conditioned medium from neonatal testis cells. The obtained structures contained oocyte-like cells expressing oocyte markers (
Fig-
α and
ZP3), indicating the possible production of female germ cells from male PSCs [
78]. However, these results were inconsistent with the proposed methods and lacked demonstration of functionality [
48]; thus, further research is required. As previously mentioned, Hayashi et al. showed that in vitro-derived PGCLCs could further differentiate into functional oocytes in reconstituted ovaries after transplantation into female mice [
4]. Combining these methods, Hayashi and colleagues, in 2016, finally derived MII oocytes from mouse ESCs in an entirely in vitro system [
79]. They derived EpiLCs from mouse ESCs and further differentiated the EpiLCs into PGCLCs, which were then aggregated with E12.5 gonadal somatic cells. These aggregates were cultured for approximately 5 weeks in three steps: in vitro differentiation, in vitro growth, and in vitro maturation [
79,
80]. The aggregates of PGCLCs and gonadal somatic cells formed a follicular structure that successfully simulated the in vivo ovarian environment and performed the key events of oogenesis in vitro, including the formation of cumulus-oocyte complexes and development of MII oocytes [
79,
80]. These in-vitro-generated oocytes could be fertilized in vitro with wild-type sperm and developed into 2-cell embryos, giving rise to healthy fertile pups after transfer to pseudopregnant surrogate mice. However, the successful full-term development rate was as low as 3.5%. This in-vitro-oocyte generation method was also valid for iPSC lines reprogrammed from mouse embryonic fibroblasts [
79]. Overall, this platform for in vitro gametogenesis by aggregating PSC-derived PGCLCs with gonadal somatic cells faithfully reconstitutes the in vivo-like gonadal environment in both males and females [
68,
72,
79,
80]. However, this approach still shows meiosis and epigenetic imperfections compared with their in vivo counterparts and requires embryonic somatic cells [
68,
79], which cannot be used in the clinic. Nevertheless, this differentiation method provides a useful in vitro system for studying the interaction between gametes and gonadal somatic cells [
81,
82].
Recently, oocyte-like cells were directly induced without specification into PGCLCs [
83]. Hamazaki et al. generated follicle structures from mouse ESCs and iPSCs aggregated with E12.5 female gonadal somatic cells through forced expression of eight transcription factors important for primordial-to-primary-follicle transition (PPT), such as
Nobox,
Figla,
Tbpl2,
Sohlh1,
Stat3,
Dynll1,
Sub1, and
Lhx8. Oocyte-like cells of the follicle structures could develop into MII oocytes, which were competent for fertilization with sperm and early embryonic development until the 8-cell stage. Overexpression of these eight transcription factors was not sufficient for the induction of oocyte-like cells from somatic cells (MEFs), suggesting that a pluripotent state is required for the induction of germ cells by overexpression of the eight transcription factors. However, it is still unknown whether different transcription factor combinations may induce germ cell specification in somatic cells.
Furthermore, follicle-like cells (FLCs) were first derived from human PSCs by forced expression of DAZL and BOULE, which are involved in in vivo germ cell specification as discussed above [
54,
84]. Germ cell commitment from hESCs was first induced by BMP4 and BMP8a treatment followed by meiosis induction by lentiviral overexpression of DAZL and BOULE with the subsequent addition of GDF9 and BMP15. Follicle-like structures with oocyte-like cells surrounded by multilayered cell aggregates appeared on day 9 after differentiation. These oocyte-like cells expressed ZP2, NOBOX, AMH, and VASA and were found to have primordial oocyte identity, as confirmed by transcriptome analysis. In addition, transplantation of FLCs into kidney capsules resulted in the formation of primordial follicle-like structures, indicating that hESC-derived FLCs are functional ovarian follicles. This in vitro FLC derivation system could be used to elucidate the early mechanism of human germline development and folliculogenesis, the details of which are still elusive.