Derivation of iPGCs from Human PSCs
Since hESCs and hiPSCs bear a primed pluripotency (Fig. S1A-B), we explored the potential of hiPSCs and hESCs to differentiate into iPGCs following the procedure showed in Fig. 1A. The morphology of hPSCs cultured in PGC medium (PGC-m) for 10 days became the flattened epithelial cell type with distinct cell-to-cell boundaries, subsequently, a small number of spherical cells and even some follicular-like cell aggregates were mostly formed at the bottom of the culture dish, and few of them floated in the culture supernatant (Fig. 1B, S2A-B). The expression of pluripotent genes OCT4, SOX2, and PRDM14 were significantly reduced during the differentiation from day 0 to day 10 (Fig. 1C, S3A). To estimate the synthesis of reproductive hormones during the differentiation, the expression of genes related to estrogen biosynthesis was determined and showed that the expressions of two crucial genes involved in estrogen biosynthesis, CYP19A1 and CYP17A1, and a gene for the follicle-stimulating hormone receptor (FSHR) were significantly increased, suggesting that the granulosa-like cells within the differentiated cells produced hormones to stimulate the development of female reproductive physiology. (Fig. 1C, S3A). Additionally, the expression level of c-KIT was increased from 0.45% in hiPSCs and 0.95% in hESCs to 50.6% and 42% in iPGCs after 10 days of differentiation, respectively (Fig. 1D, S3B). The interaction of c-KIT from PGC and SCF from somatic cells is able to modulate PGC adhesion to somatic cells and further contributes to oogenesis [11].
To further confirm iPGCs, Immunofluorescence analysis showed that cell clusters derived from both hiPSCs and hESCs expressed the key PGC transcription factors OCT4, PRDM1, SOX17, and STELLA at day 10 of differentiation (Fig. 2A, S3C). The expression of germline specific genes PRDM1, TFAP2C, and SOX17 were significantly elevated versus the early stage of differentiation (day 5), which presented the observation similar to the report in mouse PGCs [12]. The early mesoderm marker TBXT (also known as T or Brachyury) was significantly activated, indicating that cells were turned into the incipient stage of mesoderm-like cells (iMeLCs) and activated to primordial germ cell fate that was reported previously [9, 13, 14] (Fig. 2B). In the late stage of iPGCs (day 10), some cell clusters composed of iPGCs were semi–suspended above the monolayer cells in the bottom of culture dish, those iPGCs could be purified by DDX4 antibody after immunomagenetic isolation. The DDX4+ iPGCs showed the round-oval shape and large nucleus with 10–20 µm diameter (Fig. 2C). We also observed the expression of FRAGILIS, DDX4, and NOBOX after 7–10 days of induction in iFLs (Fig. 2D), which are the essential factors for folliculogenesis and regulation of oocyte development. These findings demonstrated that human iPSCs could be differentiated through iMeLCs into the late stage of PGCs and even early stage of ovarian follicle.
Transcriptome Analysis of iPGCs
To illustrate whether the iPGCs expressed the PGC specific markers, we performed the whole-genome transcriptome sequencing to analysis of the iPSCs (TAC153) and iPGCs (D5). Unsupervised hierarchical clustering of genes showed that each group clustered to its duplicate (Fig. 3A). Principal component analysis of the transcriptome datasets together with the Ovarian Kaleidoscope database (OKdb; http://ovary.stanford.edu), which provides information regarding the biological function, expression pattern, and regulation of genes that are expressed in the ovary, showed that many primordial follicle growth and development associated genes, such as YAP1, SRC, BMP4, TGFBR1, and FOXO3 etc., were extensively upregulated in iPGCs. Alternatively, the mesodermal and stem cell-related genes, such as KDR, FGF2, PPP6C, STK11, LIF, and KITLG etc., were downregulated (Fig. 3B). The heat map data show that there are many differentially expressed genes between the hiPSCs and iPGCs, in which iPGCs have the most consistent downregulation of pluripotent genes, OCT4, SOX2, NANOG, and TERT. Many genes that are involved in differentiation and reproductive processes are extensively upregulated in iPGCs (Fig. 3C), and the differentiated cells had a certain correlation with the cells in the human follicle when compared the whole transcriptome to adult ovarian six main cell clusters [15] (Fig S2C). These results indicated that the hiPSCs exited from the pluripotent state when cultured in PGC-m for 5 days and differentiated into germ cells and started to initiate the folliculogenesis.
Formation and Growth of Induced Follicle-like Structures (iFLs)
When continuing the induction from day 10 to day 15, the iFLs were formed gradually and floated in the culture medium, presenting the morphological structure similar to the follicle with granulosa cells and COC (Fig. 4A, S2A-B). At this stage, many iFLs with granulosa-like cells were released from cell aggregates, and iFLs in different sizes were observed (Fig. S2A-B). Some iFLs were attached at the surface of culture plate and some were floated in the culture medium (Fig. 4A, and video S). The iFL that was transfected by pBMP15-EGFP plasmid showed the green fluorescence (Fig. 4B), indicating that the oocyte-specific BMP15, which shares high homology with GDF9 and plays crucial role in both oocyte maturation and fertilization [3, 10], was activated in the iFL. Western blot analysis further proved that BMP15 expression was activated in iPGCs and iFLs stages, meanwhile, the OCT4 expression was significantly reduced (Fig. 4C).
To determine the existence of granulosa cells around the iFL, we discovered the expression of granulosa cell marker FOXL2 in 15-day and 25-day iFL samples, but it was absent in control (Fig. 4D). We also performed the qRT-PCR assay to measure the expression of oocyte-specific markers. Results showed that expression of DAZL, FIGLA, and IFITM3 were significantly increased (Fig. 4E, S4A). We then used the BMP15 positive iFLs to perform the immunostaining with anti-FOXL2 and anti-DDX4 antibodies, in which DDX4 is a unique protein in germ cell lineages [16]. These results revealed that a BMP15/DDX4 positive OLC was surrounded with FOXL2 positive cells (Fig. 4F). We also checked the estrogen biosynthesis related genes CYP19A1, CYP17A1, and FSHR with high level expression in iFLs compared to the control from human ovary (Fig. 4G). These results indicated that follicle-like structures derived from iPGCs expressed multiple female germline cell markers and retained the immature OLC.
OLCs Derived from Follicles and in vitro Maturation
In 15 to 20 days of induction, the follicles with large nuclei could be observed. The detached iFLs were harvested and cultured in OLC-m for 10 days. OLCs ranging from 50 to 150 µm coating zona pellucida (ZP) were detached from iFLs and surrounded by granulosa-like cells (Fig. 5A). Though the thin ZP was fragile and extremely difficult to be manipulated with a micropipette, an OLC was able to be picked up by a holding pipette (Fig. 5B). ZP2, ZP3, and BMP15 expressions in OLCs were confirmed by western blotting (Fig. 5C, S3E). We then detected the expression of SCP3 a meiosis marker as well as initiation of ZP glycoprotein 2 synthesis in OLCs, and confirmed that SCP3 as well as ZP2 was co-localized in OLCs, but not in the surrounded granulosa-like cells (Fig. 5D). And the SCP3 protein and DDX4 were also detectable in BMP15 positive iFLs derived from hESCs (Fig. S3D). Additionally, to further determine the connection between granulosa cells and OLCs, the CX37 in OLCs was low expression and showed the dot form distribution in the surface of cell membrane compare to human oocytes (Fig. 5E). Which mediates the formation of gap junction communication between oocyte and granulosa cells and is mainly expressed at the membrane of oocyte [17].
In 25-day post-induction, germinal vesicle (GV) stage OLCs with a morphologically-normal human oocytes appearance were stripped off cumulus cells (Fig. 6A). The DAPI staining indicated a distinct nucleus in OLC (Fig. 6B, S3F) and the first polar body was observed in the individual OLC (Fig. 6C). Further, the meiosis specific marker, SCP3 showed highly expression in OLCs (Fig. 6D). These results indicated that OLCs reached and arrested at metaphase II (MII) stage.
Production of Parthenogenetic embryo-like structures
To continue culturing OLCs up to 35 days in OLC-m, some OLCs developed spontaneously into cleavage stage-like embryos structures similar to preimplantation. The nuclear staining showed that two- and multiple nuclei were detected in embryo-like structures (Fig. 7A, S3G), indicating that OLCs were parthenogenetically activated, although these embryos was fragile with a thin ZP membrane (Fig. 7B). To elucidate the embryo-like structures, we performed IF analysis and showed that DDX4 and c-KIT were expressed in the parthenogenetically activated embryos (Fig. 7C, S3G), suggesting that DDX4 and c-KIT were involved in the progression of embryogenesis during differentiation, and parthenogenetic embryo-like structures had the development potential. The effectively modulates the parthenogenetic activation of OLCs and optimizes the culture condition for maintaining MII OLCs may help to reveal OLCs development ability in vitro.