Peer Review #1 of "Efficient generation of human primordial germ cell-like cells from pluripotent stem cells in a methylcellulose-based 3D system at large scale (v0.1)"

The ability to generate primordial germ cell-like (PGCLs) from induced pluripotent stem cells (iPSCs) in swine could greatly contribute to regenerative medicine. Herein, we aimed to generate porcine PGCLs (ipPGCLs) from iPSCs derived from different culture systems. Porcine (p)iPSCs from fibroblasts of stillborn animals (n=3) were transduced with lentiviral vectors containing murine OCT4, SOX2, c-MYC, and KLF4 cDNAs and maintained in iPSC medium on mouse embryonic fibroblasts (MEFs). The cells were divided into three groups: (1) supplemented with 10ngmL−1 basic fibroblast growth factor (bFGF) and murine leukemia inhibitory factor (LIF), (2) only bFGF, or (3) only LIF. The piPSC colonies were generated and characterised for pluripotency. To induce piPSCs into ipPGCLs, three or more cell lines from each culture condition (after passage 20) were differentiated into epiblast stem cell-like cells (EpiLCs) by culture with 20ngmL−1 Activin A, 12ngmL−1 bFGF, and 1% knockout serum replacement (KSR) for 2 days. Then, cells were further induced to differentiate by nonadherent culture and supplementation with 500ngmL−1 bone morphogenetic protein (BMP)4, 500ngmL−1 BMP8a, LIF, 100ngmL−1 stem cell factor (SCF), and 50ngmL−1 epidermal growth factor for 4 days. The ipPGCLs were characterised by cell morphology and detection of germ cell markers by immunofluorescence and gene expression. Statistical analysis was determined by one-way ANOVA (Prism Software). Co-location quantification was determined using the plugin Colocalization Threshold in Image J software (National Institutes of Health). On average, the efficiency rate of iPSC generation was 71% for the iPSCs-bFGF group, 17% for the LIF group, and 85% for the bFGF+LIF group. All iPSCs colonies were positive for alkaline phosphatase and OCT4, SOX2, NANOG, TRA1-60, TRA1-81, SSEA1, and SSEA4 by immunofluorescence. Embryoid body assay revealed that the piPSCs were able to differentiate into three germ layers. The culture condition did not influence the expression of OCT4, NANOG, and KLF4 based on qRT-PCR, however; SOX2 was upregulated in the LIF group (P<0.05). The ipPGCLs generated showed a round morphology. Analysis of endogenous pluripotent genes OCT4, SOX2, and NANOG throughout differentiation (fibroblasts, iPSCs, EpiLCs, and PGCLs) revealed a mild upregulation in ipPGCLs, while OCT4 was slightly downregulated in ipPGCLs from iPSCs-LIF group. PRDM14 and STELLA were not observed in ipPGCLs, although BLIMP1 was present; DAZL and VASA were mildly upregulated. The STELLA, VASA, OCT4, and SOX2 proteins were detected in ipPGCLs, and DAZL was detected only in ipPGCLs from the iPSCs-FGF group. Protein co-localization analysis showed that ipPGCLs from the iPSCs-FGF group were 100% OCT4+STELLA-positive, 55% positive for DAZL+SOX2, and 66% positive for VASA+NANOG; for the LIF group: 99.3% were OCT4+STELLA positive, DAZL was not detected, 95.2% were positive for SOX2 and 85.6% for VASA+NANOG. In the bFGF+LIF group, 95.8% were positive for OCT4+STELLA, DAZL and SOX2 were not observed, and 70% were positive for VASA+NANOG. Exogenous reprogramming factors were still expressed and did not differ between groups. These results indicate that, under our conditions, the iPSCs-FGF group may represent the best culture condition for induction into ipPGCLs. Financial support for this study was provided by FAPESP (2015/25564-0 and 2015/26818-5).

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