Previous reports have demonstrated that embryonic stem cells were capable of differentiating into primordial germ cells through the formation of embryoid bodies that subsequently generated oocyte-like cells (OLCs). Such a process could facilitate studies of primordial follicle oocyte development in vitro and regenerative medicine. To investigate the pluripotency of human amniotic fluid stem cells (hAFSCs) and their ability to differentiate into germ cells, we isolated a CD117(+)/CD44(+) hAFSC line that showed fibroblastoid morphology and intrinsically expressed both stem cell markers (OCT4, NANOG, SOX2) and germ cell markers (DAZL, STELLA). To encourage differentiation into OLCs, the hAFSCs were first cultured in a medium supplemented with 5% porcine follicular fluid for 10 days. During the induction period, cell aggregates formed and syntheses of steroid hormones were detected; some OLCs and granulosa cell-like cells could be loosened from the surface of the culture dish. Cell aggregates were collected and replated in oocyte culture medium for an additional 7-10 days. OLCs ranging from 50 to 120 μm presenting zona pellucida were observed in cumulus-oocyte complexes; some OLCs developed spontaneously into multicell structures similar to preimplantation embryos. Approximately 2% of the hAFSCs differentiated to meiotic germ cells that expressed folliculogenesis- and oogenesis-associated markers. Although the in vitro maturation and fertilization potentials are as yet unproven, short-term (<25 days) and high-efficiency (>2%) derivation of OLCs from hAFSCs might provide a new approach to the study of human germ cell development in vitro.

The generation of oocyte-like cells (OLCs) from stem cell differentiation in vitro provides an optimal approach for studying the mechanism of oocyte development and maturation. The aim of this study was to investigate the activation of bone morphogenetic protein 15 gene (BMP15) during the differentiation of human amniotic fluid stem cells (hAFSCs) into OLCs. After 15 days of differentiation, OLCs with a diameter of 50-60 μm and zona pellucida (ZP)-like morphology were observed. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the BMP15 was activated from approximately day 10 of differentiating hAFSCs and thereafter. The reporter construct pBMP15-enhanced green fluorescent protein (EGFP) was transiently transfected into the differentiated hAFSCs and the EGFP expression driven by the BMP15 promoter was positive in the OLCs. Moreover, RT-PCR analysis showed that the oocyte-specific markers including ZP1, ZP2, ZP3, and c-kit were expressed in the differentiated hAFSCs, and the immunofluorescence assay confirmed that the ZP2 was detected in the OLCs. Quantitative RT-PCR revealed that ZP2 and ZP3 were significantly elevated in the differentiated hAFSCs. Further, in the OLCs derived from hAFSCs, the BMP15 promoter directing the EGFP reporter was colocalized with ZP2. Together, these results illustrated that the BMP15 could be used as an oogenesis marker to track hAFSCs differentiation into the OLCs.

Background: Human umbilical cord-derived MSCs (hUC-MSCs) have been identified as promising seeding cells in tissue engineering and clinical applications of regenerative medicine due to their advantages of simple acquisition procedure and the capability to come from a young tissue donor over the other MSCs sources. In clinical applications, large scale production is required and optimal cryopreservation and culture conditions are essential to autologous and allogeneic transplantation in the future. However, the influence of cryopreserved post-thaw and long-term culture on hUC-MSCs remains unknown, especially in terms of specific protein expression. Therefore, biological characteristics and proteomic profiles of hUC-MSCs after cryopreserving and long-term culturing were investigated. Methods: Firstly, hUC-MSCs were isolated from human umbilical cord tissues and identified through morphology, surface markers and tri-lineage differentiation potential at passage 3, and then the biological characteristics and proteomic profiles were detected and compared after cryopreserving and long-term culturing at passage 4 and continuously cultured to passage 10 with detection occurring here as well. The proteomic profiles were tested by using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique and differential protein were confirmed by mass spectrometry. Results: The results showed no significant differences in phenotypes including morphology, surface marker and tri-lineage differentiation potential but have obvious changes in translation level, which is involved in metabolism, cell cycle and other pathways. Conclusion: This suggests that protein expression may be used as an indicator of hUC-MSCs security testing before applying in clinical settings, and it is also expected to provide the foundation or standardization guide of hUC-MSCs applications in regenerative medicine.

Type I IFNs (IFNIs) are involved in the course of antiviral and antimicrobial activities; however, robust inductions of these can lead to host immunopathology. We have reported that the Pias (protein inhibitor of activated signal transducer and activator of transcription) family member, Piasy, possesses the ability to suppress IFNI transcriptions in mouse embryonic fibroblasts (MEFs), yet the specific molecular mechanism by which it acts remains elusive. Here, we identify that the H3K4me3 levels, one activation mark of genes, in MEFs that were stimulated by poly(I:C) were impaired by Piasy in the IFN-β gene. Piasy bound to the promoter region of the IFN-β gene in MEFs. Meanwhile, retinoblastoma binding protein 2 (Rbp2) was proven to be the only known and novel H3K4me3 demethylase that interacted with Piasy. Overexpression of Rbp2, but not its enzymatically inactive mutant Rbp2, retarded the transcription activities of IFNI, whereas small interfering RNA-mediated or short hairpin RNA-mediated knockdown of Rbp2 enhanced IFNI promoter responses. Above all, coexpression of Piasy and Rbp2 led to statistically less IFNI induction than overexpression of either Piasy or Rbp2 alone. Mechanistically, Piasy bound to the Jmjc domain (451-503 aa) of Rbp2 via its PINIT domain (101-218 aa), which is consistent with the domain required for their attenuation of transcription and H3K4me3 levels of IFNI genes. Our study demonstrates that Piasy may prevent exaggerated transcription of IFNI by Rbp2-mediated demethylation of H3K4me3 of IFNI, avoiding excessive immune responses.-Yu, X., Chen, H., Zuo, C., Jin, X., Yin, Y., Wang, H., Jin, M., Ozato, K., Xu, S. Chromatin remodeling: demethylating H3K4me3 of type I IFNs gene by Rbp2 through interacting with Piasy for transcriptional attenuation.