Human pluripotent stem cell-derived DDX4 and KRT-8 positive cells participate in ovarian follicle-like structure formation.

Understanding the mechanisms of human pluripotent stem cells (hPSCs) specification, development and differentiation to gametes are useful for elucidating the causes of infertility and potential treatment. This study aims to examine whether hPSCs can be induced to DDX4 extracellularly expressing primordial germ cell-like cells (DDX4ec PGCLCs) and further into ovarian follicle stage in a combined in vitro and in vivo model. The transcriptional signatures show that these DDX4ec PGCLCs are characteristic of PGCs and express ovarian folliculogenesis markers. We also verify that keratin (KRT)-8 is highly expressed in the DDX4ec PGCLCs and plays a crucial role in germ cell migration. By co-culturing DDX4ec PGCLCs with human granulosa cells (GCs), these cells are further induced into ovarian follicle-like structures in a xenograft mice model. This approach can in the future design practical strategies for treating germ cell-associated issues of infertility.

Hyperactive CREB signaling pathway involved in the pathogenesis of polycystic ovarian syndrome revealed by patient-specific induced pluripotent stem cell modeling.

OBJECTIVE: To study whether and how the pathogenesis of polycystic ovarian syndrome (PCOS) is related to epigenetic aberrations. DESIGN: A case-control experimental study. SETTING: Tertiary university hospital. PATIENT(S): Eighteen patients with PCOS and ten non-PCOS control subjects. INTERVENTIONS(S): Patient-specific induced pluripotent stem cells (iPSCs) were obtained from skin fibroblasts through the application of nonviral episomal reprogramming and were differentiated into ovarian granulosa cells (GCs) with the use of a cocktail of growth factors. Primary ovarian GCs were collected during transvaginal oocyte retrieval surgery. MAIN OUTCOME MEASURE(S): Characterization and functional validation of iPSC-derived GCs were conducted. Whole-genomic DNA methylation profiles in women with and without PCOS in both iPSC-derived GCs and primary adult GCs were analyzed with the use of the Illumina 850K MethylationEPIC Beadchip. RESULT(S): The iPSC-derived GCs successfully expressed GC-associated genes and aromatase activity after differentiation. Whole-genomic DNA methylation analysis of the iPSC-derived GCs and adult GCs both revealed a hyperactive CREB signaling pathway in the PCOS group compared with the control group. The expression of CREB-binding protein (CBP) mRNA was significantly higher in the iPSC-derived GCs in the PCOS group, and the expression of CBP protein was also significantly higher in the primary GCs from women with PCOS. CONCLUSION(S): The combination of DNA methylomic analysis in primary adult GCs and iPSC-derived GCs showed that a preserved persistent hyperactivation of the CREB signaling pathway might be involved in the pathogenesis of PCOS. These results could have implications on the early developmental origin, inheritance nature, and environmental interaction effects of this disease.

Comparative global immune-related gene profiling of somatic cells, human pluripotent stem cells and their derivatives: implication for human lymphocyte proliferation.

Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSCs), represent potentially unlimited cell sources for clinical applications. Previous studies have suggested that hPSCs may benefit from immune privilege and limited immunogenicity, as reflected by the reduced expression of major histocompatibility complex class-related molecules. Here we investigated the global immune-related gene expression profiles of human ESCs, hiPSCs and somatic cells and identified candidate immune-related genes that may alter their immunogenicity. The expression levels of global immune-related genes were determined by comparing undifferentiated and differentiated stem cells and three types of human somatic cells: dermal papilla cells, ovarian granulosa cells and foreskin fibroblast cells. We identified the differentially expressed genes CD24, GATA3, PROM1, THBS2, LY96, IFIT3, CXCR4, IL1R1, FGFR3, IDO1 and KDR, which overlapped with selected immune-related gene lists. In further analyses, mammalian target of rapamycin complex (mTOR) signaling was investigated in the differentiated stem cells following treatment with rapamycin and lentiviral transduction with specific short-hairpin RNAs. We found that the inhibition of mTOR signal pathways significantly downregulated the immunogenicity of differentiated stem cells. We also tested the immune responses induced in differentiated stem cells by mixed lymphocyte reactions. We found that CD24- and GATA3-deficient differentiated stem cells including neural lineage cells had limited abilities to activate human lymphocytes. By analyzing the transcriptome signature of immune-related genes, we observed a tendency of the hPSCs to differentiate toward an immune cell phenotype. Taken together, these data identify candidate immune-related genes that might constitute valuable targets for clinical applications.