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( Hong-nerng Ho ) 대한산부인과학회 2017 대한산부인과학회 학술대회 Vol.103 No.-
Polycystic ovarian syndrome (PCOS) is an heterogeneous disease, though it might be inherited, however, there is no candidate gene found. By conducting various animal models, chronic anovulation and disturbed folliculogenesis in women with PCOS might be demonstrated by the exposures to exogenous androgens before or after puberty; or of various systemic disturbances as obesity, insulin resistance and chronic low-grade inflammatory status. We firstly established the PCOS-specific induced pluripotent stem cells (iPSCs) and differentiate them into ovarian granulosa- like cells. We further compared the difference of the differentiation times and function of granulosa-like cells derived from iPSCs of women with and without PCOS. Cells originating from skin fibroblasts of women with and without PCOS were reprogrammed by using origin of replication/Ebstein-Barr Nuclear Antigen-1 (ori/EBNA-1)-based episomal vectors carrying defined factors. The resulting iPSCs were then differentiated into ovarian granulosa-like cells using multistep approaches comprising in vitro treatments with cocktails of growth factors. Gene expression analyses revealed the granulosa cell specific markers including anti-Mullerian hormone (AMH), type 2 AMH receptor (AMHR2), FSH receptor, LH receptor, estrogen synthetase cytochrome P450 19A1 (CYP19A1), and forkhead transcription factor [FOXL2] all expressed in differentiated granulosa-like cells derived from PCOS and non-PCOS iPSCs. The pluripotency marker Oct4 expression along with the increase differentiation time of iPSC to granulosa cell was significantly higher in iPSCs derived from women with PCOS than those without PCOS. This implies possible delay in the differentiation potential in PCOS iPSCs. However, after FSH stimulation, the expression of Oct4 in granulosa-like cells differentiated after 12 days from PCOS iPSCs was significantly lower than those from non-PCOS iPSCs. The FSH-stimulated expressions of AMHR2, CYP19A1, and FSHR in the differentiated granulosa-like cells from PCOS iPSCs increased with increasing dose and time of FSH treatment in comparison with those from non-PCOS iPSCs. The delayed differentiation potential but sensitive response to FSH treatment in granulosa-like cell derived from iPSCs of women with PCOS mimic the clinical presentation of disturbed folliculogenesis in women with PCOS and provide further evidence to support the developmental origin and potential inherited trait in the pathogenesis of PCOS.
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Chia-Eng Wu,Chen-Wei Yu,Kai-Wei Chang,Wen-Hsi Chou,Chen-Yu Lu,Elisa Ghelfi,Fang-Chun Wu,Pey-Shynan Jan,Mei-Chi Huang,Patrick Allard,Shau-Ping Lin,Hong-Nerng Ho,Hsin-Fu Chen 생화학분자생물학회 2017 Experimental and molecular medicine Vol.49 No.-
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.
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