Generation,identification and differentiation of primary ovarian insufficiency patient-derived iPSCs |
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| Authors: | WEN Yan-fei CAI Liu-hong HE Wen ZENG Min-hui JANG Man-bo |
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| Institution: | 1. Center for Reproductive Medicine, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China;
2. Center for Reproductive Medicine, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120, China;
3. Department of Reproductive Medicine, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou 510006, China |
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| Abstract: | AIM: To generate and identify primary ovarian insufficiency (POI) patient-derived induced pluripotent stem cells (iPSCs) and to explore their differentiation potential to primordial germ cells. METHODS: Plasmid pEB-C5 expressing reprogramming factors Oct4, Sox2, c-Myc, Klf4 and Lin28, and plasmid pEB-Tg expressing SV40 T antigen, were transfected into peripheral blood mononuclear cells (PBMCs) derived from POI patients at the same time. PBMCs were reprogrammed into iPSCs, and the pluripotency of the cells was identified. After supplementation of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein 4 (BMP4), the mRNA and protein expression of primordial germ cell markers was detected by real-time PCR and Western blot, respectively. RESULTS: iPSCs derived from the PBMCs of POI patient differentiated into 3-germ layer cells and maintained pluripotency by the detection of alkaline phosphatase staining, immunofluorescence, embryoid body and teratoma formation. After addition of TGF-β1 and BMP4, the primordial germ cell markers, including stem cell growth factor receptor (c-Kit), developmental pluripotency-associated 3 (STELLA/DPPA3) and DEAD box polypeptide 4 (VASA/DDX4) were increased at mRNA level (P<0.05), and VASA/DDX4 was also up-regulated at protein level in induced group. CONCLUSION: PBMCs of POI patient are reprogrammed into integration-free iPSCs in vitro and maintain pluripotency. They differentiate into primordial germ cells by adding TGF-β1 and BMP4. |
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| Keywords: | Primary ovarian insufficiency Induced pluripotent stem cells Primordial germ cells |
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