title: Granulosa cells and retinoic acid co-treatment enrich potential germ cells from manually selected Oct4-EGFP expressing human embryonic stem cells.
abstract: Differentiation of human embryonic stem (HES) cells to germ cells may become clinically useful in overcoming diseases related to germ-cell development. Niches were used to differentiate HES cell lines, NTU1 and H9 Oct4-enhanced green fluorescence protein (EGFP), including laminin, granulosa cell co-culture or conditioned medium, ovarian stromal cell co-culture or conditioned medium, retinoic acid, stem cell factor (SCF) and BMP4-BMP7-BMP8b treatment. Flow cytometry showed that granulosa cell co-culture (P < 0.001) or conditioned medium (P = 0.007) treatment for 14 days significantly increased the percentages of differentiated H9 Oct4-EGFP cells expressing early germ cell marker stage-specific embryonic antigen 1(SSEA1); sorted SSEA1[+] cells did not express higher levels of germ cell gene VASA and GDF9. Manually collected H9 Oct4-EGFP[+] cells expressed significantly higher levels of VASA (P = 0.005) and GDF9 (P = 0.001). H9 Oct4-EGFP[+] cells developed to ovarian follicle-like structures after culture for 28 days but with low efficiency. Unlike SCF and BMP4, retinoic acid co-treatment enhanced VASA, GDF9 and SCP3 expression. A protocol is recommended to enrich differentiated HES cells with germ-cell potential by culture with granulosa cells, conditioned medium or retinoic acid, manual selection of Oct4-EGFP[+] cells, and analysis of VASA, GDF9 expression, or both.
authors.affiliation: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, College of Medicine and the Hospital, National Taiwan University, Taipei, Taiwan; Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National Taiwan University, Taipei, Taiwan; Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Hsin-Fu
authors.initials: HF
authors.lastname: Chen
authors.affiliation: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, College of Medicine and the Hospital, National Taiwan University, Taipei, Taiwan.
authors.forename: Pey-Shynan
authors.initials: PS
authors.lastname: Jan
authors.affiliation: Institute of Cellular and Organismic Biology and Genomics Research Center, Academia Sinica, Taipei, Taiwan.
authors.forename: Hung-Chih
authors.initials: HC
authors.lastname: Kuo
authors.affiliation: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, College of Medicine and the Hospital, National Taiwan University, Taipei, Taiwan.
authors.forename: Fang-Chun
authors.initials: FC
authors.lastname: Wu
authors.affiliation: Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Chen-Wei
authors.initials: CW
authors.lastname: Lan
authors.affiliation: Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Mei-Chi
authors.initials: MC
authors.lastname: Huang
authors.affiliation: Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Chung-Liang
authors.initials: CL
authors.lastname: Chien
authors.affiliation: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, College of Medicine and the Hospital, National Taiwan University, Taipei, Taiwan; Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National Taiwan University, Taipei, Taiwan; Graduate Institute of Immunology, College of Medicine, National Taiwan University, Taipei, Taiwan. Electronic address: hnho@ntu.edu.tw.
authors.forename: Hong-Nerng
authors.initials: HN
authors.lastname: Ho
mesh_terms: D002454:Cell Differentiation; D018920:Coculture Techniques; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D005854:Germ Cells / Q000166:cytology* / Q000378:metabolism; D006107:Granulosa Cells / Q000166:cytology* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D050814:Octamer Transcription Factor-3 / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014212:Tretinoin
keywords: embryonic stem cells; germ cells; granulosa cells; retinoid acid differentiation