title: Granulosa cell-derived induced pluripotent stem cells exhibit pro-trophoblastic differentiation potential.
abstract: BACKGROUND
  Human induced pluripotent stem cells (hiPSCs) have been derived from various somatic cell types. Granulosa cells, a group of cells which surround oocytes and are obtained from the (normally discarded) retrieved egg follicles of women undergoing infertility treatment, are a possible cell source for induced pluripotent stem cell (iPSC) generation. Here, we explored the possibility of using human granulosa cells as a donor cell type for iPSC reprogramming, and compared granulosa cell-derived iPSCs (iGRAs) with those derived from other cell sources, to determine the potential ability of iGRA differentiation.
  
  
  METHODS
  Granulosa cells were collected from egg follicles retrieved from women undergoing infertility treatment. After short-term culture, the granulosa cells derived from different patients were mixed in culture, and infected with retroviruses encoding reprogramming factors. The resulting iPSC clones were selected and subjected to microsatellite DNA analysis to determine their parental origin. IGRAs were subjected to RT-PCR, immunofluorescence staining, and in vitro and in vivo differentiation assays to further establish their pluripotent characteristics.
  
  
  RESULTS
  Microsatellite DNA analysis was used to demonstrate that hiPSCs with different parental origins can be simultaneously reprogrammed by retroviral transfection of a mixed human granulosa cell population obtained from multiple individuals. The iGRAs resemble human embryonic stem cells (hESCs) in many respects, including morphological traits, growth requirements, gene and marker expression profiles, and in vitro and in vivo developmental propensities. We also demonstrate that the iGRAs express low levels of NLRP2, and differentiating iGRAs possess a biased differentiation potential toward the trophoblastic lineage. Although NLRP2 knockdown in hESCs promotes trophoblastic differentiation of differentiating hESCs, it does not result in exit from pluripotency. These results imply that NLRP2 may play a role in regulating the trophoblastic differentiation of human pluripotent stem cells.
  
  
  CONCLUSIONS
  These findings provide a means of generating iPSCs from multiple granulosa cell populations with different parental origins. The ability to generate iPSCs from granulosa cells not only enables modeling of infertility-associated disease, but also provides a means of identifying potential clinical interventions through iPSC-based drug screening.
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan. cychuang@gate.sinica.edu.tw.|Institute of Cellular and Organismic Biology, Academia Sinica, 128 Academia Road, Sec. 2, Nankang District, Taipei, 115, Taiwan. cychuang@gate.sinica.edu.tw.
authors.forename: Ching-Yu
authors.initials: CY
authors.lastname: Chuang
authors.affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, 128 Academia Road, Sec. 2, Nankang District, Taipei, 115, Taiwan. kris5533@yahoo.com.tw.
authors.forename: Mei-Chi
authors.initials: MC
authors.lastname: Huang
authors.affiliation: Graduate Institute of Clinical Genomics, College of Medicine, National Taiwan University, Taipei, 115, Taiwan. hfchen@ntu.edu.tw.|Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, National Taiwan University and Hospital, Taipei, 115, Taiwan. hfchen@ntu.edu.tw.
authors.forename: Hsin-Fu
authors.initials: HF
authors.lastname: Chen
authors.affiliation: Department of Medical Genetics, National Taiwan University Hospital, Taipei, 115, Taiwan. lhtseng@ntu.edu.tw.
authors.forename: Li-Hui
authors.initials: LH
authors.lastname: Tseng
authors.affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, 128 Academia Road, Sec. 2, Nankang District, Taipei, 115, Taiwan. harrisman@gmail.com.
authors.forename: Chun-Ying
authors.initials: CY
authors.lastname: Yu
authors.affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, 128 Academia Road, Sec. 2, Nankang District, Taipei, 115, Taiwan. leestonemail@gmail.com.
authors.forename: Lee
authors.initials: L
authors.lastname: Stone
authors.affiliation: Graduate Institute of Clinical Genomics, College of Medicine, National Taiwan University, Taipei, 115, Taiwan. hh691290@gmail.com.
authors.forename: Hsiang-Po
authors.initials: HP
authors.lastname: Huang
authors.affiliation: Graduate Institute of Clinical Genomics, College of Medicine, National Taiwan University, Taipei, 115, Taiwan. hnho@ntu.edu.tw.|Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, National Taiwan University and Hospital, Taipei, 115, Taiwan. hnho@ntu.edu.tw.
authors.forename: Hong-Nerng
authors.initials: HN
authors.lastname: Ho
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan. kuohuch@gate.sinica.edu.tw.|Institute of Cellular and Organismic Biology, Academia Sinica, 128 Academia Road, Sec. 2, Nankang District, Taipei, 115, Taiwan. kuohuch@gate.sinica.edu.tw.
authors.forename: Hung-Chih
authors.initials: HC
authors.lastname: Kuo
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000328:Adult; D051017:Apoptosis Regulatory Proteins; D002454:Cell Differentiation; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D006063:Chorionic Gonadotropin / Q000032:analysis; D004958:Estradiol / Q000032:analysis; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006107:Granulosa Cells / Q000166:cytology*; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D008297:Male; D018895:Microsatellite Repeats / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D011374:Progesterone / Q000032:analysis; D034741:RNA, Small Interfering / Q000378:metabolism; D014327:Trophoblasts / Q000166:cytology* / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D006063:Chorionic Gonadotropin; C495732:NLRP2 protein, human; D034741:RNA, Small Interfering; D011374:Progesterone; D004958:Estradiol