title: Differentiation of primordial germ cells from induced pluripotent stem cells of primary ovarian insufficiency.
abstract: OBJECTIVE
  Can the induced pluripotent stem cells (iPSCs) derived from women with primary ovarian insufficiency (POI) differentiate into germ cells for potential disease modeling in vitro?
  
  
  CONCLUSIONS
  The iPSC lines derived from POI patients with 46, X, del(X)(q26) or 46, X, del(X)(q26)9qh+ could differentiate into germ cells and expressed lower levels of genes in the deletion region of the X chromosome.
  
  
  BACKGROUND
  iPSC technology has been envisioned as an approach for generating patient-specific stem cells for disease modeling and for developing novel therapies. It has also been confirmed that iPSCs differentiate into germ cells.
  
  
  METHODS
  We compared the differentiation ability of germ cells and the gene expression level of germ cell-related genes in the X chromosome deletion region of iPSC lines derived from POI patients (n = 2) with an iPSC line derived from normal fibroblasts (n = 1).
  
  
  METHODS
  We established three iPSC lines from two patients with partial Xq deletion-induced POI and normal fibroblasts by overexpressing four factors: octamer-binding transcription factor 4 (OCT4), sex-determining region Y-box 2 (SOX2), Nanog homeobox (NANOG), and lin-28 homolog (LIN28), using lentiviral vectors. We then generated stable-transfected fluorescent reporter cell lines under the control of the Asp-Glu-Ala-Asp box polypeptide 4 (DDX4, also called VASA) promoter, and selected clonal derived sublines. We induced subline differentiation into germ cells by adding Wnt3a (30 ng/ml) and bone morphogenetic protein 4 (100 ng/ml). After 12 days of differentiation, green fluorescent protein (GFP)-positive and GFP-negative cells were isolated via fluorescence-activated cell sorting and analyzed for endogenous VASA protein (immunostaining) and for germ cell markers and genes expressed in the deleted region of the X chromosome (quantitative RT-PCR).
  
  
  RESULTS
  The POI- and normal fibroblast-derived iPSCs had typical self-renewal and pluripotency characteristics. After stable transfection with the VASA-GFP construct, the sublines POI1-iPS-V.1, POI2-iPS-V.1 and hEF-iPS-V.1 produced green fluorescent cells in the differentiated cultures, and the percentage of GFP-positive cells increased over the 12 days of differentiation to a maximum of 6.9 ± 0.33%, 5.3 ± 0.57% and 8.5 ± 0.29%, respectively, of the total cell population. Immunohistochemical analysis confirmed that endogenous VASA was enriched in the GFP-positive cells. Quantitative reverse transcription-PCR revealed significantly higher expression of germ cell markers [PR domain containing 1, with ZNF domain (PRDM1, BLIMP1), developmental pluripotency-associated 3 (DPPA3, STELLA), deleted in azoospermia-like (DAZL), and VASA (DDX4)] in GFP-positive cells than in GFP-negative cells. Moreover, the GFP-positive cells from POI-iPSCs had reduced expression of the family with sequence similarity 122C (FAM122C), inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma (IKBKG), and RNA binding motif protein, X-linked (RBMX), genes located in the deleted region of the X chromosome and that are highly expressed in differentiated germ cells, compared with cells from normal iPSCs.
  
  
  CONCLUSIONS
  Gene expression profiling indicated that the germ cells differentiated from POI-iPSCs were pre-meiotic. Therefore, how the differentiated primordial germ cells could progress further to meiosis and form follicles remains to be determined in the study of POI.
  
  
  CONCLUSIONS
  Our results might provide an in vitro model for studying germ cell development in patients with POI.
  
  
  BACKGROUND
  This work was supported by grants from the Major State Basic Research Development Program of China (No. 2012CB944901), the National Science Foundation of China (No. 81222007 and 81471432), the Program for New Century Excellent Talents in University and the Fundamental Research Funds for Central Universities (No. 721500003). The authors have no competing interests to declare.
  
  
  BACKGROUND
  Not applicable.
authors.affiliation: Institute of Reproductive & Stem Cell Engineering, Central South University, Changsha 410078, China Key Laboratory of Stem Cells and Reproductive Engineering, Ministry of Health, Changsha 410078, China.
authors.forename: Lizhi
authors.initials: L
authors.lastname: Leng
authors.affiliation: Institute of Reproductive & Stem Cell Engineering, Central South University, Changsha 410078, China Key Laboratory of Stem Cells and Reproductive Engineering, Ministry of Health, Changsha 410078, China.
authors.forename: Yueqiu
authors.initials: Y
authors.lastname: Tan
authors.affiliation: Institute of Reproductive & Stem Cell Engineering, Central South University, Changsha 410078, China Key Laboratory of Stem Cells and Reproductive Engineering, Ministry of Health, Changsha 410078, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Gong
authors.affiliation: Institute of Reproductive & Stem Cell Engineering, Central South University, Changsha 410078, China Key Laboratory of Stem Cells and Reproductive Engineering, Ministry of Health, Changsha 410078, China National Engineering and Research Center of Human Stem Cell, Changsha 410078, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Hu
authors.affiliation: Institute of Reproductive & Stem Cell Engineering, Central South University, Changsha 410078, China Key Laboratory of Stem Cells and Reproductive Engineering, Ministry of Health, Changsha 410078, China National Engineering and Research Center of Human Stem Cell, Changsha 410078, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Ouyang
authors.affiliation: National Engineering and Research Center of Human Stem Cell, Changsha 410078, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Institute of Reproductive & Stem Cell Engineering, Central South University, Changsha 410078, China Key Laboratory of Stem Cells and Reproductive Engineering, Ministry of Health, Changsha 410078, China National Engineering and Research Center of Human Stem Cell, Changsha 410078, China linggf@hotmail.com lugxdirector@aliyun.com.
authors.forename: Guangxiu
authors.initials: G
authors.lastname: Lu
authors.affiliation: Institute of Reproductive & Stem Cell Engineering, Central South University, Changsha 410078, China Key Laboratory of Stem Cells and Reproductive Engineering, Ministry of Health, Changsha 410078, China National Engineering and Research Center of Human Stem Cell, Changsha 410078, China linggf@hotmail.com lugxdirector@aliyun.com.
authors.forename: Ge
authors.initials: G
authors.lastname: Lin
mesh_terms: D000328:Adult; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D005260:Female; D059247:Fertility Preservation / Q000379:methods; D020869:Gene Expression Profiling; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D010063:Ovum / Q000166:cytology* / Q000378:metabolism; D016649:Primary Ovarian Insufficiency / Q000473:pathology*
keywords: VASA; differentiation; germ cells; induced pluripotent stem cells; primary ovarian insufficiency