title: CHIR99021 combined with retinoic acid promotes the differentiation of primordial germ cells from human embryonic stem cells.
abstract: Primordial germ cells (PGCs) derived from human embryonic stem cells (hESCs) represent as a desirable experimental model as well as a potential strategy for treating male infertility. Here, we developed a simple and feasible method for differentiation of PGCs from hESCs by using CHIR99021 (an inhibitor of glycogen synthase kinase 3) and retinoic acid (RA). We firstly found that the deleted in azoospermia-like (DAZL) protein can be detected in 3 d CHIR99021 plus 9 d retinoic acid treated cultures and 12 d CHIR99021 plus retinoic acid co-treated cultures, but not expressed in single CHIR99021 treated cultures, single retinoic acid treated cultures, as well as 3 d retinoic acid plus 9 d CHIR99021 treated cultures. Next, we showed that several PGCs' markers were expressed in the 12 d CHIR99021 and retinoic acid co-treated cultures or 3 d CHIR99021 plus 9 d retinoic acid treated cultures. Moreover, meiosis was initiated in CHIR99021 and retinoic acid co-treated cultures as evidenced by a significant expression of the punctate synaptonemal complex protein 3 (SCP3). Fluorescent in situ hybridization (FISH) analysis indicated that a small percentage of putative 1N populations were formed. Mechanically, we found that β-catenin relocated into nucleus after the treatment of 3 d CHIR99021 suggesting that Wnt signaling pathway was activated. Furthermore, blockade of Wnt signaling pathway by IWR-1 can reverse CHIR99021 and retinoic acid mediated-effects. Taken together, our results indicate that CHIR99021 combined with retinoic acid can effectively differentiate hESCs into PGCs via activating Wnt signaling pathway.
authors.affiliation: Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Cheng
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Kui
authors.initials: K
authors.lastname: Zhai
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chang
authors.affiliation: Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
authors.forename: Guidong
authors.initials: G
authors.lastname: Yao
authors.affiliation: Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
authors.forename: Jiahuan
authors.initials: J
authors.lastname: He
authors.affiliation: Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Wang
authors.affiliation: Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Kong
authors.affiliation: Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
authors.forename: Hang
authors.initials: H
authors.lastname: Xin
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Huiwen
authors.initials: H
authors.lastname: Wang
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Jin
authors.affiliation: Department of Cardiac Surgery, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Gong
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Gu
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Zhiguang
authors.initials: Z
authors.lastname: Yang
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Yanyun
authors.initials: Y
authors.lastname: Wu
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Guangju
authors.initials: G
authors.lastname: Ji
authors.affiliation: Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
authors.forename: Yingpu
authors.initials: Y
authors.lastname: Sun
mesh_terms: D021581:Active Transport, Cell Nucleus; D018797:Cell Cycle Proteins; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D004268:DNA-Binding Proteins; D018507:Gene Expression Regulation, Developmental; D000071679:Glycogen Synthase Kinase 3 beta / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006238:Haploidy; D000066449:Human Embryonic Stem Cells / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D008297:Male; D008540:Meiosis / Q000187:drug effects; D009687:Nuclear Proteins / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D016601:RNA-Binding Proteins / Q000378:metabolism; D013094:Spermatozoa / Q000187:drug effects* / Q000201:enzymology; D013997:Time Factors; D014212:Tretinoin / Q000494:pharmacology*; D060449:Wnt Signaling Pathway / Q000187:drug effects; D051176:beta Catenin / Q000378:metabolism
chemical_list: C495270:CTNNB1 protein, human; D018797:Cell Cycle Proteins; C473711:Chir 99021; C101358:DAZL protein, human; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D011743:Pyrimidines; D016601:RNA-Binding Proteins; C490060:SYCP3 protein, human; D051176:beta Catenin; D014212:Tretinoin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta
keywords: CHIR99021; human embryonic stem cells; primordial germ cells; retinoic acid; β-catenin