CHIR99021 combined with retinoic acid promotes the differentiation of primordial germ cells from human embryonic stem cells

// Tingting Cheng 1 , Kui Zhai 2 , Yan Chang 2 , Guidong Yao 1 , Jiahuan He 1 , Fang Wang 1 , Huijuan Kong 1 , Hang Xin 1 , Huiwen Wang 2 , Meng Jin 2 , Bing Gong 3 , Lei Gu 2 , Zhiguang Yang 2 , Yanyun Wu 2 , Guangju Ji 2 , Yingpu Sun 1 1 Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China 2 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China 3 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 Correspondence to: Guangju Ji, email: gj28@ibp.ac.cn Yingpu Sun, email: syp2008@vip.sina.com Keywords: CHIR99021, retinoic acid, primordial germ cells, human embryonic stem cells, β-catenin Received: September 24, 2016      Accepted: December 01, 2016      Published: December 15, 2016 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.