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

Tingting Cheng,Yan Chang,Huiwen Wang,Yingpu Sun,Guangju Ji,Meng Jin,Jiahuan He,Bing Gong,Hang Xin,Guidong Yao,Fang Wang,Zhiguang Yang,Lei Gu,Kui Zhai,Huijuan Kong,Yanyun Wu

doi:10.18632/oncotarget.13958

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.

Highlights

Infertility affects approximately 10–15% of couples, with male factors accounting for 40–60% of all the cases
To access whether CHIR99021 and/or retinoic acid (RA) can induce Primordial germ cells (PGCs) from human embryonic stem cells (hESCs) (Figure 1A), deleted in azoospermia-like (DAZL) protein was initially detected by using immunofluorescence experiments
We showed that CHIR99021 combined with RA induced PGCs from hESCs

Summary

Introduction

Infertility affects approximately 10–15% of couples, with male factors accounting for 40–60% of all the cases. Germ cells have been used for studying spermatogenesis in vitro and pathological mechanisms of male infertility. Much progress has been made in the derivation of male differentiated germ cells from hESCs and induced pluripotent stem cells (iPSCs) [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17]. It is reported that male gametes derived from mESCs/iPSCs function to produce the viable offsprings suggesting www.impactjournals.com/oncotarget that there are possibilities to get germ cells in vitro from hESCs and to provide potential cures for male infertility. A comprehensive understanding of how to differentiate human male gametes is still urgent and necessary

Methods

Results

Conclusion