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.

Keywords: CHIR99021; human embryonic stem cells; primordial germ cells; retinoic acid; β-catenin.

Figure 1. CHIR99021 combined with RA can induce the PGCs from hESCs

(A) The protocol used for the differentiation of hESCs into PGCs. (B) The hESCs were treated by CHIR99021 and/or RA as indicated in (A). The DAZL positive cells were detected by using immunofluorescence staining nuclei were counterstained with DAPI. Scale bars, 25 μm. (C) Summary data showing the mean number of the DAZL positive cells in the different treatments. All data are presented as means ± SEM. *p < 0.05, **p < 0.01.

Figure 2. DAZL-positive cells have a phenotype comparable to that of migretory PGCs

(A) mRNA levels of DDX4, Blimp-1, Nanos and TFAP2C was determined by RT-qPCR. The two hESCs lines were treated by 12 d CHIR99021 plus RA co-culture and 3 d CHIR99021 plus 9 d RA. The undifferentiated hESCs were used as control. (B) The two hESCs lines were treated by 12 d CHIR99021 plus RA co-culture or 3 d CHIR99021 plus 9 d RA. The undifferentiated hESCs were used as control. Then, Acrosin, DDX4, β-catenin and β-actin were detected by Immunoblots. All data are presented as means ± SEM in three independent experiments vs undifferentiated hESCs. (C–E) Flow cytometry analysis of c-Kit⁺, CXCR4⁺ and DAZL⁺ cells. The undifferentiated hESCs were used as a control shown on the left. The percentage shown in the histogram is the rate of positive cells. The middle icon represents 3 d CHIR99021 plus 9 d RA, the right icon stands for 12 d CHIR99021 and RA co-culture treatment. All data are presented as means ± SEM in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. Putative haploid cells were formed in this induced process

(A) Immunofluorescence staining with SCP3 is shown. Scale bars, 10 μm. (B) FISH with probe against autosomal chromosome 16 and chromosome 22 in the undifferentiated hESCs group and two induced groups. Scale bars, 10 μm.

Figure 4. CHIR99021 activated Wnt signaling pathway via a long-term manner

(A) Immunofluorescence analysis showing the expression of OCT4, DAZL, and β-catenin after the treatment of 3 d CHIR99021. Scale bars, 25 μm. (B) Statistical analysis of the percentage of β-catenin in the nuclei. All data are presented as means ± SEM. *p < 0.05, **p < 0.01,***p < 0.001.

Figure 5. IWR-1 inhibited the accumulation of β-catenin in the nuclei and decreased the number of DAZL positive cells

(A) Immunofluorescence straining of β-catenin and OCT4 in 3 d CHIR99021 plus IWR-1 co-culture group and undifferentiated group. Scale bars, 50 μm. (B) Immunofluorescence straining of DAZL in 12 d CHIR99021, IWR-1 and RA co-culture group and undifferentiated group. Scale bars, 50 μm.

Figure 6. The proposed working model