VPA promoted the SSCLC induction efficiency
We firstly differentiated hiPSCs into SSCLCs using the induction medium mainly containing human GDNF, human b-FGF and VC (VC group), and the SSCLC induction efficiency was determined by the percentage of PLZF+ cells at 12 days of differentiation as described [16]. VC increased the SSCLC induction efficiency compared with the induction medium containing GDNF and b-FGF without VC (G+F group) or with half concentration of VC (0.5 VC group) (Figure 1 A, B and C, Additional file Figure 2 A). We also differentiated H1 ESCs into SSCLCs using different induction medium and observed the similar results to that of hiPSCs (Additional file Figure 1). We noticed that different cell lines showed different SSCLC induction efficiency when used the same induction medium. The SSCLC induction efficiency of VC group in our hands was around 20%, and the induction efficiency could be improved. To achieve a higher induction efficiency, we considered to increase the concentrations of GDNF and b-FGF since these two cytokines have pivotal roles in regulating SSCs. A study reported that 20 and 40 ng/ml of GDNF could strongly promote the growth of mouse SSCs [12]. However, the SSCLC induction efficiency was not significantly changed when the concentrations of GDNF and b-FGF increased either alone or both (Additional file Figure 2), indicating that 20 ng/mL GDNF and 1 ng/mL b-FGF were sufficient in the induction medium.
We introduced VPA into the SSCLC induction medium to test whether VPA could elevate the SSCLC induction efficiency. The concentration of VPA used in previous studies to exert its effects on reprogramming and differentiation varied from 0.5 mM to 3 mM (72-432 μg/mL) [17, 32], and clinically relevant concentrations of VPA (0.5 mM or 1.0 mM) were sufficient to increase gene expression in neural differentiation [32]. We examined the effects of 100 and 200 μg/mL VPA (0.5 VPA group and VPA group, respectively) on the SSCLC induction efficiency. The SSCLC induction efficiency increased in 0.5 VPA and VPA groups compared with the G+F group, and was higher than the VC group; high concentration VPA was more effective (Figure 1 A, B and C). We further examined the effects of the combination of VPA and VC on the SSCLC induction efficiency. Interestingly, the combination of high concentrations of VPA and VC (200 μg/mL VPA and VC, VPA+VC group) did not achieve a higher SSCLC induction efficiency, but the combination of low concentrations of VPA and VC (100 μg/mL VPA and VC), that is, 0.5 (VPA+VC) group, greatly elevated the SSCLC induction efficiency (Figure 1 A, B and C). RT-qPCR confirmed the expression of PLZF in different groups, and PLZF expression was highest in the 0.5 (VPA+VC) group (Figure 1 C, p<0.05). Additionally, high concentration of VPA and the combination of VPA and VC did not increase cell apoptosis during differentiation compared with the VC group (Additional file Figure 3). VPA was able to promote the SSCLC induction efficiency in the presence of GDNF and b-FGF. The combination of low concentrations of VPA and VC achieved the highest SSCLC induction efficiency in this study, which could be used as the developed SSCLC induction protocol.
To further clarify the effects of the combination of low concentrations of VPA and VC on the SSCLC induction process, we used the developed SSCLC induction protocol to differentiate hiPSCs for 22 days. At 6 days of differentiation, a very small portion of SSCLCs emerged; the percentage of SSCLCs continued to increase from day 8 to day 12; after day 12, the percentage of SSCLCs began to decrease (Figure 2 A). Immunofluorescence was used to stain the expression of SSC-related genes (PLZF, GPR125, GFRα1, VASA and PIWIL2) (Figure 2 B). The expression of PLZF and GPR125 was weak at day 8 and increased at day 12. The expression of GFRα1 was also strong at day 12. At day 16, the staining of PLZF, VASA and PIWIL2 still existed. At day 20, the expression of PLZF and GPR125 was decreased. The PLZF+ cells were GPR125, GFRα1 and PIWIL2 positive and some GPR125, GFRα1 and PIWIL2 positive cells did not express PLZF, indicating that PLZF+ cells were SSCLCs and the SSCLC induction system generated a mix of different types of cells. Moreover, PLZF+ cells had round and relatively small nuclei; they grew in an aggregated manner and formed clusters. RT-qPCR results showed the same expression tendency of PLZF as the results of flow cytometry and immunofluorescence, and the expression of spermatogonial genes (ID4, GFRα1, NANOS2, TSPAN33, LPPR3 and DMRT1) also elevated during differentiation (Figure 2 C). In addition, except for SOX2, the expression of other pluripotent genes OCT4 and NANOG decreased along with differentiation (Figure 2 C). These results showed that the SSCLC induction process elevated spermatogonial gene expression and repressed pluripotent gene expression.
A relative high concentration of VPA induced further differentiation
In the previous studies, a very few meiotic cells and haploid cells appeared in the process of SSCLC induction [11, 16]. We examined whether meiotic or haploid cells existed in differentiated cells when using our developed protocol. The expression of meiotic gene SYCP3 slightly increased at 12 to 14 days of differentiation, but another gene STRA8 hardly expressed (Figure 2 C). At 12 days of differentiation, the expression of STRA8 and SYCP3 was higher in VPA and VPA+VC groups than that in VC and 0.5 (VPA+VC) groups (Figure 3 C). We used PI to stain cell DNA and detected the percentage of haploid cells by flow cytometry. A small fraction of haploid cells was generated at 12 days of differentiation in VC and 0.5 (VPA+VC) groups. Interestingly, we observed over 5% haploid cells in VPA and VPA+VC groups (Figure 3 A). Immunofluorescence detected SYCP3+ cells representing meiotic cells, and Acrosin+ or TNP1+ cells representing haploid cells in 0.5 (VPA+VC) group at 12 days of differentiation, but these cells accounted for a very small fraction of differentiated cells (Figure 3 B). These results indicated that cells might go through meiosis in our developed SSCLC induction system and a relative high concentration of VPA induced further differentiation.
Transcriptome analysis of hiPSCs and cells differentiated from hiPSCs
We compared the transcriptome of hiPSCs (Day 0) and cells at 12 days of differentiation (Day 12) to investigate changes in gene expression during SSCLC induction. There were 7702 DEGs in Day 0 cells compared with Day 12 cells, 4489 genes and 3212 genes of which were upregulated and downregulated, respectively (Figure 4 A). Gene ontology (GO) analysis had significantly enriched upregulated genes into development (e.g. nervous system development, anatomical structure development and multicellular organism development) and Wnt signaling pathway, and enriched downregulated genes into ribosome biogenesis and RNA processing (Figure 4 B). Several GO terms were related to nervous system and neurogenesis might because that GDNF has important roles (maintaining several neuronal populations) in the central nerves system and VPA promotes neural differentiation [33]. SSCLC induction process generated a mix of different types of cells. We found that at 12 days of differentiation, Wnt signaling pathway genes, primordial germ cell (PGC)-related genes, SSC-related genes and somatic genes were upregulated but pluripotent genes were downregulated (Figure 4 C). Spermatocyte-related genes were hardly detected (Figure 4 C). Moreover, DNMT3B expression was reduced and TETs were upregulated, and these genes have roles in DNA methylation and demethylation (Figure 4 C). RT-qPCR had confirmed that the expression of genes in Wnt signaling pathway and TETs was increased accompanied with the decreased expression of DNMT3B at 12 days of differentiation (Figure 4 D), indicating that SSCLC induction might involve Wnt signaling pathway activation and the change of genome methylation.
VPA affected histone modification during differentiation
A previous study reported that genome-wide increase in H3K9ac in mouse ESCs after treated with 0.5 mM VPA and H3K9ac is correlated with gene expression [34]. In the early human germ cell development, increased H3K9ac is accompanied with decreased H3K27me3 [35]. We explored whether these two histone marks were changed during differentiation. Western blot results showed increased H3K9ac and decreased H3K27me3 in cells at 12 days of differentiation using our developed protocol (Additional file Figure 4 A). The same results were observed in the VPA group, suggesting that VPA could affect histone modifications (Additional file Figure 4 A). We also determined the gene expression of class I HDACs (HDAC1, HDAC2 and HDAC3) and KDM6B (H3K27me3 demethylase) in cells at different days of differentiation (Additional file Figure 4 B). The expression of HDAC2 was decreased while the expression of KDM6B was increased along with differentiation, which might lead to altered histone modifications.
Differentiation of hiPSCs from NOA patients into SSCLCs
We next differentiated two hiPSC lines, which were from unrelated NOA patients with unknown cause and testicular histology, into SSCLCs using different protocols. These two cell lines showed different responses to different protocols. At 12 days of differentiation, 1106 hiPSCs could be differentiated into a higher percentage of SSCLCs using our developed protocol (Figure 5 A). 1122 hiPSCs were hardly to be differentiated into SSCLCs using different protocols (Figure 5 A), indicating that this patient might have serious abnormalities of SSCs and the disease cause is associated with SSCs. Immunofluorescence detected few PLZF+/GPR125+ cells in 1106 group at 12 days of differentiation and only few GPR125+ cells were observed in 1122 group (Figure 5 B). RT-qPCR results also showed the low expression of SSC-related genes and Wnt signaling pathway genes in the 1106 group (Figure 5 C). Compared with the normal hiPSC line differentiated at 12 days (Figure 2 A and C, Figure 4 D), the SSCLC induction efficiency and the expression of genes related to SSC and Wnt signaling pathway were reduced in the NOA hiPSC lines (Figure 5 A and C), implying the defects of Wnt signaling pathway activation were associated with low SSCLC induction efficiency.