Abstract
BackgroundStudying human germ cell development and male infertility is heavily relied on mouse models. In vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells (SSCLCs) can be used as a model to study human germ cells and infertility. The current study aimed to develop the SSCLC induction protocol and assess the effects of the developed protocol on SSCLC induction.MethodsWe examined the effects of valproic acid (VPA), vitamin C (VC) and the combination of VPA and VC on the SSCLC induction efficiency and determined the expression of spermatogonial genes of differentiated cells. Haploid cells and cells expressed meiotic genes were also detected. RNA-seq analysis was performed to compare the transcriptome between cells at 0 and 12 days of differentiation and differently expressed genes were confirmed by RT-qPCR. We further evaluated the alteration in histone marks (H3K9ac and H3K27me3) at 12 days of differentiation. Moreover, the SSCLC induction efficiency of two hiPSC lines of non-obstructive azoospermia (NOA) patients was assessed using different induction protocols.ResultsThe combination of low concentrations of VPA and VC in the induction medium was most effective to induce SSCLCs expressing several spermatogonial genes from human pluripotent stem cells at 12 days of differentiation. The high concentration of VPA was more effective to induce cells expressing meiotic genes and haploid cells. RNA-seq analysis revealed that the induction of SSCLC involved the upregulated genes in Wnt signaling pathway, and cells at 12 days of differentiation showed increased H3K9ac and decreased H3K27me3. Additionally, two hiPSC lines of NOA patients showed low SSCLC induction efficiency and decreased expression of genes in Wnt signaling pathway.ConclusionsVPA robustly promoted the differentiation of human pluripotent stem cells into SSCLCs, which involved the upregulated genes in Wnt signaling pathway and epigenetic changes. hiPSCs from NOA patients showed decreased SSCLC induction efficiency and Wnt signaling pathway gene expression, suggesting that SSC depletion in azoospermia testes might be associated with inactivation of Wnt signaling pathway. Our developed SSCLC induction protocol provides a reliable tool and model to study human germ cell development and male infertility.
Highlights
Azoospermia referring to the complete lack of sperm in the ejaculate represents 10–20% cases of male infertility, and over 70% azoospermia patients are non-obstructive azoospermia (NOA) caused by the failure of spermatogenesis [1]
valproic acid (VPA) robustly promoted the differentiation of human pluripotent stem cells into Spermatogonial stem cell-like cell (SSCLC), which involved the upregulated genes in Wnt signaling pathway and epigenetic changes. Human induced pluripotent stem cell (hiPSC) from NOA patients showed decreased SSCLC induction efficiency and Wnt signaling pathway gene expression, suggesting that Spermatogonial stem cell (SSC) depletion in azoospermia testes might be associated with inactivation of Wnt signaling pathway
Using the model of differentiation of hiPSCs into SSCLCs, we found that Wnt signaling pathway was involved in SSCLC induction, and low SSCLC induction efficiency of NOA hiPSC lines might be associated with inactivation of Wnt signaling pathway
Summary
Azoospermia referring to the complete lack of sperm in the ejaculate represents 10–20% cases of male infertility, and over 70% azoospermia patients are non-obstructive azoospermia (NOA) caused by the failure of spermatogenesis [1]. In a variety of mouse models, the abnormalities of SSCs cause the absence of testicular germ cells and result in azoospermia, which is similar to that of the most severe form of NOA called Sertoli cell-only syndrome (SCOS) [3,4,5,6,7]. Less is known about the etiology and pathogenesis of human azoospermia and the development of diagnosis and therapy is faced with difficulties. Using human SSCs to study human azoospermia will provide direct and effective information. Studying human germ cell development and male infertility is heavily relied on mouse models. In vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells (SSCLCs) can be used as a model to study human germ cells and infertility. The current study aimed to develop the SSCLC induction protocol and assess the effects of the developed protocol on SSCLC induction
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