Valproic acid promotes the in vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells

Wang, Xiaotong; Qu, Mengyuan; Li, Zili; Long, Yuting; Hong, Kai; Li, Honggang

doi:10.1186/s13287-021-02621-1

Cited by 13 publications

(12 citation statements)

References 67 publications

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“…These two hiPSC lines have been described elsewhere [ 35 , 36 ]. The homozygous c.731_732delAT variant in PIWIL2 in the hiPSCs of the patient was confirmed using Sanger sequencing, and the normal hiPSCs were of wild-type at the same location [ 26 ]. The hiPSCs were seeded on Matrigel (Corning, USA)-covered plates and maintained in mTeSR 1 medium (STEMCELL Technologies, Canada) at 37 °C and 5% CO 2 , and the medium was changed every day.…”

Section: Methodsmentioning

confidence: 99%

A homozygous PIWIL2 frameshift variant affects the formation and maintenance of human-induced pluripotent stem cell-derived spermatogonial stem cells and causes Sertoli cell-only syndrome

Wang

et al. 2022

Stem Cell Res Ther

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Background The most serious condition of male infertility is complete Sertoli cell-only syndrome (SCOS), which refers to the lack of all spermatogenic cells in the testes. The genetic cause of SCOS remains to be explored. We aimed to investigate the genetic cause of SCOS and assess the effects of the identified causative variant on human male germ cells. Methods Whole-exome sequencing was performed to identify potentially pathogenic variants in a man with complete SCOS, and Sanger sequencing was performed to verify the causative variant in this man and his father and brother. The pathogenic mechanisms of the causative variant were investigated by in vitro differentiation of human-induced pluripotent stem cells (hiPSCs) into germ cell-like cells. Results The homozygous loss-of-function (LoF) variant p.His244ArgfsTer31 (c.731_732delAT) in PIWIL2 was identified as the causative variant in the man with complete SCOS, and the same variant in heterozygosis was confirmed in his father and brother. This variant resulted in a truncated PIWIL2 protein lacking all functional domains, and no PIWIL2 expression was detected in the patient’s testes. The patient and PIWIL2−/− hiPSCs could be differentiated into primordial germ cell-like cells and spermatogonial stem cell-like cells (SSCLCs) in vitro, but the formation and maintenance of SSCLCs were severely impaired. RNA-seq analyses suggested the inactivation of the Wnt signaling pathway in the process of SSCLC induction in the PIWIL2−/− group, which was validated in the patient group by RT-qPCR. The Wnt signaling pathway inhibitor hindered the formation and maintenance of SSCLCs during the differentiation of normal hiPSCs. Conclusions Our study revealed the pivotal role of PIWIL2 in the formation and maintenance of human spermatogonial stem cells. We provided clinical and functional evidence that the LoF variant in PIWIL2 is a genetic cause of SCOS, which supported the potential role of PIWIL2 in genetic diagnosis. Furthermore, our results highlighted the applicability of in vitro differentiation models to function validation experiments.

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Section: Methodsmentioning

confidence: 99%

A homozygous PIWIL2 frameshift variant affects the formation and maintenance of human-induced pluripotent stem cell-derived spermatogonial stem cells and causes Sertoli cell-only syndrome

Wang

et al. 2022

Stem Cell Res Ther

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“…These two hiPSC lines have been described elsewhere [31,32]. The homozygous c.731_732delAT variant in PIWIL2 in hiPSCs of the patient was con rmed using Sanger sequencing, and the normal hiPSCs were wild-type at the same location [22]. The hiPSCs were seeded on Matrigel (Corning, USA)-covered plates and maintained in mTeSR 1 medium (STEMCELL Technologies, Canada) at 37 °C, 5% CO 2 , and the medium was changed every day.…”

Section: Cell Culturementioning

confidence: 99%

“…In vitro differentiation of hiPSCs into PGCLCs and SSCLCs was performed as previously described [21,22].…”

Section: In Vitro Differentiation Of Hipscs Into Germ Cell-like Cellsmentioning

confidence: 99%

“…PGCLCs were dissociated and stained with ITGA6 (BD, USA) and EpCAM (BD Biosciences, USA) primary antibodies (Additional File Table 2) [21]. SSCLCs were dissociated with Accutase (Thermo Fisher Scienti c, USA) and stained with PLZF (Invitrogen, USA) primary antibody (Additional File Table 2) after xing, permeabilizing and blocking processes [22]. The percentages of PGCLCs and SSCLCs were determined by a ow cytometer (Beckman Coulter, USA).…”

Section: Flow Cytometrymentioning

confidence: 99%

“…Differentiation of human induced pluripotent stem cells (hiPSCs) into human male germ cell-like cells could provide a better in vitro disease model to study male infertility [19]. This approach bypasses the ethical and technical problems on using human primary germ cells for functional experiments, also generates large numbers of primordial germ cell-like cells (PGCLCs) and spermatogonial stem cell-like cells (SSCLCs) in a short time [20][21][22][23][24]. Furthermore, this approach could be a in vitro model to study genotype-phenotype correlations because hiPSCs and differentiated cells retain patient's genetic background.…”

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confidence: 99%

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A homozygous PIWIL2 frameshift variant affects the formation and maintenance of human induced pluripotent stem cell-derived spermatogonial stem cells and causes Sertoli cell only-syndrome

Wang

et al. 2022

Preprint

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Background The most serious condition of male infertility is complete Sertoli cell-only syndrome (SCOS), referring to lack of all spermatogenic cells in testes. Genetic cause of SCOS remains to be explored. We aimed to investigate the genetic cause of SCOS and assess the effects of the identified causative variant on human male germ cells. Methods Whole-exome sequencing was performed to identify potentially pathogenic variants in a man with complete SCOS and Sanger sequencing was performed to verify the causative variant in this man and his father and brother. The pathogenic mechanisms of the causative variant were investigated by in vitro differentiation of human induced pluripotent stem cells (hiPSCs) into germ cell-like cells. Results The homozygous loss-of function (LoF) variant p.His244ArgfsTer31 (c.731_732delAT) in PIWIL2 was identified as the causative variant in the man with complete SCOS, and the same variant in heterozygosis was confirmed in his father and brother. This variant resulted in a truncated PIWIL2 protein lacking of all functional domains, and no PIWIL2 expression was detected in the patient’s testes. The patient and PIWIL2−/− hiPSCs could be differentiated into primordial germ cell-like cells and spermatogonial stem cell-like cells (SSCLCs) in vitro, but the formation and maintenance of SSCLCs were severely impaired. RNA-seq analyses proposed inactivation of the Wnt signaling pathway in the process of SSCLC induction in the PIWIL2−/− group, which was validated in the patient group by RT-qPCR. The Wnt signaling pathway inhibitor hindered the formation and maintenance of SSCLCs during the differentiation of normal hiPSCs. Conclusions Our study revealed the pivotal role of PIWIL2 in the formation and maintenance of human spermatogonial stem cells. We provided clinical and functional evidence that the LoF variant in PIWIL2 is a genetic cause of SCOS, which supported the potential role of PIWIL2 in genetic diagnosis. Furthermore, our results highlighted the applicability of in vitro differentiation models to function validation experiments.

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DNA hypomethylation activation Wnt/TCF7L2/TDRD1 pathway promotes spermatogonial stem cell formation

Gao

Shi

Zhou

et al. 2022

Journal Cellular Physiology

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Detailed analysis of the regulatory mechanism of spermatogonia stem cell (SSCs) genesis can provide a novel strategy for the application of SSCs in the fields of transgenic animal production and regenerative medicine. Previous studies in this study showed that WNT signaling can positively regulate the formation of SSCs, but the exact regulatory mechanism is not clear. Here, we predicted the target gene of the Wnt/TCF7L2 pathway, namely TDRD1, by bioinformatics analysis. Functional studies revealed that overexpression of TDRD1 during RA-induced SSCs formation in vitro significantly upregulated the expression of reproductive marker genes (Integrinβ1 and Integrinα6), and further flow cytometric analysis also confirmed that the formation efficiency of SSCs was significantly increased after overexpression of TDRD1; while interference with TDRD1 showed the exact opposite result. The in vivo experiments were consistent with the results of the in vitro experiments.Interestingly, although Wnt/TCF7L2 can promote the formation of SSCs, its function must be dependent on the expression of TDRD1, which was also repeatedly demonstrated as a target gene of the Wnt/TCF7L2 signaling pathway. Mechanistically, we found a large number of CpG sites in the TDRD1 promoter, and BSP analysis also confirmed that DNA methylation modifications in the TDRD1 promoter were significantly higher in embryonic stem cells than in SSCs, and further dualluciferase reporter system assays revealed that low DNA methylation modification levels could enhance TDRD1 promoter activity; although previous studies demonstrated that TCF7L2 could enrich in the TDRD1 promoter region, the binding of the two was dependent on low DNA methylation modification. Taken together, we confirmed that low DNA methylation mediates Wnt/TCF7L2 regulation of TDRD1 to promote the formation of SSCs, providing a basis for SSCs in improving animal productivity.

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Valproic acid promotes the in vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells

Cited by 13 publications

References 67 publications

A homozygous PIWIL2 frameshift variant affects the formation and maintenance of human-induced pluripotent stem cell-derived spermatogonial stem cells and causes Sertoli cell-only syndrome

A homozygous PIWIL2 frameshift variant affects the formation and maintenance of human-induced pluripotent stem cell-derived spermatogonial stem cells and causes Sertoli cell-only syndrome

A homozygous PIWIL2 frameshift variant affects the formation and maintenance of human induced pluripotent stem cell-derived spermatogonial stem cells and causes Sertoli cell only-syndrome

DNA hypomethylation activation Wnt/TCF7L2/TDRD1 pathway promotes spermatogonial stem cell formation

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