Generation of male germ cells in vitro from the stem cells / 亚洲男科学杂志(英文版)

Infertility has become a serious disease since it affects 10%-15% of couples worldwide, and male infertility contributes to about 50% of the cases. Notably, a significant decrease occurs in the newborn population by 7.82 million in 2020 compared to 2016 in China. As such, it is essential to explore the effective methods of obtaining functional male gametes for restoring male fertility. Stem cells, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), spermatogonial stem cells (SSCs), and mesenchymal stem cells (MSCs), possess the abilities of both self-renewal and differentiation into germ cells. Significantly, much progress has recently been achieved in the generation of male germ cells in vitro from various kinds of stem cells under the specified conditions, e.g., the coculturing with Sertoli cells, three-dimensional culture system, the addition of growth factors and cytokines, and/or the overexpression of germ cell-related genes. In this review, we address the current advance in the derivation of male germ cells in vitro from stem cells based on the studies of the peers and us, and we highlight the perspectives and potential application of stem cell-derived male gametes in reproductive medicine.

Characterization, isolation, and culture of spermatogonial stem cells in / 亚洲男科学杂志(英文版)

Spermatogonial stem cells (SSCs) have great applications in both reproductive and regenerative medicine. Primates including monkeys are very similar to humans with regard to physiology and pathology. Nevertheless, little is known about the isolation, the characteristics, and the culture of primate SSCs. This study was designed to identify, isolate, and culture monkey SSCs. Immunocytochemistry was used to identify markers for monkey SSCs. Glial cell line-derived neurotrophic factor family receptor alpha-1 (GFRA1)-enriched spermatogonia were isolated from monkeys, namely Macaca fascicularis (M. fascicularis), by two-step enzymatic digestion and magnetic-activated cell sorting, and they were cultured on precoated plates in the conditioned medium. Reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and RNA sequencing were used to compare phenotype and transcriptomes in GFRA1-enriched spermatogonia between 0 day and 14 days of culture, and xenotransplantation was performed to evaluate the function of GFRA1-enriched spermatogonia. SSCs shared some phenotypes with rodent and human SSCs. GFRA1-enriched spermatogonia with high purity and viability were isolated from M. fascicularis testes. The freshly isolated cells expressed numerous markers for rodent SSCs, and they were cultured for 14 days. The expression of numerous SSC markers was maintained during the cultivation of GFRA1-enriched spermatogonia. RNA sequencing reflected a 97.3% similarity in global gene profiles between 0 day and 14 days of culture. The xenotransplantation assay indicated that the GFRA1-enriched spermatogonia formed colonies and proliferated in vivo in the recipient c-Kit

Inhibition of in vitro carcinogenicity by 8-bromo-7-methoxychrysin mediated by modulating miR-519d/TWIST1 axis in liver cancer stem-like cells from SMMC-7721 cell line / 中国药理学通报

Aim To determine whether 8-bromo-7-me-thoxychrysin ( BrMC) inhibits in vitro carcinogenicity via up-regulating miR-519d expression and down-regu-lating Twist1 expression in liver cancer stem-like cells ( LCSLCs) derived from SMMC-7721 cell line. Meth-ods The second generation spheroids derived from SMMC-7721 cell line were obtained by sphere-forming assay and were considered as LCSLCs . Then LCSLCs were treated with various concentrations ( 1.0, 3.0, 10.0 μmol·L-1) of BrMC. The expression level of miR-519d was detected using real-time PCR. And in vitro carcinogenicity was investigated by sphere-forming assay and clone-forming assay in agar. The transcrip-tional activity and protein expression of Twist1 were an-alyzed using luciferase reporter assay and Western blot. Moreover, the molecular mechanism of BrMC was elucidated via miR-519 mimic transfection and Twist1 gene transduction, respectively. Results Compared with SMMC-7721 cells, miR-519d-3p was low-ex-pressed and Twist1 was over expressed in LCSLCs. And the sphere-forming ratio and the clone-forming ra-tio decreased by treatment with BrMC ( 1.0, 3.0, 10.0 μmol·L-1) in a dose-dependent manner. Fur-thermore, luciferase reporter assay demonstrated miR-519d could directly target the 3′ untranslated region of Twist1 mRNA and regulate protein expression. miR-519d mimic enhanced the effects of BrMC (3.0 μmol ·L-1) . However, Twist1 gene transduction effective-ly reversed the effects of BrMC ( 3.0 μmol·L-1) . Conclusion BrMC inhibits in vivo carcinogenicity via regulating miR-519/Twist1 signal axis in LCSLCs de-rived from SMMC-7721 cell line.