The crucial role of Activin A on the formation of primordial germ cell-like cells from skin-derived stem cells in vitro

Primordial germ cells (PGCs) are founder cells of the germ cell lineage, and can be differentiated from stem cells in an induced system <i>in vitro</i>. However, the induction conditions need to be optimized in order to improve the differentiation efficiency. Activin A (ActA) is a member of the TGF-β super family and plays an important role in oogenesis and folliculogenesis. In the present study, we found that ActA promoted PGC-like cells (PGCLCs) formation from mouse skin-derived stem cells (SDSCs) in both embryoid body-like structure (EBLS) differentiation and the co-culture stage in a dose dependent manner. ActA treatment (100 ng/ml) during EBLS differentiation stage and further co-cultured for 6 days without ActA significantly increased PGCLCs from 53.2% to 82.8%, and as well as EBLS differentiation without ActA followed by co-cultured with 100 ng/ml ActA for 4 to 12 days with the percentage of PGCLCs increasing markedly <i>in vitro</i>. Moreover, mice treated with ActA at 100 ng/kg body weight from embryonic day (E) 5.5–12.5 led to more PGCs formation. However, the stimulating effects of ActA were interrupted by <i>Smad3</i> RNAi, and in an <i>in vitro</i> cultured Smad3^−/− mouse skin cells scenario. SMAD3 is thus likely a key effecter molecule in the ActA signaling pathway. In addition, we found that the expression of some epiblast cell markers, <i>Fgf5, Dnmt3a, Dnmt3b</i> and <i>Wnt3</i>, was increased in EBLSs cultured for 4 days or PGCLCs co-cultured for 12 days with ActA treatment. Interestingly, at 16 days of differentiation, the percentage of PGCLCs was decreased in the presence of ActA, but the expression of meiosis-relative genes, such as <i>Stra8, Dmc1, Sycp3</i> and <i>Sycp1</i>, was increased. In conclusion, our data here demonstrated that ActA can promote PGCLC formation from SDSCs <i>in vitro</i>, at early stages of differentiation, and affect meiotic initiation of PGCLCs in later stages

原始生殖细胞（Primordial germ cells, PGCs）是生殖细胞谱系的创始细胞，可在体外诱导体系中由干细胞分化获得。然而，为提升分化效率，仍需优化其诱导条件。激活素A（Activin A, ActA）属于转化生长因子β（TGF-β）超家族成员，在卵子发生与卵泡发生过程中发挥重要作用。本研究发现，激活素A可通过剂量依赖性方式，在类胚体结构（embryoid body-like structure, EBLS）分化阶段与共培养阶段，促进小鼠皮肤来源干细胞（mouse skin-derived stem cells, SDSCs）向原始生殖细胞样细胞（PGC-like cells, PGCLCs）分化。在类胚体分化阶段施加100 ng/ml的激活素A，随后无需激活素A继续共培养6天，可使原始生殖细胞样细胞的生成比例从53.2%显著提升至82.8%；而在类胚体分化阶段不添加激活素A，后续于100 ng/ml激活素A中进行4至12天共培养，体外条件下原始生殖细胞样细胞的比例同样显著升高。此外，在胚胎发育第5.5至12.5天以100 ng/kg体重的剂量向小鼠注射激活素A，可促使更多原始生殖细胞生成。但激活素A的促分化效应可被Smad3 RNA干扰（RNAi）阻断，且在体外培养的Smad3基因敲除（Smad3^−/−）小鼠皮肤细胞中该效应消失。因此，SMAD3极有可能是激活素A信号通路中的关键效应分子。另外，我们发现，经激活素A处理、培养4天的类胚体，或经激活素A处理、共培养12天的原始生殖细胞样细胞中，部分上胚层细胞标志物（Fgf5、Dnmt3a、Dnmt3b及Wnt3）的表达水平均有所上调。值得注意的是，在分化第16天时，激活素A处理组的原始生殖细胞样细胞比例出现下降，但减数分裂相关基因（Stra8、Dmc1、Sycp3及Sycp1）的表达水平却有所升高。综上，本研究数据表明，激活素A可在体外分化早期阶段促进小鼠皮肤来源干细胞向原始生殖细胞样细胞分化，并在分化后期影响原始生殖细胞样细胞的减数分裂起始过程。