Department for Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium.
Department for Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium
Mol Hum Reprod. 2015 May;21(5):410-23. doi: 10.1093/molehr/gav004. Epub 2015 Jan 29.
Human embryonic stem cells (hESCs) derived in the presence of Activin A (ActA) demonstrate an increased differentiation propensity toward the germ cell lineage. In addition, mouse epiblast stem cells and mouse epiblast-like cells are poised toward germ cell differentiation and are derived in the presence of ActA. We therefore investigated whether supplementation with ActA enhances in vitro hESC differentiation toward germ cell lineage. ActA up-regulated early primordial germ cell (PGC) genes STELLA/DPPA3 (developmental pluripotency associated 3) and tyrosine kinase receptor cKIT in both ActA-derived and standard-derived hESCs indicating its role in priming hESCs toward the PGC lineage. Indeed, ActA plus bone morphogenic protein 4 (BMP4) strongly increased germ cell differentiation potential of hESCs based on the high expression of late PGC markers DAZL (deleted in azoospermia-like) and VASA/DDX4 (DEAD-box polypeptide 4) at mRNA and protein level. Hence, the combination of ActA with BMP4 provides an additional boost for hESCs to develop into postmigratory germ cells. Together with increased VASA expression in the presence of ActA and BMP4, we also observed up-regulation of endoderm-specific genes GATA4 (GATA binding protein 4) and GATA6. Finally, we were able to further mature these in vitro-derived PGC-like cells (PGCLCs) by culturing them in in vitro maturation (IVM) medium, resulting in the formation of germ cell-like clusters and induction of meiotic gene expression. In conclusion, we demonstrate for the first time a synergism between ActA and BMP4 in facilitating germ cell-directed differentiation of hESCs, which is enhanced by extended culture in IVM medium, as shown by cytoplasmic VASA-expressing PGCLCs. We propose a novel relationship between the endoderm and germ cell lineage during hESC differentiation.
人胚胎干细胞（hESCs）在激活素 A（ActA）的存在下衍生，表现出向生殖细胞谱系分化的增加倾向。此外，小鼠上胚层干细胞和小鼠类上胚层细胞倾向于生殖细胞分化，并在 ActA 的存在下衍生。因此，我们研究了 ActA 是否增强了体外 hESC 向生殖细胞谱系的分化。ActA 上调了早期原始生殖细胞（PGC）基因 STELLA/DPPA3（发育多能性相关 3）和酪氨酸激酶受体 cKIT，无论是在 ActA 衍生的还是标准衍生的 hESCs 中，这表明它在启动 hESC 向 PGC 谱系分化中的作用。事实上，ActA 加骨形态发生蛋白 4（BMP4）强烈增加了 hESC 的生殖细胞分化潜力，这是基于晚期 PGC 标志物 DAZL（在无精子症样缺失）和 VASA/DDX4（DEAD 框多肽 4）在 mRNA 和蛋白水平上的高表达。因此，ActA 与 BMP4 的组合为 hESC 向迁移后生殖细胞发育提供了额外的推动力。伴随着 ActA 和 BMP4 存在下 VASA 表达的增加，我们还观察到内胚层特异性基因 GATA4（GATA 结合蛋白 4）和 GATA6 的上调。最后，我们能够通过在体外成熟（IVM）培养基中培养这些细胞，进一步使这些体外衍生的 PGC 样细胞（PGCLCs）成熟，导致生殖细胞样簇的形成和减数基因表达的诱导。总之，我们首次证明了 ActA 和 BMP4 之间在促进 hESC 向生殖细胞定向分化方面的协同作用，这种作用通过在 IVM 培养基中的延长培养得到增强，这表现为细胞质中表达 VASA 的 PGCLCs。我们提出了 hESC 分化过程中内胚层和生殖细胞谱系之间的新关系。
