Exogenous supplementation of Activin A enhances germ cell differentiation of human embryonic stem cells
Exogenous supplementation of Activin A enhances germ cell differentiation of human embryonic stem cells
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DOI：
10.1093/molehr/gav004
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发表时间：
2015-05-01
影响因子：
4
通讯作者：
De Sutter, Petra
中科院分区：
文献类型：
--
作者：
Duggal, Galbha;Heindryckx, Bjorn;De Sutter, Petra
在激活素A（ActA）存在下衍生的人胚胎干细胞（hESC）表现出向生殖细胞谱系的增加的分化倾向。此外，小鼠外胚干细胞和小鼠外胚样细胞正准备向生殖细胞分化，并在ActA存在的情况下衍生。因此，我们研究了补充ActA是否增强体外hESC向生殖细胞谱系的分化。ActA上调早期原始生殖细胞（PGC）基因STELLA/DPPA 3（发育多能性相关3）和酪氨酸激酶受体cKIT在ActA衍生的和标准衍生的hESC表明其在启动hESC向PGC谱系的作用。事实上，ActA加骨形态发生蛋白4（BMP 4）基于晚期PGC标志物DAZL（在无精子症样中缺失）和VASA/DDX 4（DEAD盒多肽4）在mRNA和蛋白质水平的高表达强烈增加hESC的生殖细胞分化潜能。因此，ActA与BMP 4的组合为hESC发育成迁移后生殖细胞提供了额外的促进。与ActA和BMP 4存在下VASA表达增加一起，我们还观察到内胚层特异性基因GATA 4（加塔结合蛋白4）和GATA 6的上调。最后，我们能够通过在体外成熟（IVM）培养基中培养这些体外衍生的PGC-like细胞（PGCLC）来进一步成熟，从而形成生殖细胞样簇并诱导减数分裂基因表达。总之，我们第一次证明了ActA和BMP 4之间的协同作用，促进生殖细胞定向分化的hESC，这是通过在IVM培养基中延长培养增强，如细胞质VASA表达PGCLC所示。我们提出了一种新的内胚层和生殖细胞谱系之间的关系在hESC分化。
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.