Helmholtz-Institute for Biomedical Engineering, Medical Faculty of RWTH Aachen University, 52074 Aachen, Germany; Institute for Stem Cell Biology, University Hospital of RWTH Aachen, 52074 Aachen, Germany.
Helmholtz-Institute for Biomedical Engineering, Medical Faculty of RWTH Aachen University, 52074 Aachen, Germany; Institute for Stem Cell Biology, University Hospital of RWTH Aachen, 52074 Aachen, Germany; PL BioScience, Technology Centre Aachen, 52068 Aachen, Germany.
Biomater Adv. 2023 Mar;146:213308. doi: 10.1016/j.bioadv.2023.213308. Epub 2023 Jan 25.
Induced pluripotent stem cells (iPSCs) form aggregates that recapitulate aspects of the self-organization in early embryogenesis. Within few days, cells undergo a transition from epithelial-like structures to organized three-dimensional embryoid bodies (EBs) with upregulation of germ layer-specific genes. However, it is largely unclear, which signaling cascades regulate self-organized differentiation. The Yes-associated protein 1 (YAP1) is a downstream effector of the Hippo pathway and essential mechanotransducer. YAP1 has been suggested to play a crucial role for early embryo development, but the relevance for early germ layer commitment of human iPSCs remains to be elucidated. To gain insights into the function of YAP1 in early cell-fate decisions, we generated YAP1 knockout (YAP) iPSC lines with CRISPR/Cas9 technology and analyzed transcriptomic and epigenetic modifications. YAP iPSCs showed increased expression of several YAP1 targets and of NODAL, an important regulator of cell differentiation. Furthermore, YAP1 deficiency evoked global DNA methylation changes. Directed differentiation of adherent iPSC colonies towards endoderm, mesoderm, and ectoderm could be induced, albeit endodermal and ectodermal differentiation showed transcriptomic and epigenetic changes in YAP lines. Notably, in undirected self-organized YAP EBs germ layer specification was clearly impaired. This phenotype was rescued via lentiviral overexpression of YAP1 and also by NODAL inhibitors. Our results demonstrate that YAP1 plays an important role during early germ layer specification of iPSCs, particularly for the undirected self-organization of EBs, and this is at least partly attributed to activation of the NODAL signaling.
诱导多能干细胞（iPSCs）形成聚集体，重现早期胚胎发生中的自我组织的某些方面。在几天内，细胞从上皮样结构转变为具有上调的胚层特异性基因的组织三维胚状体（EBs）。然而，调控自我组织分化的信号级联在很大程度上尚不清楚。Yes 相关蛋白 1（YAP1）是 Hippo 通路的下游效应物，也是必需的机械转导蛋白。YAP1 被认为对早期胚胎发育起关键作用，但对于人类 iPSCs 早期胚层承诺的相关性仍有待阐明。为了深入了解 YAP1 在早期细胞命运决定中的功能，我们使用 CRISPR/Cas9 技术生成了 YAP1 敲除（YAP）iPSC 系，并分析了转录组和表观遗传修饰。YAP iPSCs 显示出几个 YAP1 靶基因的表达增加，以及细胞分化的重要调节剂 NODAL 的表达增加。此外，YAP1 缺失引起了全局 DNA 甲基化变化。可以诱导贴壁 iPSC 集落向内胚层、中胚层和外胚层的定向分化，尽管在 YAP 系中，内胚层和外胚层分化显示出转录组和表观遗传变化。值得注意的是，在未定向的自我组织的 YAP EB 中，胚层特化明显受损。这种表型通过 YAP1 的慢病毒过表达和 NODAL 抑制剂得到挽救。我们的结果表明，YAP1 在 iPSCs 的早期胚层特化中起重要作用，特别是对于 EB 的未定向自我组织，这至少部分归因于 NODAL 信号的激活。
