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Abstract：
Optimizing the efficiency of definitive endoderm (DE) differentiation is necessary for the generation of diverse organ-like structures. In this study, we used the small molecule inhibitor saracatinib (SAR) to enhance DE differentiation of human embryonic stem cells and induced pluripotent stem cells. SAR significantly improved DE differentiation efficiency at low concentrations. The interaction between SAR and Focal Adhesion Kinase (FAK) was explored through RNA-seq and molecular docking simulations, which further supported the inhibition of DE differentiation by p-FAK overexpression in SAR-treated cells. In addition, we found that SAR inhibited the nuclear translocation of Yes-associated protein (YAP), a downstream effector of FAK, which promoted DE differentiation. Moreover, the addition of SAR enabled a significant reduction in activin A (AA) from 50 to 10 ng/mL without compromising DE differentiation efficiency. For induction of the pancreatic lineage, 10 ng/ml AA combined with SAR at the DE differentiation stage yielded a comparative number of PDX1+/NKX6.1+ pancreatic progenitor cells to those obtained by 50 ng/ml AA treatment. Our study highlights SAR as a potential modulator that facilitates the cost-effective generation of DE cells and provides insight into the orchestration of cell fate determination.
摘要：
优化定形内胚层（DE）分化的效率对于产生不同的器官样结构是必要的。在这项研究中,我们使用小分子抑制剂saracatinib(SAR)增强人胚胎干细胞的DE分化和诱导多能干细胞。SAR在低浓度下显着提高了DE分化效率。通过RNA-seq和分子对接模拟探讨了SAR与粘着斑激酶(FAK)的相互作用,这进一步支持在SAR处理的细胞中通过p-FAK过表达抑制DE分化。此外,我们发现SAR抑制Yes相关蛋白（YAP）的核易位，FAK的下游效应器，这促进了DE分化。此外，SAR的添加使得活化素A(AA)从50ng/mL显著降低至10ng/mL,而不损害DE分化效率。为了诱导胰腺谱系，在DE分化阶段，10ng/mlAA与SAR结合产生的PDX1/NKX6.1胰腺祖细胞数量与通过50ng/mlAA处理获得的细胞数量相当。我们的研究强调了SAR作为一种潜在的调节剂，可以促进DE细胞的成本效益产生，并提供了对细胞命运决定的编排的见解。
