Generation of insulin-producing cells from human pluripotent stem cells (hPSCs) in vitro would be useful for drug discovery and cell therapy in diabetes. Three-dimensional (3D) culture is important for the acquisition of mature insulin-producing cells from hPSCs, but the mechanism by which it promotes β cell maturation is poorly understood. We established a stepwise method to induce high-efficiency differentiation of human embryonic stem cells (hESCs) into mature monohormonal pancreatic endocrine cells (PECs), with the last maturation stage in 3D culture. To comprehensively compare two-dimensional (2D) and 3D cultures, we examined gene expression, pancreas-specific markers, and functional characteristics in 2D culture-induced PECs and 3D culture-induced PECs. The mechanisms were considered from the perspectives of cell–cell and cell–extracellular matrix interactions which are fundamentally different between 2D and 3D cultures. The expression of the pancreatic endocrine-specific transcription factors PDX1, NKX6.1, NGN3, ISL1, and PAX6 and the hormones INS, GCG, and SST was significantly increased in 3D culture-induced PECs. 3D culture yielded monohormonal endocrine cells, while 2D culture-induced PECs co-expressed INS and GCG or INS and SST or even expressed all three hormones. We found that focal adhesion kinase (FAK) phosphorylation was significantly downregulated in 3D culture-induced PECs, and treatment with the selective FAK inhibitor PF-228 improved the expression of β cell-specific transcription factors in 2D culture-induced PECs. We further demonstrated that 3D culture may promote endocrine commitment by limiting FAK-dependent activation of the SMAD2/3 pathway. Moreover, the expression of the gap junction protein Connexin 36 was much higher in 3D culture-induced PECs than in 2D culture-induced PECs, and inhibition of the FAK pathway in 2D culture increased Connexin 36 expression. We developed a strategy to induce differentiation of monohormonal mature PECs from hPSCs and found limited FAK-dependent activation of the SMAD2/3 pathway and unregulated expression of Connexin 36 in 3D culture-induced PECs. This study has important implications for the generation of mature, functional β cells for drug discovery and cell transplantation therapy for diabetes and sheds new light on the signaling events that regulate endocrine specification.

中文翻译：

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通过抑制粘着斑激酶增强人多能干细胞向3D培养中的胰腺内分泌细胞的分化

从人多能干细胞（hPSC）体外产生胰岛素的细胞可用于糖尿病的药物开发和细胞治疗。三维（3D）培养对于从hPSC中获取成熟的产生胰岛素的细胞非常重要，但是对于促进β细胞成熟的机制了解甚少。我们建立了一种逐步的方法，以诱导人类胚胎干细胞（hESCs）高效分化为成熟的单激素胰腺内分泌细胞（PECs），并在3D培养中达到最后一个成熟阶段。为了全面比较二维（2D）和3D培养物，我们检查了2D培养物诱导的PEC和3D培养物诱导的PEC中的基因表达，胰腺特异性标记和功能特性。从细胞-细胞和细胞-细胞外基质相互作用的角度考虑了这种机制，这在2D和3D培养之间是根本不同的。在3D培养诱导的PEC中，胰腺内分泌特异性转录因子PDX1，NKX6.1，NGN3，ISL1和PAX6以及激素INS，GCG和SST的表达显着增加。3D培养产生单激素内分泌细胞，而2D培养诱导的PEC共表达INS和GCG或INS和SST，甚至表达所有三种激素。我们发现在3D培养物诱导的PECs中，黏着斑激酶（FAK）磷酸化显着下调，选择性FAK抑制剂PF-228的治疗改善了2D培养物诱导的PECs中β细胞特异性转录因子的表达。我们进一步证明3D文化可能会通过限制FAD依赖的SMAD2 / 3途径的激活来促进内分泌作用。而且，在3D培养物诱导的PEC中间隙连接蛋白连接蛋白36的表达比在2D培养物诱导的PEC中高得多，并且在2D培养物中抑制FAK途径增加了连接蛋白36的表达。我们制定了从hPSCs诱导单激素成熟PECs分化的策略，发现3D培养物诱导的PECs中有限的FAK依赖性SMAD2 / 3途径激活和连接蛋白36的表达不受调节。这项研究对于糖尿病药物开发和细胞移植疗法的成熟，功能性β细胞的产生具有重要意义，并为调节内分泌指标的信号传递活动提供了新的思路。