Interfering with DNA replication improves beta cell differentiation and maturation from human pluripotent stem cells

Cell cycle progression plays an important role in mediating the transition from a differentiated state to a pluripotent stem cell. Conversely, establishing limitations in proliferative potential may be important to achieve functional maturity as well as to prevent abnormal growths after transplantation of stem cell derived products. Here we induced exit from the cell cycle in pancreatic progenitors by interfering with the progression of DNA replication and determined growth potential, differentiation and maturation to insulin producing endocrine cells. Treatment with the DNA polymerase inhibitor aphidicolin, as well as with antineoplastic agents, etoposide and cisplatin arrested cell cycle progression in G1 and promoted differentiation of pancreatic progenitor cells towards C-peptide positive cells. G1 arrest by aphidicolin did not induce DNA damage response or apoptosis, improved the functional maturity of stem cell-derived cells and protected mice from diabetes, without the formation of cystic growths. Therefore, induction of G1 arrest is an efficient way to promote precursor cell differentiation and generate insulin-producing cells with a stable identity and predictable growth potential after transplantation. Gene expression differences between differentiated ECs with and without APH

细胞周期进程在介导分化细胞状态向多能干细胞（pluripotent stem cell）转化的过程中发挥关键作用。反之，限制增殖潜能对于实现细胞功能成熟，以及防止干细胞衍生制剂移植后发生异常增殖同样至关重要。本研究通过干扰DNA复制进程，诱导胰腺祖细胞退出细胞周期，并对其生长潜能、向胰岛素分泌内分泌细胞的分化与成熟情况进行了检测。采用DNA聚合酶抑制剂阿非迪霉素（aphidicolin），以及抗肿瘤药物依托泊苷（etoposide）和顺铂（cisplatin）处理细胞，可使细胞周期阻滞于G1期（G1 phase），并促进胰腺祖细胞向C肽阳性细胞分化。阿非迪霉素诱导的G1期阻滞不会引发DNA损伤应答或细胞凋亡，能够改善干细胞衍生细胞的功能成熟度，且在不形成囊性增生的前提下，可保护小鼠免于罹患糖尿病。因此，诱导G1期阻滞是促进前体细胞分化、获取具有稳定细胞身份特征且移植后生长潜能可预测的胰岛素分泌细胞的有效手段。经与未经APH处理的分化内分泌细胞（ECs，endocrine cells）之间的基因表达差异。