Enzymatic dissociation of human pluripotent stem cells (hPSCs) into single cells during routine passage leads to massive cell death. Although the Rho-associated protein kinase inhibitor, Y-27632 can enhance hPSC survival and proliferation at high seeding density, dissociated single cells undergo apoptosis at clonal density. This presents a major hurdle when deriving genetically modified hPSC lines since transfection and genome editing efficiencies are not satisfactory. As a result, colonies tend to contain heterogeneous mixtures of both modified and unmodified cells, making it difficult to isolate the desired clone buried within the colony. In this study, we report improved clonal expansion of hPSCs using a retinoic acid analogue, TTNPB. When combined with Y-27632, TTNPB synergistically increased hPSC cloning efficiency by more than 2 orders of magnitude (0.2% to 20%), whereas TTNPB itself increased more than double cell number expansion compared to Y-27632. Furthermore, TTNPB-treated cells showed two times higher aggregate formation and cell proliferation compared to Y-27632 in suspension culture. TTNPB-treated cells displayed a normal karyotype, pluripotency and were able to stochastically differentiate into all three germ layers both in vitro and in vivo. TTNBP acts, in part, by promoting cellular adhesion and self-renewal through the upregulation of Claudin 2 and HoxA1. By promoting clonal expansion, TTNPB provides a new approach for isolating and expanding pure hPSCs for future cell therapy applications.

在常规传代过程中，人多能干细胞 (hPSC) 酶解成单细胞会导致大量细胞死亡。尽管 Rho 相关蛋白激酶抑制剂 Y-27632 可以在高接种密度下增强 hPSC 的存活和增殖，但在克隆密度下解离的单细胞会发生凋亡。由于转染和基因组编辑效率不令人满意，这在获得转基因 hPSC 系时构成了主要障碍。因此，集落往往含有修饰和未修饰细胞的异质混合物，使得很难分离埋藏在集落内的所需克隆。在这项研究中，我们报告了使用视黄酸类似物 TTNPB 改善了 hPSC 的克隆扩增。当与 Y-27632 组合时，TTNPB 协同将 hPSC 克隆效率提高 2 个数量级以上（0.2% 至 20%），而与 Y-27632 相比，TTNPB 本身增加了两倍以上的细胞数量扩增。此外，与悬浮培养中的 Y-27632 相比，TTNPB 处理的细胞显示出两倍高的聚集体形成和细胞增殖。 TTNPB 处理的细胞显示出正常的核型、多能性，并且能够在体外和体内随机分化为所有三个胚层。 TTNBP 的作用部分是通过上调Claudin 2和HoxA1来促进细胞粘附和自我更新。通过促进克隆扩增，TTNPB 提供了一种分离和扩增纯 hPSC 的新方法，用于未来的细胞治疗应用。