Division of Cardiology , The First Affiliated Hospital of Nanjing Medical University , Nanjing 210029 , China.
Department of R&D , HELP Stem Cell Therapeutics , Nanjing 210010 , China.
ACS Appl Mater Interfaces. 2019 Jan 30;11(4):3679-3689. doi: 10.1021/acsami.8b17090. Epub 2019 Jan 22.
The development of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provides significant advances to cell therapy, disease modeling, and drug screening applications. However, the current differentiation protocol is inefficient in mimicking biophysical and biochemical characteristics of cardiac niche. Hence, immature cardiomyocytes are often generated. In this study, hiPSC-CMs were generated on a new family of substrates called monolayer binary colloidal crystals (BCCs). Four BCCs were fabricated with different sizes (2 or 5 or 0.4 or 0.2 μm) and materials [Si or polystyrene (PS) or poly(methyl methacrylate)] abbreviated as 2PS, 5PS, 2PM, and 5PM. BCCs have complex surface micro-/nanotopographies and heterogeneous chemistries which are important modulators in microenvironments in vitro. The results showed that hiPSCs formed adhered spheroids with strong pluripotent markers ( Oct4, Nanog, and Sox2) on PM surfaces compared to PS and flat surfaces. After 30-day differentiation, hiPSC-CMs on PM surfaces showed markedly improved myofibril ultrastructures, Ca handling, and electrophysiological properties, indicating that more mature hiPSC-CMs were generated. hiPSC-CMs generated on 5PM are more similar to adult heart tissue compared to other surfaces in terms of genes ( ACTC1, TNNT2, RYR2, SERCA2a, SCN5a, KCNJ2, CACNA1c, ITGB1, GJA1, MYH6, and MYH7) and protein (ssTnI and cTnI) expressions. We further demonstrated that 5PM surfaces facilitated cadherin switching (from E- to N-) during cardiac differentiation and mature N-cadherin expression, which were positively correlated with the cardiogensis markers ( GATA4, MEF2c, and NKX2.5). This study illuminated that a tailored surface nanotopography was beneficial in hiPSC culture and in situ cardiac differentiation. This one-step approach and BCCs can be a next-generation tool for hiPSC expansion and CM differentiation.
人诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）的发展为细胞治疗、疾病建模和药物筛选应用提供了重大进展。然而，目前的分化方案在模拟心脏生态位的生物物理和生化特征方面效率不高。因此，通常会产生不成熟的心肌细胞。在这项研究中，hiPSC-CMs 是在一种称为单层二元胶体晶体（BCC）的新基质家族上生成的。使用不同的尺寸（2 或 5 或 0.4 或 0.2μm）和材料[硅或聚苯乙烯（PS）或聚甲基丙烯酸甲酯]制造了四个 BCC，分别缩写为 2PS、5PS、2PM 和 5PM。BCC 具有复杂的表面微/纳米形貌和异质化学性质，这些是体外微环境中的重要调节剂。结果表明，与 PS 和平面相比，PM 表面上的 hiPSCs 形成了附着的球体，具有强烈的多能标记物（Oct4、Nanog 和 Sox2）。在 30 天的分化后，PM 表面上的 hiPSC-CMs 表现出明显改善的肌原纤维超微结构、Ca 处理和电生理特性，表明生成了更成熟的 hiPSC-CMs。在基因（ACTC1、TNNT2、RYR2、SERCA2a、SCN5a、KCNJ2、CACNA1c、ITGB1、GJA1、MYH6 和 MYH7）和蛋白（ssTnI 和 cTnI）表达方面，与其他表面相比，5PM 上生成的 hiPSC-CMs 更类似于成人心脏组织。我们进一步证明，在心脏分化过程中，5PM 表面促进了钙粘蛋白的转换（从 E-到 N-）和成熟的 N-钙粘蛋白表达，这与心脏发生标记物（GATA4、MEF2c 和 NKX2.5）呈正相关。这项研究表明，定制的表面纳米形貌有利于 hiPSC 培养和原位心脏分化。这种一步法和 BCC 可以成为 hiPSC 扩增和 CM 分化的下一代工具。
