Hazeltine Laurie B, Badur Mehmet G, Lian Xiaojun, Das Amritava, Han Wenqing, Palecek Sean P
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA.
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA.
Acta Biomater. 2014 Feb;10(2):604-12. doi: 10.1016/j.actbio.2013.10.033. Epub 2013 Nov 4.
A significant clinical need exists to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes, enabling tissue modeling for in vitro discovery of new drugs or cell-based therapies for heart repair in vivo. Chemical and mechanical microenvironmental factors are known to impact the efficiency of stem cell differentiation, but cardiac differentiation protocols in hPSCs are typically performed on rigid tissue culture polystyrene (TCPS) surfaces, which do not present a physiological mechanical setting. To investigate the temporal effects of mechanics on cardiac differentiation, we cultured human embryonic stem cells (hESCs) and their derivatives on polyacrylamide hydrogel substrates with a physiologically relevant range of stiffnesses. In directed differentiation and embryoid body culture systems, differentiation of hESCs to cardiac troponin T-expressing (cTnT+) cardiomyocytes peaked on hydrogels of intermediate stiffness. Brachyury expression also peaked on intermediate stiffness hydrogels at day 1 of directed differentiation, suggesting that stiffness impacted the initial differentiation trajectory of hESCs to mesendoderm. To investigate the impact of substrate mechanics during cardiac specification of mesodermal progenitors, we initiated directed cardiomyocyte differentiation on TCPS and transferred cells to hydrogels at the Nkx2.5/Isl1+ cardiac progenitor cell stage. No differences in cardiomyocyte purity with stiffness were observed on day 15. These experiments indicate that differentiation of hESCs is sensitive to substrate mechanics at early stages of mesodermal induction, and proper application of substrate mechanics can increase the propensity of hESCs to differentiate to cardiomyocytes.
将人类多能干细胞（hPSC）分化为心肌细胞存在重大临床需求，这有助于进行组织建模，以便在体外发现新药或用于体内心脏修复的细胞疗法。已知化学和机械微环境因素会影响干细胞分化的效率，但hPSC中的心脏分化方案通常在刚性组织培养聚苯乙烯（TCPS）表面上进行，而该表面不存在生理力学环境。为了研究力学对心脏分化的时间效应，我们将人类胚胎干细胞（hESC）及其衍生物培养在具有生理相关刚度范围的聚丙烯酰胺水凝胶底物上。在定向分化和胚状体培养系统中，hESC向表达心肌肌钙蛋白T（cTnT+）的心肌细胞的分化在中等刚度的水凝胶上达到峰值。在定向分化第1天，短尾相关转录因子（Brachyury）的表达在中等刚度水凝胶上也达到峰值，这表明刚度影响了hESC向中胚层内胚层的初始分化轨迹。为了研究中胚层祖细胞心脏特化过程中底物力学的影响，我们在TCPS上启动定向心肌细胞分化，并在Nkx2.5/Isl1+心脏祖细胞阶段将细胞转移到水凝胶上。在第15天未观察到心肌细胞纯度随刚度的差异。这些实验表明，hESC的分化在中胚层诱导的早期阶段对底物力学敏感，并且适当应用底物力学可以增加hESC分化为心肌细胞的倾向。
