Department of Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA.
Circ Res. 2012 Oct 12;111(9):1125-36. doi: 10.1161/CIRCRESAHA.112.273144. Epub 2012 Aug 21.
Cardiomyocytes (CMs) differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research, including disease modeling, and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix is also fundamentally involved in cardiac development from the earliest morphogenetic events, such as gastrulation.
We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using extracellular matrix in combination with growth factors known to promote cardiogenesis.
PSCs were cultured as monolayers on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, bone morphogenetic protein 4, and basic fibroblast growth factor) generated CMs with high purity (up to 98%) and yield (up to 11 CMs/input PSC) from multiple PSC lines. The resulting CMs progressively matured over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial, and ventricular CMs were observed, and monolayers of electrically coupled CMs modeled cardiac tissue and basic arrhythmia mechanisms.
Dynamic extracellular matrix application promoted epithelial-mesenchymal transition of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.
由人多能干细胞（PSCs）分化而来的心肌细胞（CMs）越来越多地用于心血管研究，包括疾病建模，并有望应用于临床。目前的心脏分化方案在不同的 PSC 系之间显示出可变的成功率，并且主要基于生长因子的应用。然而，细胞外基质也从最早的形态发生事件（如原肠胚形成）中根本涉及心脏发育。
我们试图通过使用细胞外基质结合已知促进心脏发生的生长因子，开发一种更有效的人类 PSC 心脏分化方案。
PSCs 在 Matrigel（一种细胞外基质制剂）上培养为单层，随后在 Matrigel 上覆盖。基质夹层促进了上皮-间充质转化，类似于原肠胚形成，产生 N-钙粘蛋白阳性间充质细胞。将基质夹层与生长因子（激活素 A、骨形态发生蛋白 4 和碱性成纤维细胞生长因子）的顺序应用相结合，从多种 PSC 系中产生了高纯度（高达 98%）和产量（高达 11 个 CMs/输入 PSC）的 CMs。在培养物中，基于肌丝表达模式和有丝分裂活性，生成的 CMs 在 30 天内逐渐成熟。观察到典型的胚胎节点、心房和心室 CMs 的动作电位，并且电耦合 CMs 的单层模拟了心脏组织和基本心律失常机制。
动态细胞外基质应用促进了人 PSCs 的上皮-间充质转化，并补充了生长因子信号，以实现强大的心脏分化。
