Abstract：
Chemically defined, suspension culture conditions are a key requirement in realizing clinical translation of engineered cardiac tissues (ECTs). Building on our previous work producing functional ECT microspheres through differentiation of biomaterial encapsulated human induced pluripotent stem cells (hiPSCs), here we establish the ability to use chemically defined culture conditions, including stem cell media (E8) and cardiac differentiation media (chemically defined differentiation media with three components, CDM3). A custom microfluidic cell encapsulation system was used to encapsulate hiPSCs at a range of initial cell concentrations and diameters in the hybrid biomaterial, poly(ethylene glycol)-fibrinogen (PF), for the formation of highly spherical and uniform ECT microspheres for subsequent cardiac differentiation. Initial microsphere diameter could be tightly controlled, and microspheres could be produced with an initial diameter between 400 and 800 µm. Three days after encapsulation, cardiac differentiation was initiated through small molecule modulation of Wnt signaling in CDM3. Cardiac differentiation occurred resulting in in situ ECT formation; results showed that this differentiation protocol could be used to achieve cardiomyocyte (CM) contents greater than 90%, although there was relatively high variability in CM content and yield between differentiation batches. Spontaneous contraction of ECT microspheres initiated between Days 7 and 10 of differentiation and ECT microspheres responded to electrical pacing up to 1.5 Hz. Resulting CMs had well-defined sarcomeres and the gap junction protein, connexin 43, and had appropriate temporal changes in gene expression. In summary, this study demonstrated the proof-of-concept to produce functional ECT microspheres with chemically defined media in suspension culture in combination with biomaterial support of microsphere encapsulated hiPSCs.
摘要：
化学定义，悬浮培养条件是实现工程化心脏组织（ECT）临床转化的关键要求。在我们以前的工作基础上，通过生物材料封装的人诱导多能干细胞（hiPSCs）的分化来生产功能性ECT微球，在这里，我们建立了使用化学定义的培养条件的能力，包括干细胞培养基（E8）和心脏分化培养基（化学成分确定的分化培养基，具有三种成分，CDM3)。定制的微流控细胞封装系统用于在混合生物材料中封装一系列初始细胞浓度和直径的hiPSC。聚（乙二醇）-纤维蛋白原（PF），用于形成高度球形和均匀的ECT微球，用于随后的心脏分化。初始微球直径可以严格控制，和微球可以生产的初始直径在400和800μm之间。封装后三天，通过小分子调节CDM3中的Wnt信号启动心脏分化.心脏分化发生导致原位ECT形成；结果表明，这种分化方案可用于实现心肌细胞（CM）含量大于90％，尽管不同批次之间的CM含量和产量差异相对较高。在分化的第7天至第10天之间开始的ECT微球的自发收缩，并且ECT微球对高达1.5Hz的电起搏有反应。产生的CM具有明确的肌节和间隙连接蛋白，连接蛋白43，基因表达有适当的时间变化。总之,这项研究证明了在悬浮培养中使用化学定义的培养基与微球封装的hiPSCs的生物材料支持相结合来生产功能性ECT微球的概念验证。
