Data_Sheet_1_Combinatorial Treatment of Human Cardiac Engineered Tissues With Biomimetic Cues Induces Functional Maturation as Revealed by Optical Mapping of Action Potentials and Calcium Transients.pdf

Although biomimetic stimuli, such as microgroove-induced alignment (μ), triiodothyronine (T3) induction, and electrical conditioning (EC), have been reported to promote maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), a systematic examination of their combinatorial effects on engineered cardiac tissue constructs and the underlying molecular pathways has not been reported. Herein, human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) were used to generate a micro-patterned human ventricular cardiac anisotropic sheets (hvCAS) for studying the physiological effects of combinatorial treatments by a range of functional, calcium (Ca2+)-handling, and molecular analyses. High-resolution optical mapping showed that combined μ-T3-EC treatment of hvCAS increased the conduction velocity, anisotropic ratio, and proportion of mature quiescent-yet-excitable preparations by 2. 3-, 1. 8-, and 5-fold (>70%), respectively. Such electrophysiological changes could be attributed to an increase in inward sodium current density and a decrease in funny current densities, which is consistent with the observed up- and downregulated SCN1B and HCN2/4 transcripts, respectively. Furthermore, Ca2+-handling transcripts encoding for phospholamban (PLN) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) were upregulated, and this led to faster upstroke and decay kinetics of Ca2+-transients. RNA-sequencing and pathway mapping of T3-EC-treated hvCAS revealed that the TGF-β signaling was downregulated; the TGF-β receptor agonist and antagonist TGF-β1 and SB431542 partially reversed T3-EC induced quiescence and reduced spontaneous contractions, respectively. Taken together, we concluded that topographical cues alone primed cardiac tissue constructs for augmented electrophysiological and calcium handling by T3-EC. Not only do these studies improve our understanding of hPSC-CM biology, but the orchestration of these pro-maturational factors also improves the use of engineered cardiac tissues for in vitro drug screening and disease modeling.

尽管仿生刺激——如微沟槽诱导排列（microgroove-induced alignment，μ）、三碘甲状腺原氨酸（triiodothyronine，T3）诱导及电刺激（electrical conditioning，EC）——已被报道可促进人多能干细胞衍生心肌细胞（human pluripotent stem cell-derived cardiomyocytes，hPSC-CMs）的成熟，但针对这些刺激的联合作用对工程化心脏组织构建体的系统性探究，及其潜在分子通路的解析，迄今尚未见报道。本研究采用人胚胎干细胞衍生心室心肌细胞（human embryonic stem cell-derived ventricular cardiomyocytes，hESC-VCMs）构建微图案化人类心室各向异性心肌片（micro-patterned human ventricular cardiac anisotropic sheets，hvCAS），通过一系列功能学、钙（calcium，Ca2+）处理及分子生物学分析，探究联合处理的生理学效应。高分辨率光学标测结果显示，对hvCAS联合应用μ-T3-EC处理，可使其传导速度、各向异性比及静息但可兴奋标本的比例分别提升2.3倍、1.8倍和5倍（增幅超过70%）。这类电生理变化可归因于内向钠电流密度的升高与起搏电流（funny current）密度的降低，这与分别观察到的SCN1B基因上调及HCN2/4基因下调的结果一致。此外，编码受磷蛋白（phospholamban，PLN）与肌浆网/内质网Ca2+-ATP酶（sarco/endoplasmic reticulum Ca2+-ATPase，SERCA）的钙处理相关转录本均出现上调，这使得钙瞬变的上升相和衰减相动力学过程加快。对经T3-EC处理的hvCAS进行RNA测序（RNA-sequencing）及通路注释分析发现，转化生长因子β（TGF-β）信号通路出现下调；TGF-β受体激动剂TGF-β1与拮抗剂SB431542可分别部分逆转T3-EC诱导的静息状态，并降低自发性收缩频率。综上，我们认为仅单独使用地形学信号即可使心脏组织构建体获得致敏性，使其可通过T3-EC联合处理进一步增强电生理功能与钙处理能力。本研究不仅加深了我们对hPSC-CM生物学特性的理解，同时通过整合这些促成熟因子，进一步优化了工程化心脏组织在体外药物筛选与疾病模型构建中的应用。