Department of Biomedical Engineering, School of Medicine, School of Engineering, The University of Alabama at Birmingham , Birmingham, Alabama.
Am J Physiol Heart Circ Physiol. 2018 Aug 1;315(2):H327-H339. doi: 10.1152/ajpheart.00688.2017. Epub 2018 Apr 6.
The microenvironment of native heart tissue may be better replicated when cardiomyocytes are cultured in three-dimensional clusters (i.e., spheroids) than in monolayers or as individual cells. Thus, we differentiated human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CMs) and allowed them to form spheroids and spheroid fusions that were characterized in vitro and evaluated in mice after experimentally induced myocardial infarction (MI). Synchronized contractions were observed within 24 h of spheroid formation, and optical mapping experiments confirmed the presence of both Ca transients and propagating action potentials. In spheroid fusions, the intraspheroid conduction velocity was 7.0 ± 3.8 cm/s on days 1- 2 after formation, whereas the conduction velocity between spheroids increased significantly ( P = 0.003) from 0.8 ± 1.1 cm/s on days 1- 2 to 3.3 ± 1.4 cm/s on day 7. For the murine MI model, five-spheroid fusions (200,000 hiPSC-CMs/spheroid) were embedded in a fibrin patch and the patch was transplanted over the site of infarction. Later (4 wk), echocardiographic measurements of left ventricular ejection fraction and fractional shortening were significantly greater in patch-treated animals than in animals that recovered without the patch, and the engraftment rate was 25.6% or 30% when evaluated histologically or via bioluminescence imaging, respectively. The exosomes released from the spheroid patch seemed to increase cardiac function. In conclusion, our results established the feasibility of using hiPSC-CM spheroids and spheroid fusions for cardiac tissue engineering, and, when fibrin patches containing hiPSC-CM spheroid fusions were evaluated in a murine MI model, the engraftment rate was much higher than the rates we have achieved via the direct intramyocardial injection. NEW & NOTEWORTHY Spheroids fuse in culture to produce structures with uniformly distributed cells. Furthermore, human cardiac lineage-induced pluripotent stem cells in cardiomyocytes in adjacent fused spheroids became electromechanically coupled as the fusions matured in vitro, and when the spheroids were combined with a biological matrix and administered as a patch over the infarcted region of mouse hearts, the engraftment rate exceeded 25%, and the treatment was associated with significant improvements in cardiac function via a paracrine mechanism, where exosomes released from the spheroid patch.
当心肌细胞在三维细胞簇（即球体）中培养时，其所在的微环境可能比在单层或单个细胞中更接近天然心脏组织。因此，我们将人诱导多能干细胞分化为心肌细胞（hiPSC-CM），并让它们形成球体和球体融合，然后对这些球体进行体外特征描述，并在实验性心肌梗死（MI）后在小鼠体内进行评估。在球体形成后 24 小时内观察到同步收缩，并且光学映射实验证实存在钙瞬变和传播动作电位。在球体融合中，融合后第 1-2 天的球体内部传导速度为 7.0±3.8cm/s，而球体之间的传导速度显著增加（P=0.003），从融合后第 1-2 天的 0.8±1.1cm/s 增加到第 7 天的 3.3±1.4cm/s。对于小鼠 MI 模型，将 5 个球体融合（每个球体 20 万个 hiPSC-CM）嵌入纤维蛋白贴片，并将贴片移植到梗死部位。之后（4 周），在接受贴片治疗的动物中，左心室射血分数和缩短分数的超声心动图测量值明显大于未接受贴片治疗的动物，并且组织学或生物发光成像评估的植入率分别为 25.6%或 30%。从球体贴片释放的外泌体似乎增加了心脏功能。总之，我们的研究结果确立了使用 hiPSC-CM 球体和球体融合进行心脏组织工程的可行性，并且当在小鼠 MI 模型中评估含有 hiPSC-CM 球体融合的纤维蛋白贴片时，植入率远高于我们通过直接心肌内注射获得的植入率。创新与亮点球体在培养中融合以产生具有均匀分布细胞的结构。此外，在体外融合成熟过程中，相邻融合球体中的心肌细胞中的人诱导多能干细胞成为电机械耦联，并且当球体与生物基质结合并作为贴片施用于小鼠心脏的梗死区域时，植入率超过 25%，并且该治疗通过旁分泌机制与心脏功能的显著改善相关，其中球体贴片释放的外泌体。
