Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.
Biomaterials. 2011 Dec;32(35):9180-7. doi: 10.1016/j.biomaterials.2011.08.050. Epub 2011 Sep 8.
Recent advances in pluripotent stem cell research have provided investigators with potent sources of cardiogenic cells. However, tissue engineering methodologies to assemble cardiac progenitors into aligned, 3-dimensional (3D) myocardial tissues capable of physiologically relevant electrical conduction and force generation are lacking. In this study, we introduced 3D cell alignment cues in a fibrin-based hydrogel matrix to engineer highly functional cardiac tissues from genetically purified mouse embryonic stem cell-derived cardiomyocytes (CMs) and cardiovascular progenitors (CVPs). Procedures for CM and CVP derivation, purification, and functional differentiation in monolayer cultures were first optimized to yield robust intercellular coupling and maximize velocity of action potential propagation. A versatile soft-lithography technique was then applied to reproducibly fabricate engineered cardiac tissues with controllable size and 3D architecture. While purified CMs assembled into a functional 3D syncytium only when supplemented with supporting non-myocytes, purified CVPs differentiated into cardiomyocytes, smooth muscle, and endothelial cells, and autonomously supported the formation of functional cardiac tissues. After a total culture time similar to period of mouse embryonic development (21 days), the engineered cardiac tissues exhibited unprecedented levels of 3D organization and functional differentiation characteristic of native neonatal myocardium, including: 1) dense, uniformly aligned, highly differentiated and electromechanically coupled cardiomyocytes, 2) rapid action potential conduction with velocities between 22 and 25 cm/s, and 3) significant contractile forces of up to 2 mN. These results represent an important advancement in stem cell-based cardiac tissue engineering and provide the foundation for exploiting the exciting progress in pluripotent stem cell research in the future tissue engineering therapies for heart disease.
最近多能干细胞研究的进展为研究人员提供了丰富的心肌细胞来源。然而，将心脏祖细胞组装成具有生理相关电传导和力产生能力的排列整齐的三维（3D）心肌组织的组织工程方法仍然缺乏。在这项研究中，我们在纤维蛋白基水凝胶基质中引入了 3D 细胞排列线索，以从基因纯合的小鼠胚胎干细胞来源的心肌细胞（CMs）和心血管祖细胞（CVPs）中构建具有高度功能的心脏组织。首先优化了 CM 和 CVP 的衍生、纯化和在单层培养中的功能分化程序，以产生稳健的细胞间耦合并最大限度地提高动作电位传播速度。然后，应用一种多功能软光刻技术可重复性地制造具有可控尺寸和 3D 结构的工程心脏组织。虽然当补充支持非心肌细胞时，纯化的 CMs 才能组装成功能 3D 合胞体，但纯化的 CVPs 分化为心肌细胞、平滑肌和内皮细胞，并自主支持功能心脏组织的形成。在与小鼠胚胎发育（21 天）相似的总培养时间后，工程心脏组织表现出前所未有的 3D 组织和功能分化水平，类似于天然新生儿心肌，包括：1）密集、均匀排列、高度分化和电机械耦联的心肌细胞；2）动作电位传导速度在 22 至 25cm/s 之间；3）高达 2mN 的显著收缩力。这些结果代表了基于干细胞的心脏组织工程的重要进展，并为未来利用多能干细胞研究的激动人心进展为心脏病的组织工程疗法提供了基础。
