Bauwens Celine L, Toms Derek, Ungrin Mark
Institute of Biomaterials and Biomedical Engineering, University of Toronto.
Department of Comparative Biology and Experimental Medicine, University of Calgary.
J Vis Exp. 2016 Sep 25(115):54308. doi: 10.3791/54308.
Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.
人类多能干细胞（hPSC）的心脏分化通常在悬浮细胞聚集体中进行。hPSC的传统聚集体形成涉及将细胞集落解离成较小的团块，通过移液细胞悬液来粗略控制团块大小，直到达到所需的团块大小。基于传统聚集体的hPSC心脏分化的主要挑战之一是培养异质性和空间无序导致心肌细胞产量可变且效率低下。我们和其他人之前曾报道，人类胚胎干细胞（hESC）聚集体大小可以进行调节以优化心脏诱导效率。我们通过采用一种可扩展的、基于微孔的方法来控制聚集体形成的物理参数，特别是聚集体大小和形状，解决了这一挑战。我们在此描述的方法包括在微孔中强制聚集确定数量的hPSC，然后在引导心脏诱导的条件下培养这些聚集体。该方案可根据所用孔的大小和数量轻松扩展。使用这种方法，我们能够始终如一地获得心肌细胞频率大于70%的培养结果。
