Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, 37232, USA.
Sci Rep. 2019 Nov 4;9(1):15917. doi: 10.1038/s41598-019-52467-0.
Cardiac muscle cells lack regenerative capacity in postnatal mammals. A concerted effort has been made in the field to determine regulators of cardiomyocyte proliferation and identify therapeutic strategies to induce division, with the ultimate goal of regenerating heart tissue after a myocardial infarct. We sought to optimize a high throughput screening protocol to facilitate this effort. We developed a straight-forward high throughput screen with simple readouts to identify small molecules that modulate cardiomyocyte proliferation. We identify human induced pluripotent stem cell-derived cardiomyocytes (hiCMs) as a model system for such a screen, as a very small subset of hiCMs have the potential to proliferate. The ability of hiCMs to proliferate is density-dependent, and cell density has no effect on the outcome of proliferation: cytokinesis or binucleation. Screening a compound library revealed many regulators of proliferation and cell death. We provide a comprehensive and flexible screening procedure and cellular phenotype information for each compound. We then provide an example of steps to follow after this screen is performed, using three of the identified small molecules at various concentrations, further implicating their target kinases in cardiomyocyte proliferation. This screening platform is flexible and cost-effective, opening the field of cardiovascular cell biology to laboratories without substantial funding or specialized training, thus diversifying this scientific community.
心肌细胞在出生后哺乳动物中缺乏再生能力。该领域已经做出了协同努力，以确定心肌细胞增殖的调节剂，并确定诱导分裂的治疗策略，最终目标是在心肌梗塞后再生心脏组织。我们试图优化高通量筛选方案以促进这一努力。我们开发了一种简单的高通量筛选方法，具有简单的读数，可以识别调节心肌细胞增殖的小分子。我们将人诱导多能干细胞衍生的心肌细胞（hiCM）鉴定为这种筛选的模型系统，因为 hiCM 的一小部分具有增殖的潜力。hiCM 增殖的能力与细胞密度有关，细胞密度对增殖的结果（有丝分裂或双核化）没有影响。筛选化合物文库揭示了许多增殖和细胞死亡的调节剂。我们提供了全面灵活的筛选程序和每个化合物的细胞表型信息。然后，我们提供了在进行此筛选后遵循的步骤示例，使用三种已识别的小分子在不同浓度下，进一步表明它们的靶激酶参与心肌细胞增殖。该筛选平台灵活且具有成本效益，使心血管细胞生物学领域向没有大量资金或专门培训的实验室开放，从而使该科学领域多样化。
