The Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.
Department of Pathology, University of Washington School of Medicine, Seattle, Washington, USA.
Nat Protoc. 2017 Jan;12(1):15-31. doi: 10.1038/nprot.2016.153. Epub 2016 Dec 1.
Human pluripotent stem cells (hPSCs) provide a valuable model for the study of human development and a means to generate a scalable source of cells for therapeutic applications. This protocol specifies cell fate efficiently into cardiac and endothelial lineages from hPSCs. The protocol takes 2 weeks to complete and requires experience in hPSC culture and differentiation techniques. Building on lessons taken from early development, this monolayer-directed differentiation protocol uses different concentrations of activin A and bone morphogenetic protein 4 (BMP4) to polarize cells into mesodermal subtypes that reflect mid-primitive-streak cardiogenic mesoderm and posterior-primitive-streak hemogenic mesoderm. This differentiation platform provides a basis for generating distinct cardiovascular progenitor populations that enable the derivation of cardiomyocytes and functionally distinct endothelial cell (EC) subtypes from cardiogenic versus hemogenic mesoderm with high efficiency without cell sorting. ECs derived from cardiogenic and hemogenic mesoderm can be matured into >90% CD31/VE-cadherin definitive ECs. To test the functionality of ECs at different stages of differentiation, we provide methods for assaying the blood-forming potential and de novo lumen-forming activity of ECs. To our knowledge, this is the first protocol that provides a common platform for directed differentiation of cardiomyocytes and endothelial subtypes from hPSCs. This protocol yields endothelial differentiation efficiencies exceeding those of previously published protocols. Derivation of these cell types is a critical step toward understanding the basis of disease and generating cells with therapeutic potential.
人多能干细胞（hPSCs）为研究人类发育提供了有价值的模型，并为治疗应用提供了可扩展的细胞来源。本方案专门针对从 hPSCs 高效诱导分化为心脏和内皮谱系细胞。本方案需要 2 周时间完成，并且要求具备 hPSC 培养和分化技术方面的经验。该单层定向分化方案基于早期发育过程中的经验教训，使用不同浓度的激活素 A 和骨形态发生蛋白 4（BMP4）将细胞极化为中胚层亚型，这些亚型反映了中原始条纹心脏中胚层和后原始条纹造血中胚层。这种分化平台为生成不同的心血管祖细胞群体提供了基础，从而能够高效地从心脏中胚层和造血中胚层中衍生出具有高细胞纯度的心肌细胞和功能不同的内皮细胞（EC）亚型，而无需细胞分选。从心脏中胚层和造血中胚层衍生的 EC 可以成熟为>90% CD31/VE-cadherin 定型 EC。为了测试分化过程中不同阶段 EC 的功能，我们提供了用于检测 EC 造血潜能和新管腔形成活性的方法。据我们所知，这是第一个提供从 hPSCs 定向分化心肌细胞和内皮亚型的通用平台的方案。该方案产生的内皮分化效率超过了先前发表的方案。这些细胞类型的衍生是理解疾病基础和生成具有治疗潜力的细胞的关键步骤。
