Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation, while fine-tuning of BA conditions facilitated definitive endoderm commitment. After prolonged differentiation in vitro or in vivo, BA pretreatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be adapted for use in mesoderm and endoderm differentiation.

人多能干细胞（hPSC）是组织工程，发育研究和疾病建模中必不可少的细胞来源。在干细胞生物学领域中，用于快速谱系诱导hPSC的有效，广泛适用的方案引起了人们的极大兴趣。我们描述了一种简单，可靠的方法，用于在定义条件下利用单细胞播种（SCS）和BMP4和激活素A（BA）分化为hPSC到中胚层。BA处理很容易被整合到现有的软骨和内皮祖细胞分化方案中，而对BA条件的微调则促进了内胚层的定型。体外或体内长时间分化后，BA预处理导致较高的中胚层和内胚层水平，但以形成外胚层为代价。这些数据表明，采用BA处理的SCS是诱导中胚层的有力方法，可适用于中胚层和内胚层的分化。