Transcriptomically-guided mesendoderm induction of human pluripotent stem cells using a systematically defined culture scheme

Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient and broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple yet robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding and BMP4 and Activin A (BA) treatment. Gene sets and gene ontology terms related to mesoderm and endoderm differentiation were enriched after 48 hours of BA treatment. After 14 days of differentiation, BA-treated cells expressed genes indicative of mesoderm and endoderm-derived tissue types, including lung, kidney and adipose tissue. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation. Teratomas formed from BA-treated cells contained higher ratios of mesoderm and endoderm to ectoderm tissue compared to PSC-derived teratomas. Collectively, these data demonstrate that single-cell seeding with BA treatment is a powerful method for induction of mesendoderm that can be integrated into protocols for mesoderm and endoderm differentiation. Overall design: 35 samples total. BA (mesendoderm) samples: 6hr, 12hr, 18hr, 24hr x 3, 30hr, 36hr, 42hr, 48hr x 3. E8 (pluripotency control) samples (6hr, 12hr, 18hr, 24hr, 30hr, 36hr, 42hr, 48hr x 3). E6 (spontaneous differentiation control) samples: 6hr, 12hr, 18hr, 24hr, 30hr, 36hr, 42hr, 48hr x 3. time_zero control