Single-cell RNA-seq of human cardiovascular progenitor cells generated with a chemically defined, xeno-free laminin based differentiation protocol

Regeneration of injured human heart muscle is limited and an unmet clinical need. There are no methods for the reproducible generation of clinical quality stem-cell-derived cardiovascular progenitors (CVPs). We identified laminin-221 (LN-221) as the most likely expressed cardiac laminin. We produced it as human recombinant protein, and showed that LN-221 promotes differentiation of pluripotent hESCs towards cardiomyocyte lineage and downregulates pluripotency and teratoma associated genes. We developed a chemically defined, xeno-free laminin-based differentiation protocol to generate CVPs. Single-cell RNA sequencing of CVPs derived from hESC lines supported reproducibility and identified three main progenitor subpopulations. These CVPs were transplanted into myocardial infarction mice, where heart function was measured by echocardiogram and human heart muscle bundle formation was identified histologically. This method may provide clinical quality cells for use in regenerative cardiology. We generated four independent single-cell RNA-sequencing datasets with day 9 and 11 cardiovascular progenitor cells derived from HS1001 and H1 hES cells respectively. H1 and HS1001 cells were cultured for 9 and 11 days in wells coated with LN-521+221 from day 0. We used nutristem to maintain the cells. Differentiation commenced when cells reached confluency. Small molecule inhibitor CHIR99021 (inhibitor of GSK3) was added at day 4 followed by IWP2 (inhibitor of Wnt production) at day 7.