SRA

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. We show high reproducibility of the differentiation protocol using time-course bulk RNA sequencing developed from different hESC lines. 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. Overall design: Human embryonic stem cells HS1001 were cultured in wells coated with LN-521 and LN-221 using RPMI1460 supplemented with B21 without insulin medium. Differentiation commenced when cells reached confluency. Small molecule inhibitor CHIR99021 (inhibitor of GSK3) was added at day 0 followed by IWP2 (inhibiot of Wnt production) at day 3. Cells continue to be cultured in RPMI1460 supplemented with B21 without insulin medium for 10 days with medium change every other day. After day 10, medium was changed to RPMI1460 supplemented with B21 CTS grade medium. RNA was extracted from triplicates wells at day 0, 1, 3, 11, 14, 20, 30 and 90 and from 5 wells at day 5, 7 and 9.