Cardiac pericytes (CPs), a major mural cell type maintaining homeostasis, integrity, and perfusion of the coronary microvasculature, remain the most enigmatic and underappreciated cell population in the heart. Accumulating evidence suggests that CPs play a key role in cardiovascular complications such as coronary vasospasm, no reflow after myocardial infarction, and cancer drug–induced cardiotoxicity. 1 However, the lack of unequivocal cell markers and specific tools for characterization, lineage tracing, and conditional targeting of CPs has precluded a comprehensive understanding of their pathogenic role in coronary microvascular dysfunction. Here, we report a stepwise approach to generate first-of-its-kind CPs from human induced pluripotent stem cells (iPSCs), which were shown to transcriptionally and functionally resemble their primary counterparts. To derive pure iPSC-CPs, we first generated epicardial cells (EPIs), the predominant progenitor cells giving rise to CPs, in a stepwise fashion (Figure [A]). We found that temporal activation or inhibition of key morphogens at different differentiation stages enabled the generation of pure EPIs (Figure [B, i]). 2 In contrast, using an extant protocol that only manipulated Wnt signaling throughout the entire differentiation process (referred to as the GiWiGi protocol), 3 we observed a large variation of EPI induction efficiency (ranging from 3% to 87%) even among iterative differentiations (Figure [B, ii]). Moreover, our stepwise protocol generated more mature EPIs (ALDH1A2 [aldehyde dehydrogenase 1 family member A2], UPK3B [uroplakin 3B], and ANXA8 [annexin A8]) than did the

GiWiGi protocol (Figure [C]). Finally, we performed single-cell ATAC (assay for transposase-accessible chromatin) sequencing on stepwise and GiWiGi EPIs and projected them onto human fetal heart single-cell ATAC sequencing data (Figure [D, i]) based on their chromatin landscape similarities. 4 Our data showed that> 30% of GiWiGi EPIs acquired a fibroblast fate (Figure [D, ii]), precluding the generation of pure CPs for cell type–specific studies. In contrast, stepwise EPIs overlapped with primary EPIs and endocardial cells, 2 progenitors that can give rise to CPs (Figure [D, iii]). 5 Next, we differentiated stepwise EPIs in a commercial pericyte medium and observed that it took at least 12 days to generate iPSC-CPs (Figure [E]) with low efficiency (Figure [F]). Since pericyte–endothelial cell crosstalk and PDGFR (platelet-derived growth factor receptor) signaling are critical for pericyte development, we successfully generated pure iPSC-CPs (PDGFRβ+/CD146+/NG2+/CD13+) with exogenous PDGFBB (platelet-derived growth factor BB), a ligand predominantly secreted by endothelial cells (Figure [F]). Next, we confirmed comparable expression levels of cell markers between primary and iPSC-CPs using quantitative reverse transcription polymerase chain reaction (Figure [G]). Finally, we showed that iPSC-CPs are negative for smooth muscle cell markers and not reactive to antifibroblast antibody TE-7 (Figure [H]), further confirming their cell-type specificity.