Extended Data Fig. 9: Gating strategy for sorting hESC-cardiomyocytes from microtissues for bulk RNA sequencing.

(a) Microtissues (HT-Biowires) were seeded with hESC-cardiomyocytes and fibroblasts with or without hESC-macrophages for bulk RNA sequencing of hESC-cardiomyocytes on day 3 post-seeding. Representative gating strategy shown. DAPI⁻ live single cells were first gated, followed by CD45⁻CD14⁻CD90⁻RFP⁻ cells. (b) hESC-cardiomyocytes, fibroblasts and hESC-macrophages were independently stained and acquired for flow cytometry. Data was overlaid into a single plot. CD45⁻CD14⁻ cells were hESC-cardiomyocytes, CD45^dimCD14^dim cells were fibroblasts, and CD45⁺CD14⁺ cells were hESC-macrophages, confirming the gating strategy utilized in (A). (c) CD45 expression of populations shown in (B), showing that hESC-cardiomyocytes are CD45⁻, fibroblasts are CD45^dim and hESC-macrophages are CD45⁺. (d) hESC-cardiomyocytes or fibroblasts were pre-labeled with CFSE prior to seeding microtissues. On day 3, flow cytometry was performed showing that the CFSE⁺ hESC-cardiomyocytes were CD14⁻RFP⁻, whereas CFSE⁺ fibroblasts were CD14^dimRFP^dim, confirming the gating strategy utilized in (A). CM: cardiomyocyte; FB: fibroblast; MF: macrophage.