Supplementary Figure 1: Characterization of NKX2-5^-SIRPA⁺ SANLPCs.

(a) Representative flow cytometric analyses showing the proportion of PDGFRα⁺KDR⁺ mesodermal cells at day 4 of differentiation and cTNT⁺ cells at day 20. (b) qRT-PCR analysis of mesoderm, pan-cardiomyocyte and pacemaker genes in whole embryoid body populations at the indicated times of differentiation. Values represent expression levels relative to the housekeeping gene TBP (n = 4). (c) Representative flow cytometric analyses of cTNT expression in day 20 NKX2-5⁺SIRPA⁺ and NKX2-5^-SIRPA⁺ sorted populations. (d) Representative flow cytometric analyses of MLC2V expression in the day 20 NKX2-5⁺SIRPA⁺ and NKX2-5^-SIRPA⁺ sorted populations, bar graph indicates average proportion of MLC2V⁺cTNT⁺ cells from multiple experiments (n = 3). (e,f) Photomicrographs showing immunostaining of (e) the atrial/posterior cardiomyocyte marker COUP-TFII and (f) the pacemaker transcription factor TBX3 in NKX2-5⁺SIRPA⁺ and NKX2-5^-SIRPA⁺ cells isolated from day 20 cultures. Cells were counterstained with cTNT to visualize all cardiomyocytes and DAPI to visualize all cells. Scale bars represent 100 μm. Error bars represent s.e.m. t-test: **P < 0.01 vs Nkx2-5⁺SIRPA⁺ cells.