Supplementary Figure 2: Characterization of NKX2-5^-SIRPA⁺ SANLPCs after 30 days of additional culture.

(a-d) qRT-PCR analysis of the expression levels of: pan-cardiomyocyte and ventricular cardiomyocyte (a), SAN pacemaker (b), AVN pacemaker (c) and cardiac ion channel (d) genes in day 20 NKX2-5⁺SIRPA⁺ and NKX2-5^-SIRPA⁺ sorted cells cultured for an additional 30 days (n = 4). Values represent expression levels relative to housekeeping gene TBP. (e) Beating rate of day 20 NKX2-5⁺SIRPA⁺ and NKX2-5^-SIRPA⁺ sorted cells following additional 30 days of culture (n = 5). (f) Photomicrographs showing immunostaining of cTNT in day 20 NKX2-5⁺SIRPA⁺ and NKX2-5^-SIRPA⁺ sorted cells following additional 30 days of culture. NKX2-5:GFP expression was visualized to demonstrate that the cardiomyocytes that were isolated based on their lack of NKX2-5 expression (NKX2-5^-SIRPA⁺) remain NKX2-5 negative following the additional 30 day culture period. Scale bars represent 100 μm. (g) Representative flow cytometric analyses showing the proportion of NKX2-5:GFP⁺cTNT⁺ cells in the day 20 NKX2-5⁺SIRPA⁺ and NKX2-5^-SIRPA⁺ populations following additional 30 days of culture. Error bars represent s.e.m. t-test: *P < 0.05, **P < 0.01 vs Nkx2-5⁺SIRPA⁺ cells.