(A) Schematic showing the location of markers examined on the mitochondrial genome, adapted from Figure 1 of Uhler and Falkenberg, 2015. (B) Relative abundance of mitochondrial markers ND1, ND4, ND6, and mtCOX2 in cardiac organoid-derived cfDNA, obtained using ddPCR. Shown are the average copies/μl of n = 3 biological replicates, each normalized to the values on day 1, to correct for batch variation. Samples on day 2 were compared to day 1 using a one-sample t-test comparing to a hypothetical value of 1.0. Day 6 samples were compared to day 2 values using an unpaired t-test with Welch’s correction. *p<0.05; **p<0.01; ***p<0.001; ns, nonsignificant. (C) Ratio of the values obtained in panel (B) for each marker to mtCOX2. Significant deviations from a ratio of 1.0 indicating unequal marker abundance were determined using a one-sample t-test comparing to hypothetical value of 1.0. Shown are the averages of n = 3 biological replicates, + SD. (D) Expression of mitochondrial DNA markers at the tissue level in cardiac organoids during differentiation obtained using ddPCR, normalized to β-actin. (E) Western blot showing protein expression of the mitochondrial protein VDAC/Porin and loading control vinculin in n = 3 biological replicates of cardiac organoids taken at different timepoints during differentiation. (Vinculin loading control is the same for Figure 4D.) (F) Quantification of VDAC protein expression from the western blot in panel (E), normalized to vinculin. Samples were further normalized to day 1 and compared using a one-sample t-test with hypothetical value of 1.0 to test for significant deviation from day 1. (G) Concentration of cfDNA extracted from media conditioned by cardiac organoids on growth days 2 or 6 after ultracentrifugation (100,000 × g for 1 hr) compared to control (no ultracentrifugation). Abundance of mitochondrial markers ND1 (H) and mtCOX2 (I) in cfDNA derived from ultracentrifuged media conditioned by cardiac organoids during development, obtained using ddPCR. Graphs show average concentrations + SD. Samples were compared using an unpaired t-test with Welch’s correction. *p<0.05; **p<0.01; ***p<0.001; ns, nonsignificant.