## 3 Results
\begin{table}
\begin{tabular}{c c c c c c c} \hline
**Position** & **Gene/Region** & **Coverage** & **Reference Allele** & **Ref (\%)** & **Alternative Allele** & **Alt (\%)** \\ \hline
307 & D-loop & 112 & C & 90.2 & T & 8.9 \\ \hline
308 & D-loop & 90 & C & 65.6 & T & 34.4 \\ \hline
309 & D-loop & 81 & T & 67.9 & C & 32.1 \\ \hline
624 & tRNA-Phe & 35 & G & 57.1 & A & 42.9 \\ \hline
2000 & 165 rRNA & 74 & C & 89.2 & T & 10.8 \\ \hline
2129 & 165 rRNA & 50 & A & 94.0 & G & 6.0 \\ \hline
13406 & ND5 & 129 & A & 91.5 & G & 8.5 \\ \hline
14247 & ND6 & 97 & C & 91.8 & T & 8.2 \\ \hline \end{tabular} Note: This table presents heteroplasmy sites detected in mitochondrial DNA from the same MSC cell line using whole genome sequencing. The D-loop region (positions 16024-576) contains three adjacent sites (307-309) with moderate to high heteroplasmy levels. Position 624 in the tRNA-Phe gene shows the highest heteroplasmy level at 42.9%. Coverage depth ranges from 35\(\times\) to 129\(\times\), with the highest coverage observed at position 13406 in the ND5 gene.  
\end{table} 
Table 4: Heteroplasmy sites detected by whole genome sequencing. Figure 6: Mitochondrial DNA heteroplasmy analysis using AS data. **(A)** Distribution of heteroplasmy frequencies across positions in the mitochondrial genome, with color indicating alternate allele frequency. **(B)** Heatmap showing variant frequencies across identified clusters at heteroplasmic sites. Yellow indicates high allele frequency (approaching 1.0), while blue indicates low frequency. **(C)** Principal component analysis of mtDNA reads based on heteroplasmic variants, showing separation between Cluster 0 (17 reads) and Cluster 1 (5 reads). **(D)** Circular representation of mtDNA heteroplasmy with gene regions shown as colored segments in the outer ring. Points in the center represent heteroplasmic sites, with color intensity corresponding to alternate allele frequency. Figure 7: Mitochondrial DNA heteroplasmy analysis from whole genome sequencing. **(A)** Distribution of heteroplasmy frequencies across positions in the mitochondrial genome, with variant frequencies reaching up to 0.40 at specific loci. **(B)** Heatmap displaying variant frequencies across the two identified clusters at eight heteroplasmic sites, revealing distinct patterns of allele distribution. **(C)** Principal component analysis visualizing the clustering of mtDNA reads based on heteroplasmic variants, with clear separation between Cluster 0 (16 reads) and Cluster 1 (13 reads). **(D)** Circular representation of mtDNA heteroplasmy showing the functional gene regions as colored segments in the outer ring, with inner points representing heteroplasmic sites colored according to their alternate allele frequency. Complementary Sequencing Approaches Reveal Extensive Mitochondrial DNA Heteroplasmy and Hierarchical Evolutionary Dynamics in Human Mesenchymal Stem Cells  
Mitochondrial DNA (mtDNA) typically exhibits homoplasmy (a single mtDNA type per individual) due to maternal transmission, yet heteroplasmy--the presence of multiple mtDNA types--occurs across many species. This phenomenon arises through new somatic mutations, inheritance from heteroplasmic mothers, or rare paternal mtDNA leakage during fertilization. Heteroplasmy creates cellular mixtures of \"major\" and \"minor\" populations that differ by one or several mutations. In organ-on-chip models, mtDNA haplotype analysis provides insights into cellular evolutionary origins and inheritance patterns. This approach enables the assessment of population-level diversity that influences bioenergetic function and drug responses across tissue models. Though traditionally a concept for nuclear chromosomes, it adapts effectively to mtDNA, as heteroplasmic populations can be clustered into distinct subpopulations with characteristic variant profiles. Our study identified these mitochondrial signatures, which can serve as valuable markers for quality control and standardization of OoC systems.