The Tkemaladze Method maps cell lineage with mutant mitochondrial transfer

Abstract

The comprehensive mapping of cellular lineages from the zygote to a fully formed organism remains a fundamental and unresolved challenge in developmental biology. While modern single-cell technologies offer snapshots of cellular heterogeneity, they lack the inherent, permanent markers required to trace progeny through the complex events of asymmetric division and migration over time. This work introduces the Tkemaladze Method, a novel lineage-tracing approach that utilizes mutant mitochondrial DNA (mtDNA) as a stable, inheritable genetic label. The method involves the isolation of mitochondria from cytoplasts harboring known pathogenic mtDNA mutations and their microinjection into murine embryonic stem cells (mESCs). We confirmed successful transfer and functional integration via fluorescence microscopy and quantitative PCR. These labeled progenitor cells were used to generate chimeric embryos, where we demonstrated stable heteroplasmy and faithful inheritance of the mutant mtDNA in clonal progeny throughout development. Using fluorescent reporters, we visualized the fate of individual progenitors, enabling the quantitative construction of a detailed cytogenealogical map across tissues like the central nervous system, liver, and myocardium. A key finding was the tissue-specific segregation of mitochondrial tags, revealing selective pressure in high-energy-demand tissues. The Tkemaladze Method thus provides an unprecedented, powerful tool for fundamental developmental biology, disease modeling, and tracking the fate of transplanted cells in regenerative medicine.