Single-cell mitochondrial lineage tracing decodes fate decision and spatial clonal architecture in human hematopoietic organoids

The ability to perform lineage tracing at the single-cell level is critical to reconstructing dynamic transitions during cell differentiation. However, prospective tracing approaches inevitably encounter outstanding challenges including barcoding precision, barcode diversity, and detection efficiency, which can skew inferred lineage relationships. Human pluripotent stem cells (hPSCs) even face risks of DNA-damage-induced toxicity-related cell death. We explored the use of naturally occurring somatic mutations in mitochondrial transcripts detected in single-cell RNA-seq as genetic lineage barcodes in hPSCs. In this study, we used an enrichment of scRNA-seq mitochondrial reads and a robust computational method to identify clonally relevant mitochondrial variants as endogenous genetic barcodes for clonal tracking of early embryonic hematopoiesis from hPSC. We modeled the development of embryonic tissues from hPSCs and delineated cell fate specification by integrating synthetic barcoding with mitochondrial lineage tracing. Using a biophysical model, we reconstructed the sequential transcriptional logic of fate specification and its underlying regulatory network. We further applied mitochondrial lineage tracing to spatial transcriptomics, which enabled us to identify the spatial clonal architecture of human embryonic organoids. Our analysis revealed that this spatial zonation was orchestrated by NOTCH-mediated crosstalk between stromal cells and hematopoietic progenitors. Our multi-modal framework links clonal dynamics with niche-specific fate decisions, providing a generalizable approach to dissecting tissue organization in development and disease. This study underscores the utility of mitochondrial variants as endogenous markers for high-resolution spatial clonal tracking in stem cell-derived organoid models of human development.