Single‐Cell Mitochondrial Lineage Tracing Decodes Fate Decision and Spatial Clonal Architecture in Human Hematopoietic Organoids

ABSTRACT Lineage tracing at single‐cell resolution is vital for mapping cell fate decisions, yet synthetic barcoding faces limitations in precision, diversity, and toxicity—especially in human pluripotent stem cells (hPSCs). Here, we repurpose naturally occurring somatic mutations in mitochondrial transcripts from single‐cell RNA sequencing as endogenous genetic barcodes. By enriching mitochondrial reads and applying a robust computational pipeline, we identified clonally informative variants to trace hematopoietic lineage emergence from hPSCs during early embryogenesis. Integrating mitochondrial barcoding with synthetic lineage tracing, we modeled embryonic tissue development and reconstructed the transcriptional logic and regulatory networks driving fate specification using a dynamical systems model. Extending this approach to spatial transcriptomics, we mapped the clonal architecture of human embryonic organoids, revealing spatial zonation orchestrated by NOTCH‐mediated crosstalk between stromal cells and hematopoietic progenitors. This multimodal strategy links clonal dynamics with niche‐driven fate decisions, offering a scalable, non‐invasive method to dissect tissue organization in development and disease. Together, our work establishes a scalable, non‐invasive multimodal framework that leverages endogenous mitochondrial DNA variants to reconstruct high‐resolution spatiotemporal clonal dynamics and decode niche‐driven fate decisions in a human stem cell‐derived model. This approach provides a powerful strategy for dissecting tissue self‐organization in development and disease.