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

## TL;DR

This study uses natural mitochondrial DNA mutations to track cell lineage in human stem cell-derived organoids, revealing how cells organize and specialize during early blood development.

## Contribution

The novel approach repurposes endogenous mitochondrial DNA variants as barcodes for non-invasive lineage tracing in human hematopoietic organoids.

## Key findings

- Mitochondrial DNA mutations were used to trace clonal dynamics during hematopoietic development.
- NOTCH-mediated crosstalk between stromal cells and hematopoietic progenitors orchestrates spatial zonation.
- A multimodal framework was established to reconstruct spatiotemporal clonal dynamics in human 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.

This study repurposes mitochondrial DNA mutations as endogenous barcodes for lineage tracing in human pluripotent stem cell‐derived organoids. Integrated with transcriptomic and spatial data, it reveals NOTCH‐mediated stromal‐progenitor crosstalk orchestrates clonal dynamics and spatial zonation during early hematopoietic development, offering a non‐invasive method to dissect tissue organization.