Elucidating the Origins and Drivers of Clonal Dynamics in Hematopoiesis - The human body produces hundreds of billions of blood cells daily, replenished by hematopoietic stem cells (HSCs) in the bone marrow. Over time, HSC clones—populations derived from a single HSC—fluctuate in size, with some clones expanding while others dwindle. Clonal dynamics have been studied by reconstructing HSC phylogenic trees from somatic mutations they have accrued using whole-genome sequencing of single-cell- derived colonies. Our group pioneered the study of clonal dynamics in myeloproliferative neoplasms (MPNs), showing that driver mutations, such as JAK2, arise decades before diagnosis and confer a fitness advantage, enabling mutant clones to dominate the population. Strikingly, similar clonal dominance is observed in aging healthy individuals, though only ~20% of expansions can be attributed to known driver mutations. Understanding why certain HSC clones expand, especially in the absence of clear genetic causes, remains a fundamental unanswered question in hematopoiesis. Clonal expansion of HSCs may result from cell-intrinsic factors, as not all HSCs are equivalent. We would like to understand how during development a heterogenous population of HSCs is generated. Extrinsic factors, such as signals from the niche or systemic inflammation, may also drive clonal expansion. However, we lack basic knowledge of the drivers of clonal dynamics in native hematopoiesis because (1) reconstructing clonal history using single-cell phylogenies is not scalable—whole-genome sequencing of colonies is invasive, slow, and costly; and (2) mouse models, while useful for perturbing clonal dynamics, fail to recapitulate human clonal dynamics. This is because, despite the fitness advantages of certain HSC clones, the short lifespan of mice does not allow sufficient time for these clones to expand and dominate the stem cell population. To resolve clonal expansions in mice, we need scalable methods to reconstruct the phylogenetic history of all HSCs, not just a subset. We propose a comprehensive research program for developing new technologies to address these challenges and uncover the drivers of HSC clonal dynamics. First, we will create a non-invasive, rapid, and cost-effective method to reconstruct HSC clonal histories using long-read bulk sequencing of methylation patterns in blood cells, reducing the cost per sample from $100,000 to $1,000 and enabling large-scale human studies. Second, we will engineer mice to record lineage and key signaling histories of HSCs directly in their own DNA by extending lineage-recording mouse models we previously developed. Phylogenetic trees of all HSCs can then be reconstructed efficiently by sequencing specific target regions instead of entire genomes. By integrating signaling activity with lineage history, we will decorate tree branches with molecular events that drive clonal expansion. These engineered mice will enable mapping the developmental origins of HSC heterogeneity and quantifying the impact of extrinsic factors on clonal dynamics. Together, these approaches will address fundamental questions in stem cell regulation and aging, improve prognosis and treatment of hematological disorders, and provide transformative tools for studying blood.
