Discovery and manipulation of transcription factors to restore long term stem cell repopulation in aged bone-marrow

Project Summary Aging has a complex underlying biology characterized by a progressive loss of cellular and physiological function and this deterioration is strongly correlated with degenerative disease. In the bone marrow, aging markedly reduces the capacity of hematopoietic stem cells (HSCs) to self-renew and differentiate into lymphoid lineages,

resulting in hindered immune function and systemic effects on multiple tissues, such as muscle repair after injury. Bone marrow from young recipients has been shown to rejuvenate aged bone marrow as well as systemically in other tissues. However, the exact HSC and progenitor cell states as well as other factors that drive the

rejuvenating effects are not well understood. Profiling of HSCs and other bone marrow cell types during aging and an understanding of the transcription factors (TFs)-that control HSC self-renewal and their changes in activity during aging could provide new therapeutic approaches with the potential to reverse both blood-specific and

whole-animal effects of aging. TF-based interventions, such as partial reprogramming, have shown promise to promote stem-cell cycling and regeneration. However, current approaches are limited to a small set of predetermined TFs, commonly the Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc, and have only been

demonstrated in a select set of tissues. Furthermore, the diversity of the HSC population, containing both senescent cells and long-term renewing state (LT-HSC) among other subtypes, makes discovery of master regulators challenging, and existing approaches do not address this heterogeneity. We hypothesize that high-

resolution single cell RNA sequencing (scRNA-seq) of HSCs from mice of different ages will reveal putative TF regulators of the aging process, and that these candidates can reprogram aged HSCs towards LT-HSC and quiescent states capable of niche restoration to reverse age-associated phenotypes. We will profile molecular

signatures of aging in HSCs at single cell resolution and use these data to both develop metrics for aging and nominate TFs to promote LT-HSC restoration and rejuvenation. We will synthesize selected TFs for pooled screening allowing for rapid evaluation of their reprogramming effects in vitro and in vivo. Coupling these pooled

perturbations in vivo with scRNA-seq readouts will allow for evaluation of HSC rejuvenation via our aging signatures and measurement of lymphoid/myeloid skew. Candidate TFs that demonstrate the strongest potential for rejuvenation of LT-HSCs will be tested individually and in combination for modification of whole-organism

phenotypes, including increased repopulation potential, reduction of inflammatory factors, and improvement of muscle repair in response to injury. The repurposing of novel TFs regulating HSC rejuvenation as new therapeutics for aging-associated disease provides a new framework for cellular engineering. This proposal,

coupling transcriptomic readouts and screening for discovery of new regulators of cell states, serves as the foundation for TF-based interventions for disease, both in aging and in broader human health.