CHARACTERIZATION OF THE AGE-DEPENDENT CHANGES IN HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS

Abstract

Biological aging leads to a reduced capacity of hematopoietic stem/progenitor cells (HSPCs) to efficiently produce blood and immune cells, which contributes to various age-related pathologies. Further understanding of this aging process in HSPCs may provide insight into novel strategies for diagnosis and pharmacological interventions aimed towards minimizing the detrimental effects of aging, thereby promoting a longer and healthier lifespan. Past studies have highlighted multiple changes in the characteristics of the HSPC compartment in both mice and humans, as well as various mechanisms underlying these progressive deteriorations. However, major knowledge gaps remain, which prevent a definitive conclusion to be drawn regarding the age-dependent changes in human HSPCs (CD34+ cells). This thesis is aimed towards investigating the potential age-dependent changes in the proliferation, clonogenicity, cell cycle activity, and mitochondrial activity of human HSPCs. For the first objective, HSPCs were enriched from human cord blood (young) and bone marrow (aged; 50 – 90 years old), followed by in vitro functional characterizations. Aged HSPCs produced fewer myeloid but not erythroid colonies in the colony-forming cell assay. Cell cycle and proliferation analyses showed that aged HSPCs contained fewer proliferating cells (Ki67+), albeit with more cells in S-G2-M phases, and display more asynchronous proliferation in vitro compared to young HSPCs. Additionally, fluorescence microscopy indicated a significant increase in active mitochondria (p < 0.0001) in aged HSPCs. For the second objective, next-generation sequencing was employed to detect somatic mutations that are associated with age-related clonal hematopoiesis (ARCH) in mature hematopoietic cells (CD34-) collected from the same bone marrow donors. The mutational status of each individual was correlated to the clonogenicity of their HSPCs. Mutations in the epigenetic modifiers DNMT3A and TET2 were observed in 28.6% of individuals above 50 years old (VAF > 2%). HSPCs derived from individuals with these mutations produced more colonies (p = 0.0137), with a bias for granulocytic-monocytic-immature erythroid lineages, compared to those without mutations. This study has further explored the age-dependent changes in human HSPCs and provided preliminary evidence of a positive correlation between ARCH and the clonogenicity of HSPCs in the elderly.