3057 – HEMATOPOIETIC STEM CELL-DERIVED PLATELET PROGENITORS POSSESS STRIKING HETEROGENEITY THAT CHANGES WITH AGE

Hematopoietic stem cells differentiate into all blood cells, including Megakaryocyte progenitors (MkPs) and subsequent platelets. Understanding platelet generation informs cardiovascular and thrombotic disease, a leading global health concern among the aged population. We sought to uncover underappreciated heterogeneity among young, steady-state MkPs, and any changes with age. Recently, our lab uncovered functional differences between MkP populations. We defined two phenotypic subpopulations: canonical (cMkPs), and non-canonical (ncMkPs). We discovered that MkP heterogeneity changes with age, indicated by a dramatic increase in frequency and number of ncMkPs. First, we asked what surface proteins define young MkPs using targeted flow cytometry informed by bulk and single cell RNAseq. We found that specific surface proteins were differentially expressed among steady-state MkPs, indicating greater heterogeneity than previously observed. We combined nine candidate markers that demonstrated variable expression and applied dimensionality reduction analysis (UMAP) to standardized flow cytometry data. We observed heterogeneity among young MkPs and defined clusters of phenotypically-unique subpopulations. Second, we compared old and young MkP heterogeneity in mice. Aged MkPs demonstrated greater heterogeneity at both individual marker and combinatorial (UMAP) levels. Specific subclusters of MkPs were expanded with age. Finally, we assessed human MkPs for features analogous to the mouse. We found that, similar to mice, human MkPs possess phenotypic heterogeneity that changes with age. These new data define phenotypically distinct subpopulations of MkPs in both mice and humans that change with age. Advancing our understanding of platelet generation by specific MkP subpopulations may contribute to understanding the underlying biology of thrombotic and cardiovascular disease.