3134 – CHARACTERIZATION OF THE HEMATOPOIETIC STEM CELLS AND BONE MARROW MICROENVIRONMENT IN SICKLE CELL DISEASE PATIENTS

Abstract

Little is known about the status of the bone marrow (BM) in Sickle Cell Disease (SCD) patients. In this study, we characterized the hematopoietic stem and progenitors (HSPC) and the stromal cells from BM aspirates of eight SCD patients before transplant in comparison with age-matched BM aspirates from healthy individuals. By using multiparametric flow analysis, we observed a group of SCD patients (n=4) with characteristics similar to controls, and a distinct group of SCD patients (n=4) that exhibited lower average frequency of CD34+ cells, HSCs, multipotent and lymphoid progenitors. Methylcellulose colony assays showed a decreased ability of the BM cells of these patients to form colonies compared to control BM. scRNA-seq analysis of sorted BM CD34+ cells from SCD patients indicated changes in the DNA damage and cell cycle regulatory pathways compared to controls. Interestingly, all SCD patients displayed an increase in the BM non-hematopoietic cells including CD45-/CD31+ and CD45-/CD105+ subsets compared to controls, suggesting a change in their BM stromal niche. In vitro, SCD BM-derived stromal cells revealed distinct gene expression patterns including an increase in inflammatory cytokine (IL-6) & cell death-related genes (p16 and Bax) but decrease expression of a pluripotent gene (Oct4). We also detected an increase of inflammatory regulators, miR-155, and miR-146 in all BM-derived stromal cells from SCD patients, which correlates with increased inflammatory gene expression in these cells. Finally, analysis of the BM biopsies revealed alterations in the stromal components as evidenced by an overall decrease in cellularity in some patients, and increased iron deposits and activated macrophages. Taken together, these preliminary data suggest that the BM cells and BM microenvironment of SCD patients are phenotypically & molecularly different from control BM. Further studies will clarify whether there are correlations between the severity of the disease and BM alterations and whether the BM status at pre-transplant may help in predicting engraftment and outcomes of BM transplants in SCD patients.