SINGLE CELL RNA-SEQ REVEALED CLONAL REPOPULATION DYNAMICS BY MOUSE HEMATOPOIETIC STEM CELLS

Abstract

Single hematopoietic stem cells (HSCs) can reconstitute the entire hematopoietic system after transplantation. However, how HSCs reconstitute hematopoietic cells is poorly understood. We used single-cell RNA sequencing (scRNA-seq) to identify cells and study their kinetics. Single CD201+CD150+CD48-Lineage- cells from GFP-transgenic B6 mice were transplanted into lethally irradiated mice with competitor cells. From BM and spleen of recipients, single GFP+ cells were isolated 2-3 weeks after transplantation, and single GFP+ CD34-KSL cells were isolated 1, 4, 8, and 12 months after transplantation. These cells were subjected to scRNA-seq. Based on transcriptome-defined (t) cell types under homeostasis, donor-derived single cells were classified into tHSCs, tMPPs, tCPs (committed progenitors, CMP, MEP, GMP and CLP), tMEs, tGMs, and tLymphocytes (B, T, and NK cells). We detected tMEs and tGMs as the major populations 2 weeks after transplantation. We first detected tHSCs 3 weeks after transplantation. Based on the absolute number of tHSCs, their maximum expansion rate was detected between 3 and 4 weeks after transplantation. We also detected tMEs, tGMs, and tLymphocytes from 1 to 12 months, and these cells increased more 8 and 12 months after transplantation. Donor cells were assigned as a and b cells based on the ratio of myeloid cells (GM) and lymphoid cells (T+B) in peripheral blood 4 month after transplantation. a cells gave rise to tHSC1 and 2 while b cells gave rise to tHSC1, 2, and 3. These data supported the original function definition of these cells. In summary, this study not only illustrates the early differentiation phase, followed by the expansion phase of HSC after transplantation. Deep analysis of the key regulation network in clonal hematopoiesis will provide more information for molecular mechanism of HSC self-renewal and lineage differentiation.