Differences in Cell Cycle Status Underlie Transcriptional Heterogeneity in the HSC Compartment

Hematopoietic stem cells (HSCs) are considered a heterogeneous cell population. To further resolve the HSC compartment, we characterized a retinoic acid (RA) reporter mouse line. Sub-fractionation of the HSC compartment in RA-CFP reporter mice demonstrated that RA-CFP-dim HSCs were largely non-proliferative and displayed superior engraftment potential in comparison with RA-CFP-bright HSCs. Gene expression analysis demonstrated higher expression of RA-target genes in RA-CFP-dim HSCs, in contrast to the RA-CFP reporter expression, but both RA-CFP-dim and RA-CFP-bright HSCs responded efficiently to RA in vitro. Single-cell RNA sequencing (RNA-seq) of > 1,200 HSCs showed that differences in cell cycle activity constituted the main driver of transcriptional heterogeneity in HSCs. Moreover, further analysis of the single-cell RNA-seq data revealed that stochastic low-level expression of distinct lineage-affiliated transcriptional programs is a common feature of HSCs. Collectively, this work demonstrates the utility of the RA-CFP reporter line as a tool for the isolation of superior HSCs.