Expansion of Quiescent Hematopoietic Stem Cells under Stress and Nonstress Conditions in Mice

Hematopoietic stem cells (HSCs) are maintained in the quiescent state for protection from stress. How quiescent HSCs expand in vivo under stress and nonstress conditions, however, is poorly understood. Using the fluorescent ubiquitination-based cell cycle indicator (Fucci) mice, we analyzed quiescent and cycling HSCs in the bone marrow after transplantation and during development and aging. The cell cycle of HSCs in Fucci mice were analyzed by flow cytometry. Single-cell colony assays suggested that cycling cells were likely in the process of differentiation. Long-term competitive repopulation and limiting dilution assays revealed that given a higher frequency of functional HSCs in quiescent cells, durable self-renewal potential was greater in quiescent cells than cycling cells. In the bone marrow, functional HSC pool, represented by quiescent HSCs, was rapidly re-established by three weeks after transplantation, significantly expanded by three weeks of age in development, and gradually accumulated with aging. Single-cell RNA-sequencing with flow cytometric index sorting suggested that high levels of CD201 and Sca-1 expression and a low level of mitochondrial activity were associated with quiescent HSCs. A set of candidate quiescent genes in HSCs were also provided. This study implied that controlling quiescence in HSCs is important for their in vivo expansion and maintenance.