Abstract: Hematopoietic stem cells (HSCs) reside at the apex of the differentiation hierarchy and provide the lifelong supply of all blood cell lineages. While decades of work have revealed HSCs' differentiation pathways and kinetics to maintain homeostasis and ensure regeneration, our knowledge about their immediate cell fates following various stress conditions remains limited. Here, we determined the daily proliferation and survival states of murine HSCs following 5-fluorouracil (5-FU)-induced myeloablation during a 14-day recovery process. We further investigated their differentiation kinetics in this period using complementary approaches of scRNA-Seq and lineage tracing. Using the high expression of endothelial protein C receptor (EPCR) as the primary marker for HSC identification, our work revealed a partial but immediate depletion of the most primitive HSC population following 5-FU treatment. Notably, this partial depletion was not attributed to increased HSC cell death or migration. Instead, it was caused by an abrupt increase in their differentiation, as suggested by the results of a lineage tracing analysis using an inducible EPCR-directed Cre labeling model. scRNA-Seq and mathematical modeling further revealed that HSC produced the multipotent progenitor (MPP) compartment through an atypical differentiation pathway that is active under the stress condition but latent at the steady state. Unexpectedly, HSC atypical differentiation declined rapidly after this initial surge, and HSCs contributed mainly to the primed HSC compartment and the the megakaryocyte-erythroid biased MPP subtype during the bulk of the recovery period. Collectively, our work uncovers sequential differentiation patterns adopted by HSCs upon extensive myeloablation, which provides a framework to elucidate the molecular mechanisms controlling HSC-mediated regeneration and ultimately helps to design novel strategies to facilitate hematopoietic recovery in myelosuppressed patients.
