Hematopoietic Flt3+ Multipotent Progenitors Are Organized in Discrete Functionally and Hierarchically Distinct Populations

Blood production in the bone marrow (BM) is classically viewed as a constant hematopoietic flux originating from a small pool of self-renewing hematopoietic stem cells (HSCs). In this model, quiescent HSCs regularly differentiate and give rise to a complex set of transient multipotent progenitors (MPPs) that serve as compartments of amplification through division, while undergoing progressive lineage specification Recent studies exploiting sophisticated genetic marking for in vivo cell-fate tracking have challenged this paradigm and suggested that HSCs have a limited contribution to steady state blood production (Sun, Nature 2014, Busch, Nature 2015; Rodriguez-Fraticelli, Nature 2018). The interpretation of these results and the extent of the HSC contribution to native hematopoiesis remains a matter of debate (Pucella, Annu Rev Cell Dev Biol 2020; Rodriguez-Fraticelli, Curr. Opin. Hematol 2021). Beyond this debate, these studies have highlighted the underestimated contribution of the MPP compartment to the daily hematopoietic output in unperturbed conditions. This new understanding raises questions about the functional properties of the MPP compartment in steady state condition and its ability to independently sustain prolonged hematopoietic cell production.

Here we revisited the early hematopoietic hierarchy present in the Lin^- Kit⁺ Sca1⁺ (LSK) fraction that includes all HSC and MPP (MPP1-5) compartments. Highly-multiplexed CITE-Seq single cell analyses with over 60 antibodies identified a novel LSK CD62L^- Flt3⁺ population (CD62L^- MPP4) that clusters within the MPP compartments but displays a cell cycle gene signature similar to the one found in quiescent HSCs. Analysis of the Mki67 reporter mouse model and nucleotide analogue incorporation assays directly confirmed the existence of this unexpected cell cycle diversity in MPP4 during unperturbed hematopoiesis. Consistent with the molecular data, these analyses characterized a resting CD62L^- and a proliferative CD62L⁺ MPP4 compartments. These two populations showed similar cloning efficiency in methylcellulose assays indicating that both populations behave as intermediary MPP compartments. When assessed in short-term culture, both populations were responsive to Flt3L stimulation but displayed specific kinetics of division. They also showed distinct short-term reconstitution ability upon transplantation in correlation with their cell cycle activity. Analysis of these populations in native condition using the H2B-GFP label-retaining system further confirmed the specific properties of the resting CD62L^- MPP4 cells, distinct from the other proliferative MPP2, MPP3 and CD62L⁺ MPP4 compartments. Finally, these studies uncovered a novel early path of differentiation that involves the MPP5 cells (LSK Flt3⁺ CD48^- CD150^-) sequentially acquiring Flk2 and CD48 expression to generate the resting CD62L^- MPP4 compartment, which in turn gives rise to cycling CD62L⁺ MPP4 cells. Altogether, these results challenge the accepted dichotomy between quiescent HSCs and dividing progenitors. While difference in lineage potential has been historically a major criterium to classify MPP populations, we established a new dimension at the MPP diversity, based on their proliferative activity at steady state. These results particularly highlight a new path of differentiation directly linking MPP5, a quiescent compartment with residual self-renewal capacity, to the resting CD62L^- Flt3+ MPP cells. Based on these results, we proposed that slow-dividing Flt3+ MPPs act as a key transitional compartment that can buffer the hematopoietic cell production and therefore regulate hematopoietic homeostasis at steady state. We envision that this novel compartment could be a key intermediary for hematopoietic malignancies driven by the acquisition of Flt3 mutations and a cellular target for intervention.

Cancelas:Hemanext LLC: Consultancy; TerumoBCT: Consultancy; Vascular Solutions/Teleflex Inc: Consultancy; Cytosorbents Inc.: Consultancy; Westat Inc: Research Funding; Cytosorbents Inc: Research Funding; Fresenius-Kabi: Research Funding; TerumoBCT: Research Funding; Becton-Dickinson: Research Funding; Platefuse Inc.: Other: Co-Ownership; Preservation Bio Inc: Other: Co-Ownership.