CD9 Marks Flt3+ Multipotent Hematopoietic Progenitors within Lsk Cells

Hematopoietic Stem Cells (HSCs) that give rise to all lineages of the blood are defined as a lineage-negative (Lin^-), c-Kit⁺, Sca-1⁺(LSK) population in mouse. The expression of Flt3 on LSK cells is accompanied by loss of self-renewal capacity, and transition to lymphoid-primed multipotent progenitors (LMPPs). The magnitude of Flt3 surface abundance has been shown to be correlated with lineage potential, where megakaryocyte/erythroid (MegE) potential is incompatible with high Flt3 levels (Buza-Vidas et al. 2011). However, it is unclear whether MegE capacity is marked by Flt3 itself or restricted to a specific population within the Flt3 spectrum. Interestingly, it has been shown that commitment to MegE lineages is an early event in HSC differentiation and that MegE progenitors share several cell-surface markers with HSCs. One such marker is CD9, which is highly expressed on megakaryocyte progenitors (Nakorn, Miyamoto, and Weissman 2003)as well as on the most primitive stem cells in mouse (Karlsson et al. 2013). In fact, we have recently shown that CD9 captures all long-term reconstitution activity of the murine LSK population, even in cell fractions that lacks some of the most established HSC markers .To compare CD9- and Flt3 expression we performed FACS analysis of the LSK compartment and plotted CD9 high- and low cells on top of the CD34/Flt3 expression profile. Interestingly we found that even if CD9 expression were predominantly high in LT-(CD34^-Flt3^-) and ST-HSCs (CD34⁺Flt3^-), CD9^highcells reached all the way into the Flt3⁺population but were lost in LMPPs, as defined by the 25% highest Flt3 expressing cells. To addresswhether CD9^highcells represent a distinct subpopulation within Flt3^intcells, LSKCD34⁺Flt3^int(Flt3^int) and LSKCD34⁺FLT3^intCD9^high(CD9^high) populations, were subjected to single-cell RNA-Sequencing analysis. Interestingly,our results demonstratethatCD9^highcells are enriched in the most primitive cluster of the Flt3^intpopulation. This was further validated in transplantation assays where only CD9^highFlt3^intcells reconstituted irradiated recipients. However, long-term reconstitution was limited to lymphoid lineages, while myeloid engraftment was only observed at early time points and self-renewal activity was absent. To address the clonal capacity of CD9^highcells, we designed a protocol to assess the potential of single-cells to generate B-, Myeloid (My)- and Erythroid (Er) cells in vitro.In these conditions 68% of clones generated from CD9^highcells were bipotent (55% My/B and 13% My/Er). In contrast, the majority of CD9^lowcells were unipotent (32% My- and 52% B cells).Moreover,the colony forming capacity for the CD9^highpopulation was significantly higher compared to CD9^lowcells and when assayed for colony-forming unit-megakaryocyte (CFU-MK) capacity, Meg potential was exclusively observed in LT-HSCs, ST-HSCs as well as the CD9^highFlt3^intpopulation, while neither CD9^lownor LMPPs generated any megakaryocyte colonies. In an attempt to improve the efficiency of MegE differentiation, single-cell clonogenic assays were performed in suspension cultures. Here,multipotency including MegE potential was gradually down regulated with increasing Flt3 cell surface expression, and decreasing CD9 expression. Together our results shows thatCD9 captures the most primitive cells within the LSKFlt3⁺population, including multipotent cells with MegE differentiation potential, thus resolving the heterogeneity of Flt3⁺hematopoietic progenitors and identifying a multipotent LSKCD9⁺Flt3⁺population distinct from LMPPs.