Low-Dose Epo after Tgfβ Blockade Triggers Robust Erythropoiesis and Increased RBC Production

Background: Therapy for chronic anemias is limited to RBC transfusions and Erythropoiesis Stimulating Agents (ESA) which often work on only transiently or not at all. New approaches to treat chronic anemia are needed but development has been limited by our incomplete understanding of erythropoiesis, most of which relates to the terminal maturation of erythroid precursors. Erythropoietin (Epo) acts during a very narrow window of erythropoiesis, well after progenitor commitment to an exclusively erythroid fate. It is not known if the final steps of RBC maturation are coupled to the earlier stages of hematopoietic stem and progenitor cell (HSPC) differentiation; a process that begins almost three weeks earlier when an HSC starts its march towards committed RBC precursors via a series of branching cell fate decisions.We searched for independent control and compartmentalization of erythropoiesis that could couple early hematopoiesis to terminal erythroid commitment and maturation. Results: We deleted TGFβ1 in megakaryocytes (TGFβ1ΔMk/ΔMk) and found that peripheral blood counts were normal in TGFβ1ΔMk/ΔMkmice compared to TGFβ1FL/FLcontrols despite the pool of primitive hematopoietic cells being expanded (Fig. 1a). Similarly total bone marrow cellularity was normal in TGFβ1ΔMk/ΔMkmice (Fig. 1b). Excess HSCs in TGFβ1ΔMk/ΔMkmice appeared capable of robust differentiation because the number of immature lineage-negative (Linneg) hematopoietic progenitor cells was increased in the marrows of TGFβ1ΔMk/ΔMkmice (Fig. 1c). Thus, it remained unexplained why the expanded number of HSPCs (Fig. 1d) do not increase blood counts and marrow cellularity. We hypothesized that the excess progenitors observed in the TGFβ1ΔMk/ΔMkmice failed to increase blood counts because their progeny were unneeded, and inadequately supported by homeostatic levels of late-acting cytokines. Indeed, bone marrow apoptosis was increased in the TGFβ1ΔMk/ΔMkmice compared to controls, as reported by AnnexinV (AV) binding (Fig. 1e-f). Apoptosis of lineage-marker negative (Linneg), Kit+Sca1neg(LKSneg) HPCs and LKS+HSPCs was rare in both TGFβ1ΔMk/ΔMkmice and littermate controls (Fig. 1g). These results suggest that excess, hematopoietic precursors present in the TGFβ1ΔMk/ΔMkmice are pruned by apoptosis during hematopoietic differentiation. We found 10-fold apoptosis in TGFβ1ΔMk/ΔMkprecursors populations BM (Fig. 1h). Epo levels were normal in the serum of these mice, we reasoned that the excess, unneeded cel