Notch2 Signaling Inhibits Differentiation and Promotes Self Renewal of Hematopoietic Stem and Progenitor Cells During Marrow Regeneration.

Abstract 2544

Poster Board II-521

Notch regulates numerous lineage choices during vertebrate development, and although ex vivo studies suggest that Notch regulates hematopoietic stem cell (HSC) and multipotential progenitor (MPP) differentiation, a functional role for Notch in HSC/MPP self renewal in vivo remains controversial. We previously reported a Notch2 signaling role during bone marrow (BM) recovery following injection with chemotherapeutic agent 5-fluorouracil (5FU), where Notch2 signaling impedes myeloid differentiation, allowing for generation of sufficient numbers of progenitor cells. Herein, we examine a Notch2 signaling role in HSC as well as progenitor cell self renewal by enumerating generation of HSC and short term repopulating cells in lethally irradiated recipients (Ly5.1⁺) transplanted with a limiting number (5 × 10⁵) of BM cells from either control mice or from mice bearing Cre-LoxP-inducible Notch2 deletions (Ly5.2⁺). In recipient mice transplanted with control BM, recovery was evident from Day11 to Day13 post transplant when significantly more than the initial post-irradiation number of 9.0 × 10⁶ BM cells was seen in the recovering marrow. In recovering mice, recipients receiving control cells generated more BM cells than did recipients receiving Notch2-deficient cells. Furthermore, mice receiving control cells generated significantly more donor Sca-1⁺c-kit⁺ (SK⁺) cells than recipients receiving Notch2-deficient BM cells [44.4×10³ (s.e.m.+/− 14×10³) vs 8.2×10³ (s.e.m.+/−1.5×10³), respectively, p=0.001]. To quantitate the generation of short term repopulating cells, secondary radioprotection assays were performed. Irradiated secondary recipient mice received 1×10⁶ BM cells from the primary recipients previously transplanted with either control cells or Notch2-deficient cells. Secondary recipients receiving cells from primary control transplants survived significantly longer than those receiving cells from primary Notch2-deficient transplants or than irradiated mice receiving no cells (n=4, p=0.01), indicating Notch2 is required to generate sufficient numbers of cells to provide radioprotection. To quantitate long term HSC generated in the recovering marrow, competitive repopulating units (CRU) were enumerated by performing secondary transplants in which 4-doses of BM cells ranging from 4 × 10⁴ to 5 × 10⁶ cells from primary transplants were injected into secondary recipients along with 1 × 10⁵ Ly5.1⁺ competing cells. Enumeration of CRU at 2 weeks post transplant confirmed the number of short term repopulating cells was significantly decreased in mice transplanted with Notch2-deficient cells compared to mice transplanted with control cells [(1.3 CRU vs 8.8 CRU / 1×10⁶ BM cells, respectively), p=0.0004)]. Enumeration of CRU at 9 weeks post transplant indicated HSC numbers were also significantly decreased in mice transplanted with Notch2-deficient cells compared to mice transplanted with control cells [(0.1 CRU vs 0.7 CRU / 1×10⁶ BM cells, respectively), p=0.02]. Taken together, our results demonstrate a role for Notch2 in enhancing generation of long term HSC as well as short term repopulating cells and suggests that Notch2 signaling regulates a hierarchy of events to assure the initial repopulation by HSC and MPP, while delaying myeloid differentiation during hematopoietic regeneration.