Pleiotrophin Signaling Is Necessary and Sufficient for Hematopoietic Stem Cell Self-Renewal In Vivo

Abstract 404

Several signaling pathways have been elucidated which regulate hematopoietic stem cell self-renewal, including the Notch, Wnt, HOX and BMP signaling pathways. However, several of these pathways (e.g. Notch, Wnt) may not be necessary for maintenance of HSCs in vivo. We recently demonstrated that treatment of murine and human HSCs with the heparin binding growth factor, pleiotrophin (PTN), was sufficient to induce self-renewal of murine and human HSCs in culture (Himburg, Nat Med, 2010). In order to determine if PTN signaling is necessary for HSC self renewal and normal hematopoiesis in vivo, we examined the bone marrow HSC content and hematopoietic profile of mice bearing a constitutive deletion of PTN (PTN−/− mice) as well as mice bearing constitutive deletion of the PTN receptor, receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ) (courtesy of Dr. Gonzalo Herradon, Spain and Dr. Sheila Harroch, L'Institut Pasteur, Paris, FR). PTN−/− mice demonstrated no significant differences in total bone marrow (BM) cells or BM colony forming cells (CFCs) but had significantly decreased bone marrow CD34(-)c-kit(+)sca-1(+)lin(-) (34-KSL) cells compared to littermate controls which retained PTN (PTN+/+) mice (0.007% vs. 0.02%, p=0.03). Consistent with this phenotype, PTN−/− mice also contained 2–fold decreased CFU-S12 compared to control PTN+/+ mice (p= 0.003). PTN−/− mice also demonstrated an 11-fold reduction in long-term repopulating HSC content compared to PTN+/+ mice as measured via competitive repopulating assay (12 week CRU frequency: 1 in 6 cells vs. 1 in 66 cells). Taken together, these data demonstrate that PTN signaling is necessary for maintenance of the BM HSC pool in vivo. Since PTN is known to antagonize the phosphatase activity of RPTPβ/ζ, we hypothesized that deletion of RPTPβ/ζ would increase BM HSC self-renewal and result in expansion of the BM HSC pool in vivo. Consistent with this hypothesis, RPTPβ/ζ−/− mice displayed a 1.3-fold increase in total BM cells (p= 0.04), 1.8-fold increase in BM 34-KSL cells (p=0.03), 1.6-fold increase in BM CFCs (p= 0.002) and 1.6–fold increase in BM CFU-S (p< 0.0001). RPTPβ/ζ−/− mice also demonstrated 1.4–fold higher long-term repopulating capacity (12 weeks) following competitive repopulating assay compared to RPTPβ/ζ+/+ mice (Donor CD45.1+ cell engraftment: 4.2% vs. 1.5%). Interestingly, RPTPβ/ζ −/− mice had significantly increased PB white blood cell counts, hemoglobin and platelet counts compared to RPTPβ/ζ+/+ mice coupled with splenomegaly. The RPTPβ/ζ−/− mice also had significantly increased BM vascular density (via quantitative mouse endothelial cell antigen staining) compared to RPTPβ/ζ+/+ mice, suggesting that PTN/RPTPβ/ζ signaling may augment the HSC pool size directly and also indirectly via activation of the BM vascular niche. These results demonstrate that PTN signaling is necessary and sufficient for induction of HSC self-renewal in vivo.