Novel Role for Host-Derived Megakaryocytes In Facilitating Stem Cell Engraftment through Enhancement of Osteoblastic Niche Restoration Following Radioablation

Abstract 558

The osteoblastic niche is a critical site of engraftment following stem cell transplantation. We have previously demonstrated that the osteoblastic niche expands within the first 48 hours following marrow radioablation. This expansion is accompanied by relocalization of megakaryocytes from their homeostatic location within the central marrow space to the endosteal surface at sites of osteoblast expansion, suggesting that these relocalized megakaryocytes may play a key role in reconstitution of the niche. To determine whether megakaryocytes contribute to the radioablation-induced osteoblast expansion and consequently assist in facilitating engraftment, we have examined osteoblast expansion and stem cell engraftment in wildtype (WT) mice, thrombopoietin receptor (c-Mpl) deficient mice (mpl−/−) that have less than 20% of normal megakaryocyte numbers, and in mice treated with an anti-CD41 blocking antibody (MWREG30). BrdU incorporation and TUNEL assays demonstrated that megakaryocyte relocalization following radioablation occurs through active migration of viable megakaryocytes. mpl−/− mice or anti-CD41 treated WT mice developed less than 20% of the megakaryocyte endosteal migration seen in untreated WT mice 48 hours after radioablation (*P<0.001), and anti-CD41 treatment of irradiated mpl−/− mice reduced endosteal megakaryocytes to baseline pre-radiation numbers. Moreover, mpl−/− mice developed less than 50% of the osteoblast expansion seen in WT mice following irradiation; thus, abrogating megakaryocyte activity blocks critical signals required for osteoblast expansion. Bone marrow expression of IGF-1, a recognized osteoblast growth factor, increased 5-fold within 48 hours after radiation relative to pre-radiation levels in WT mice, but this IGF-1 spike is completely blocked by c-Mpl deficiency, suggesting that megakaryocytes may induce osteoblast expansion through a pathway in which c-Mpl signaling leads to IGF-1 expression. To test the functional significance of radiation-induced megakaryocyte migration and osteoblast expansion, we transplanted lethally irradiated WT or mpl−/− mice with or without anti-CD41 treatment using bone marrow from GFP-transgenic mice. Initial bone marrow engraftment of WT GFP+ donor cells within 24h of transplantation was significantly reduced (*P<0.01 for all groups) in anti-CD41 treated WT (51% of engraftment seen in untreated wildtype recipients), untreated mpl−/− (45%), and anti-CD41 treated mpl−/− (35%) recipients. Expansion of engrafted WT GFP+ donor cells at 3, 5 and 7 days post-transplant was also significantly reduced in untreated mpl−/− recipients (36%, 53%, 63% of untreated WT recipients at 3, 5, and 7 days, respectively, *P<0.05) and anti-CD41 treated mpl−/− recipients (25%, 33%, and 30% at 3, 5, and 7 days, respectively, *P<0.05), with prominent deficits specifically in the reconstitution of the B lymphocyte lineage. Bone marrow cellularity remained significantly reduced in anti-CD41 treated WT and untreated or anti-CD41 treated mpl−/− recipients by 35–45% relative to untreated WT recipients at least out to 3 weeks post-transplant (*P<0.01). Using competitive repopulation secondary transplantation assays performed with marrow harvested from primary recipients at 24h after primary transplantation, we showed that progenitor and short term hematopoietic stem cell (HSC) engraftment was significantly decreased in mpl−/− versus WT primary recipients (*P<0.05). Secondary transplant assays performed with marrow harvested from primary recipients 3 weeks after initial transplantation demonstrated that engraftment and expansion of long term-HSC (*P <0.05) and B cell reconstitution (*P<0.005) are significantly impaired in anti-CD41 treated mpl−/− versus untreated WT recipients. Taken together, our findings demonstrate that host megakaryocytes migrate to the endosteal surface following marrow radioablation where, through the enhancement of osteoblast niche expansion and potential osteoblast-independent effects, they play a pivotal role in facilitating efficient HSC engraftment following transplantation. Further understanding of these stem cell niche restoration pathways may reveal novel therapeutic targets to improve engraftment efficiency in the clinical setting.