IGF1-Mediated Osteoblastic Niche Expansion After Marrow Ablation Enhances Long-Term Hematopoietic Stem Cell Engraftment and Hematopoietic Reconstitution After Bone Marrow Transplantation

Abstract 557

The capacity for hematopoietic stem cells (HSCs) to engraft in bone marrow (BM) and reconstitute long-term multilineage hematopoiesis after transplantation depends on their interaction with a specific microenvironment, the stem cell niche. We previously demonstrated a profound reorganization of the marrow endosteal microenvironment after lethal total body irradiation (TBI) of mice that leads to a rapid restoration of the osteoblastic HSC niche, followed by a transient, reversible expansion of this niche. Here, we show that the niche restoration and expansion is driven by a small number of highly proliferative osteoprogenitors via an IGF1 dependent mechanism, that directly facilities long-term hematopoietic stem cell homing and engraftment. At homeostasis, BM osteoblasts (OB) are organized as a single layer of flattened cells at the endosteal surface. Within 3 h of TBI administered to C57BL6 mice, few OB were identified at the endosteal surface. However, by 6 h post-TBI, a new single layer of OB was evident, followed by a marked increase in OB proliferation resulting in multiple layers of cells at the endosteum. The OB proliferation showed half maximal expansion at 24 h post-TBI and maximal expansion at 48 h, at which time the expanded OB constituted significantly more cell tiers than in unirradiated controls (p<0.005). BrdU incorporation/Ki67 expression dual marker studies, conducted over 48 h post-TBI, revealed clusters of highly proliferative cells that expand to repopulate the endosteum and to drive the osteoblastic niche expansion, suggesting a pool of radioresistant osteoprogenitors as the source of the niche restoration and expansion. Real-time qPCR of BM cells harvested after TBI showed an increased gene expression of Sdf1, Pdgfβ, TGFβ, Fgf2, and notably, Igf1, that peaks at 48 h. Using an ELISA, we, similarly, found an increase in protein expression, relative to unirradiated controls, that also peaks at 48 h (SDF1, 2.3-fold increase, p<0.005; PDGFβ, 6.1-fold, p<0.005; TGFβ, 4.4-fold, p<0.005; FGF2, 3.2-fold, p=0.05; IGF1, 5.3-fold, p<0.005). We propose that this extensive niche remodeling, closely correlated with increased expression of highly relevant cytokines, serves to optimize the osteoblastic niche for homing and engraftment of HSCs. To test the hypothesis, we used competitive repopulation secondary transplantation assays to assess the capacity of the osteoblastic niche to foster HSC engraftment. Primary recipients were transplanted with GFP-transgenic BM (3×10⁶ cells/mouse) at 0.5 h, 24 h, or 48 h (maximal niche expansion) after TBI. At 24 h after transplantation, BM cells (representing 12.5% of the total marrow population) harvested from the primary recipients were transplanted into lethally irradiated secondary recipients with 2×10⁵ competitor (unlabeled) BM cells. Secondary recipients, receiving marrow from primary recipients transplanted at 48 h post TBI, showed greater engraftment at 3 (p<0.05), 6 (p<0.001), and 18 (p<0.05) weeks after BMT compared with the 0.5 h and 24 h groups, indicating that 48 h of niche expansion facilitates homing/early engraftment of short- and long-term hematopoietic repopulating cells. To further define the importance of niche expansion and to determine the contribution of IGF1 signaling, we performed a competitive repopulation secondary transplantation assay with and without picropodophyllin (PPP), a specific IGF1 receptor tyrosine kinase inhibitor. Treated mice were either sacrificed for evaluation of niche expansion or transplanted with GFP-transgenic BM cells at 48 h post TBI. The short half-life of PPP (3 h) indicates that the drug will be eliminated prior to transplantation, 36 h after the last PPP dose. Histological analysis of irradiated, PPP-treated mice revealed that the osteoblastic niche was restored but did not expand to multiple layers at 48 h. Secondary recipients, receiving marrow from PPP-treated primary recipients, showed no evidence of short-term or long-term hematopoiesis in contrast to untreated controls (p<0.05). These data indicate that IGF1 mediated effects on the BM stem cell niche are essential for niche expansion and for engraftment of transplanted HSCs. Collectively, our data indicate that IGF1 mediated osteoblastic niche expansion is a pivotal component of HSC homing and engraftment. Targeting niche expansion may enhance HSC engraftment after clinical BMT, especially if HSC dose is limiting.