Abstract
Normal function of adult hematopoietic stem cells (HSC) is dependent upon the bone marrow microenvironment, which is comprised of multiple cell types, including fibroblasts, endothelial cells, osteoblasts and stromal progenitors. We hypothesized that canonical Wnt signaling, which is necessary for the development of mesenchymal tissue, regulates the cellular structure and function of the microenvironment. We tested this hypothesis using an in vitro model of bone marrow stroma that is deficient in β-catenin, the critical transactivator of canonical Wnt signals. β-catenin−/ − Dexter stroma cultures were established from whole bone marrow harvested from β-cateninlox/lox Mx-cre+/cre mice treated with 7–10 doses of 300 μg pIpC. PCR analysis of stromal cell DNA showed nearly 100% deletion of β-catenin. Confluent stroma cultures were irradiated and seeded with 4 x 104 lin cells/cm2. We have reported (Nemeth, et al, Blood, 108, 29a) that β-catenin−/ − stroma exhibit decreased ability to support CFU formation and generate osteoblasts. The reduction in CFU-C correlates with a 75% increase in the percentage of lin− progenitors cultured on β-catenin−/ − stroma undergoing apoptosis (23.6 ± 3.4%) compared to wild-type (13.6 ± 1.3% ; n = 3; p < .01). To determine the mechanism by which canonical Wnt signaling regulates microenvironment function, we used a cytokine antibody array to analyze protein levels of 30 different hematopoietic growth factors and adhesion molecules. We observed decreased amounts of the soluble factors bFGF (3.3 ± 0.6-fold) and SCF (2.3 ± 0.3-fold), and the adhesion factor VCAM-1 (2.7 ± 0.3-fold) (n = 3; p < .01) in β-catenin−/ − stroma. The decrease in VCAM-1 corresponded with decreased percentages of VCAM-1+ osteoblasts (26.8 ± 0.9% vs. 43.9 ± 5.7%) and endothelial cells (31.6 ± 5.7% vs. 76.5 ± 10.9%) in β-catenin−/ − stroma compared to wild-type (n = 4, p < .01). From these data, we hypothesized that β-catenin is necessary for maintaining the stromal cells that support HSCs in vivo. We tested this hypothesis by transplanting 2 x 106 wild-type whole bone marrow cells (CD45.1) into lethally-irradiated β-cateninlox/lox Mx-cre+/cre mice and littermate controls. 8 weeks later, transplanted mice were treated with pIpC, resulting in mice with a wild-type hematopoietic system and a β-catenin−/ − microenvironment. Two weeks after the final treatment, we observed a 2.7 ± 0.1-fold reduction in the percentage of long- and short-term HSCs (lin−, c-kitHI, Sca-1HI) in bone marrow from mice with a β-catenin−/ − microenvironment compared to wild-type (n = 4, p < .05). We performed a competitive repopulation assay, transplanting 1 x 106 whole bone marrow cells harvested from primary recipients with a wild-type or β-catenin−/ − microenvironment with 1 x 106 CD45.2 whole bone marrow cells into lethally-irradiated secondary recipient mice. After 16 weeks, there was no difference in mean repopulation by bone marrow cells from the β-catenin−/ − microenvironment (9.2 ± 2.8%) compared to wild-type (10.7 ± 0.6%), indicating that self-renewal was unaffected. However, we did observe a significant 4-fold increase in variability of repopulation by bone marrow cells from the β-catenin−/ − microenvironment (F-test = .01). Since smaller numbers of HSCs will yield greater variability in repopulation than larger numbers, this is consistent with the observation that the β-catenin−/ − microenvironment supports fewer HSCs. From these data, we propose a model in which canonical Wnt signaling in the microenvironment is necessary for hematopoietic proliferation and differentiation.
Notch-Wnt signal crosstalk regulates proliferation and differentiation of osteoprogenitor cells during intramembranous bone healing
