Abstract
The canonical Wnt signaling pathway is defined by Wnt ligand-mediated stabilization and nuclear translocation of β-catenin to induce target gene expression. This pathway has been demonstrated to regulate differentiation of mesenchymal tissue, which includes the cell types (e.g. osteoblasts, myofibroblasts, adipocytes) that comprise the stromal cells of the hematopoietic microenvironment. We hypothesized that loss of canonical Wnt signaling would result in disruption of the ability of stromal cells to support hematopoiesis. To test this hypothesis, we generated transgenic mice that expressed conditonal loss of function β-catenin alleles along with Cre-recombinase under the control of the inducible Mx1 promoter, which is active in bone marrow. We induced excision of β-catenin by injecting β-cateninlox/lox Mx-cre+/cre mice with 10 doses of 300 mg/ml pIpC. Whole bone marrow from treated (KO) and untreated (WT) animals was used to establish Dexter stromal cultures with an input of 1 × 106 cells/cm2 culture surface area. PCR performed on DNA isolated from KO stromal cells showed that nearly 100% deletion of β-catenin occurred with this regimen. To determine the ability of KO stroma to support hematopoiesis, irradiated KO and WT stromal cultures were seeded with 4 × 104 normal lin− cells/cm2. There were no differences in cell expansion, cell cycle activity, or apoptosis between hematopoietic cells cultured on WT vs. KO stroma. We determined the capacity of β-catenin deficient stroma to maintain hematopoietic progenitors by measuring myeloid CFU formation after 1, 2, and 3 weeks in culture. After 1 week, hematopoietic cells cultured on WT stroma contained 5-fold more CFU-GM (151.7 ± 21.4 CFU-GM/1×104 cells) than cells cultured on KO stroma (28.7 ± 4.9; n = 6, p < .001). Similar differences in CFU-GM formation were observed after 2 weeks (WT 46.5 ± 8.0 vs. KO 10.3 ± 1.7; n = 6, p< .001) and 3 weeks (WT 16.5 ± 2.8 vs. KO 2.6 ± 1.5; n = 6, p < .001) in culture. This decrease in the production of hematopoietic progenitor cells was not due to decreased numbers of stromal cells as the average number of KO stromal cells (4.8 ± 0.07 × 104/cm2) was greater than WT (3.7 ± 0.7 × 104/cm2; n = 3, p = .05). We also determined the ability of WT and KO mesenchymal progenitors to generate fibroblast colonies (CFU-F) and found no difference between WT (17 ± 1.8 CFU-F/1 × 106 bone marrow cells) and KO (15.8 ± 3.5; n = 4, p = .54). Canonical Wnt signaling has been proposed to regulate the differentiation of mesenchymal stem cells into osteoblasts. Since osteoblasts contribute to the proper regulation of hematopoiesis, we hypothesized that the depletion of hematopoietic progentiors in KO stromal cultures is due to a reduction in the number of osteoblasts. To detect osteoblasts in vitro, we performed histochemical staining to detect alkaline phosphatase (ALP) activity in WT and KO stromal cultures and scored the positive cells. We observed a significant 50% reduction in the percentage of ALP+ cells in KO stroma (13.2 ± 4.8%) compared to WT (28.0 ± 7.9%) (n = 3, p = .05). In summary, these data indicate that loss of canonical Wnt signaling results in decreased support of hematopoietic progenitors and osteoblasts. From these data, we propose a model in which canonical Wnt signaling is necessary to maintain normal numbers of osteoblasts within the bone marrow stroma and that loss of β-catenin leads to a decrease in the number of osteoblasts and a subsequent reduction in the ability of the stroma to support hematopoiesis.
Phenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells