Bone Morphogenetic Protein 4 Regulates Hematopoietic Stem Cell Maintenance in Vivo.

The secreted signaling molecule Bone Morphogenetic Protein 4 (BMP4) is expressed by osteoblasts and other cell types that comprise the hematopoietic microenvironment. Therefore, we hypothesized that BMP4 may play an important regulatory role in the maintenance and function of hematopoietic stem cells (HSCs). As the deletion of BMP4 is lethal during early embryogenesis, we exploited a viable BMP4 hypomorph in which a point mutation in BMP4 reduces, but does not abolish the amount of active BMP4 ligand. In these mutants, peripheral blood cell lineages and bone marrow cellularity are normal during steady state conditions. However, consistent with our hypothesis, 40% fewer c-kit+, Sca-1+, lineage- (KSL) cells were present in the femurs of mutants (7.4 x10³ ± 0.4 x10³ SEM) than in age-and sex-matched wild-type (WT) controls (12.4 x10³ ± 1.3 x10³, p<0.005). Transplantation of mutant KSL cells produced levels of hematopoietic engraftment indistinguishable from transplanted WT KSL cells, consistent with a stem cell extrinsic effect of BMP4. To functionally assess the mutant hematopoietic microenvironment, unfractionated WT bone marrow cells (BM) were transplanted into lethally irradiated mutants or WT controls. Although WT cells could engraft mutant primary recipients to the same degree as WT recipients, serial transplantation of these WT cells into secondary WT hosts revealed a marked depletion of hematopoietic reconstitution activity. Specifically, nearly a 4-fold reduction of donor cells was found in the peripheral blood of secondary recipients that received BM from reconstituted mutant hosts (p<0.0005). To determine whether such defects in the mutant microenvironment exist in the absence of myeloablative conditioning and transplantation, a parabiotic mouse model was employed. CD45.1 WT mice were joined to CD45.2 BMP4 hypomorphs for 8 weeks and then separated. As early as one month following separation, a greater than 2- fold reduction in circulating WT donor cells was detected in mutant hosts relative to the number of mutant donor cells detected in the WT hosts (p<0.005). Analysis of BM at 24 weeks following separation revealed a striking 23- fold reduction in WT donor hematopoietic cells in the mutant host BM compared to the number of mutant hematopoietic cells detected in WT host BM. Together, our findings reveal a novel, critical role for BMP4 in maintaining HSCs in vivo.