P190-B RhoGAP Is Critical for Hematopoietic Stem Cell Niche Regulation

Abstract 221

Hematopoiesis is regulated by components of the stromal microenvironment, so-called niche. Although the concept of hematopoietic stem and progenitor cell (HSC/P) niche is well known, its molecular regulation remains ill-defined. Here, we provide evidence that p190-B GTPase Activating Protein (p190-B), a negative regulator of Rho activity, is a regulator of mesenchymal/stromal cell functions necessary for normal hematopoiesis during fetal development. Mice lacking p190-B die before birth. At day 14.5 post coitum, p190-B^−/− embryos are paler than WT embryos with a 40% lower hematocrit. Cellularity and numbers of burst forming-unit (BFU) erythroid (E), colony forming-unit (CFU)-granulo-monocytic and CFU-erythroid per p190-B^−/− fetal livers (FL) were 50% lower than WT. In addition, the frequency of LongTerm-HSC (Lin^negSca^posKit^posCD150^posCD48^neg), ShortTerm-HSC (LSK-CD150^posCD48^pos), and progenitors (LK) appeared significantly reduced in the bone marrow of e17.5/18 embryos. These data suggest a defect in fetal hematopoiesis. However, p190-B^−/− FL cells were able to fully reconstitute hematopoiesis of adult irradiated recipients (Xu, Blood 2009). Mice reconstituted with p190-B^−/− FL cells exhibited bone marrow content, white blood count, red blood count and hematocrit similar to those reconstituted with WT cells. Furthermore, hematopoietic recovery of p190-B^−/− reconstituted animals following 5-Fluorouracil or phenylhydrazine-induced stress was comparable to that of WT. Therefore, FL HSC/P retain hematopoietic potential, which suggests a non-cell autonomous defect in p190-B^−/− embryos. In the present study, we examined in more detail this possibility. The numbers of CFU-fibroblast (CFU-F) were 2-fold lower in p190-B^−/− FL compared to WT FL. We next examined the hematopoietic supportive capacity of p190-B^−/− FL microenvironment using stromal cell cultures derived from e14.5 FL. No noticeable differences were observed during the establishment of the stromal cell cultures. p190-B^−/− stromal cells exhibited a spindle-like shape and expressed CD90, CD44 and Sca-1 but not CD45, CD31 or CD11b similarly to WT stromal cells. Cobblestone Area Forming Cell assays (CAFC) was performed in co-culture between various number of WT BM cells and p190-B^−/− and WT stromal cells. We examined 5 independent stromal cell cultures from both genotypes. Frequency of CAFC was 75% lower on p190-B^−/− stroma than WT stroma at both 1 week (1 in 5 × 10 ³ versus 1 in 21 × 10 ³, n = 5 p < 0.01) and 2 weeks (1 in 21 × 10 ³ versus 1 in 87 × 10 ³, n = 4 p < 0.01) in culture. CFU with cells recovered from one week coculture was also performed. Cocultures with p190-B^−/− stroma gave rise to 2-fold less CFU than with WT stroma (538+90 versus 255+81, p < 0.01, one representative experiment of 4). Using competitive repopulation assay, we then assessed the in vivo repopulation ability of CD45.2⁺ hematopoietic cells co-cultured for one week on each stroma. Fifteen weeks following transplantation, the frequency of CD45.2⁺ cells in the peripheral blood of mice that received cells cocultured on p190-B^−/− stroma was dramatically reduced compared to mice that received cells cocultured on WT stroma (0.43% + 0.37 versus 32.4% + 7.5, n = 6, p < 0.01), equivalent to a 100-fold difference in calculated competitive repopulation unit. Therefore, p190-B^−/− stroma exhibited defective hematopoietic supportive activity. Interestingly, p190-B^−/− stromal cells, like WT, showed characteristic of mesenchymal stem cells (MSC), i.e. ability to give rise to CFU-F and to differentiate to adipocytes and osteoblasts. They express relatively high level of nestin and osteopontin. At a mechanistic level, gene expression analysis of molecules known to play a critical role in HSC maintenance in the niche indicated 200-fold downregulation of Kit-ligand and Wnt3a in p190-B^−/− stroma compared to WT stroma (p<0.05). Surprisingly, BMP4, angiopoietin and CXCL12 were upregulated in p190-B^−/− stroma, 35-fold, 14-fold and 16-fold, respectively (p<0.05). Hence, p190-B expression in MSC/stromal cell microenvironment fine-tunes niche regulatory pathways to maintain normal hematopoiesis. Therefore, p190-B may be a critical regulator of the mesenchymal stem/hematopoietic stem and progenitor niche.