Abstract 1962: CXCR4-mediated Bone Marrow Stem Cell Homing and Niche Maintenance Is Modulated by the Functional Status of c-kit

Abstract

Objective: The bone marrow (BM) “niche”, where the stem cells are dynamically maintained, is an attractive target for cell-based cardiovascular repair. Both CXCR4 antagonist and c-kit blocking antibody can rapidly mobilize BM stem cells; however, the functional interaction between CXCR4 and c-kit remains unclear. We hypothesize that CXCR4 crosstalk with c-kit to maintain BM stem cell niche. Methods and Results: We designed an in vitro adhesion assay to mimic BM niche. Mouse total BM cells were harvested and applied onto VCAM1-coated plates for 15 min, and followed by addition of CXCR4 agonist SDF-1 or antagonist AMD3100. The adhesion of BM cells to VCAM1 resulted in marked c-kit phosphorylation. Interestingly, AMD3100 significantly decreased while SDF-1 augmented the c-kit phosphorylation, suggesting that CXCR4 may regulate the functional status of c-kit. To determine the role of c-kit in CXCR4-regulated stem cell niche maintenance, we developed an in vivo homing assay. The c-kit mutant (c-kit W/W-V ) and WT mice (n=5) were first treated with AMD3100 to disrupt CXCR4-mediated niche, and 2 h later, transplanted with eGFP-transgenic BM cells. After 3 h, BM cells were isolated and analyzed by FACS. The percentages of donor-derived eGFP+, eGFP+Lin+, and eGFP+Lin− cells were similar between the two groups. However, the donor-derived primitive stem cells, including eGFP+Lin-Sca1+ and eGFP+Lin-ckit+ cells, were significantly fewer in c-kit W/W-V mice than in WT mice (c-kit W/W-V vs. WT: 94.1% vs. 96.0%, p<0.05, and 33.82% vs. 55.27%, p<0.01, respectively), indicating that c-kit-deficient BM niche specifically affects primitive stem cell homing and maintenance. Importantly, although donor-derived CXCR4-expressing total (eGFP+CXCR4+) cells were similar in both groups, CXCR4-expressing stem cells, including eGFP+Lin-CXCR4+, eGFP+Lin-Sca1+CXCR4+, and eGFP+Lin-ckit+CXCR4+ cells, were much fewer in c-kit W/W-V than in WT mice (c-kit W/W-V vs. WT: 19.7% vs. 30.6%, p<0.01, 20.3% vs. 29.1%, P<0.05, and 6.7% vs. 17.9%, p<0.001, respectively), suggesting c-kit deficiency reduces the homing and maintenance of stem cells that express CXCR4. Conclusion: CXCR4-mediated BM stem cell homing and niche maintenance is modulated by the functional status of c-kit. This research has received full or partial funding support from the American Heart Association, AHA National Center.