Attenuation of CXCR4/SDF-1 Axis in Bone Marrow Mesenchymal Stromal Cells Impairs Hematopoietic Niche Activity and Promotes Stem Cell Aging

Aging impairs hematopoietic stem cell (HSC) function by reducing their regeneration potential and skewing differentiation towards the myeloid lineage, leading to anemia, decreased immune function, and increased propensity for hematologic malignancies. While stem cell intrinsic mechanisms are known to contribute to HSC aging, it is not well understood whether age-related changes in bone marrow niches also contribute to HSC aging. Recent studies have shown that mesenchymal stem cells (MSC) form a crucial component of the HSC niche, promoting HSC quiescence and balanced differentiation. We recently discovered that deficiency of CXCR4 expression on bone marrow stromal cells and Nestin+ MSCs reduces hematopoietic regeneration after HSC transplantation. Whether impairment of CXCR4/SDF-1 signaling in the aged marrow contributes to loss of HSC regenerative potential is not known. Analysis of bone marrow from aged (22-25 month) and young (3-4 month) C57BL/6 mice revealed ~55% reduction in CXCR4 expression on MSC (CD45-Ter119-CD31-CD51+PDGFR+Nestin+) and 40% reduction in CXCR4 expression on total bone marrow stromal cells (CD45-Ter119-CD31-) as measured by flow cytometry. In addition, MSC and total stromal cells from aged mice demonstrated lower expression of CXCR4 transcripts compared to MSC derived from young mice. The reduced expression of CXCR4 on MSC from aged mice was accompanied by increased expression of total and mitochondrial ROS (3-fold and 3.5-fold higher, respectively), senescence markers including p16 and b-galactosidase (2.5-fold and 2.1-fold higher, respectively) and DNA damage (2-fold higher Kap-1 phosphorylation). Aged mice demonstrated fewer total MSC in the bone marrow (2.5-fold lower) and reduced ex vivo clonal expansion (5-fold lower) measured by CFU-F formation. Furthermore, the marrow niche in aged mice produced significantly lower amounts of the HSC maintenance factors SDF-1, SCF and b-FGF. In vitro exposure of SLAM LSK cells from young mice to MSC from aged mice resulted in a higher cycling of the HSCs and reduced HSC engraftment potential by 3.2 fold, with myeloid biased differentiation observed at 6 months after competitive transplantation compared to SLAM LSK cells from young mice cultured on MSC from young mice. These data suggest that reduced expression of CXCR4 on MSC associated with aging may have functional consequences on HSC niche function leading to an aged HSC phenotype. To determine whether the loss of CXCR4 in bone marrow stromal cells/MSC drive HSC aging, we created chimeric mice by transplanting wild-type bone marrow donor cells either into tamoxifen-inducible conditional CXCR4 knockout mice or into wild-type recipients. Interestingly, bone marrow SLAM-LSK cells of chimeric mice made with CXCR4 deficient stroma demonstrated an aged phenotype including increased cycling (35% higher) and myeloid skewed differentiation compared to chimeric mice made with wild-type stroma. Furthermore, CXCR4 deletion exclusively in nestin+ MSCs also produced an aged HSC phenotype. In addition, similar to our observations with MSC from aged mice, CXCR4 deficient MSC showed higher expression of cellular and mitochondrial ROS. Since aged and CXCR4 deficient MSC exhibited higher expression of ROS, we explored whether high levels of MSC-derived ROS contributes to HSC aging. Treatment of aged or CXCR4 deficient MSC with N-acetyl-L-cysteine (NAC) for 1 week, improved their niche supporting activity including CFU-F formation and SDF-1 production and attenuated the HSC aging phenotype. In conclusion, our studies suggest that age-associated reduction in CXCR4 expression on bone marrow MSC impairs niche activity and increases ROS production, driving an HSC aging phenotype. These findings suggest that modulation of CXCR4/SDF-1 axis in MSC may lead to novel intervention to alleviate stem cell aging, thereby improving the age-associated decline in immune/hematopoietic function.