Factors Influencing Short Term In Vivo Biodistribution of Human Bone Marrow Derived Mesenchymal Stem Cells (hMSCs) in a Xenogeneic Animal Model.

Human MSCs are potential agents for tissue regeneration, enhancing haemopoietic stem cell transplantation and delivering genes of therapeutic interest. To successfully implement any of these therapeutic strategies, we need a better understanding of the tissue localisation of systemically administered hMSCs and of the factors that critically influence their biodistribution. Bone marrow (BM) derived hMSCs were isolated from normal donors, cultured using previously described protocols and characterised by phenotype and multi-lineage differentiation. HMSCs were labelled with PKH-26 and injected by tail vein into b2m/NOD/SCID animals (2x10⁶ cells /animal). Analysis of murine tissue mononuclear cells by flow cytometry 24 hours later consistently detected a proportion of transplanted cells in the BM (327 ± 31), spleen (645 ± 147) and lungs (1485 ± 138) in all experiments. This was also confirmed by flow cytometry using human-specific HLA-Class I mAb and FISH analysis. Homing of infused hMSCs to the BM and spleen of b2m/NOD/SCID animals was significantly higher in younger (<10 weeks) when compared with older (>10 weeks) animals (p<0.0001). [BM (424 ± 24 vs 209 ± 20), spleen (1175 ± 154 vs 292 ± 30), lungs (2760 ± 321 vs 1225 ± 269) respectively]. Prolonged ex vivo expansion of hMSCs also impaired their homing. Homing to the BM and spleen was significantly lower (p<0.022, and p<0.015 respectively) for late passage (> P14) when compared with earlier passage hMSCs (< P10). Lentiviral transduction of hMSCs with eGFP did not alter homing when compared to unmanipulated cells.

Tissue homing was markedly enhanced by prior irradiation of animals (325Gy). Numbers of PKH positive cells detected by flow cytometry were significantly higher in the BM of irradiated recipients (425.3 ± 42 vs 110 ± 20 for unirradiated mice, p=0.001). Irradiation also increased homing to the lungs (4722 ± 1220 vs 495 ± 70, p=0.0327). Overexpression of CXCR4 by lentiviral gene transfer in hMSCs significantly enhanced their migration to SDF-1 in vitro. CXCR4 overexpression did not influence short term homing to the BM and other tissues of unconditioned b2 m NOD/SCID. In contrast, CXCR4 transduced hMSCs demonstrated significantly higher levels of homing (compared with eGFP-control transduced hMSCs) when transplanted into irradiated b2 m NOD/SCID. This was particularly marked in the lungs (p=0.007).

Conclusion: In vivo homing of hMSCs is critically influenced by animal age, length of ex vivo cell manipulation and is enhanced in the irradiated-stressed animal model. Overexpression of CXCR4 does not significantly alter hMSCs homing in the unconditioned animal model but does so in the irradiated recipients. Identifying these factors and strategies to improve tissue homing is of great importance for the successful application of hMSCs in cell and gene therapy.