Human CD34(+)CXCR4(-) sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation.

Homing and repopulation of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice by enriched human CD34(+) stem cells from cord blood; bone marrow; or mobilized peripheral blood are dependent on stromal cell-derived factor 1 (SDF-1)/CXCR4 interactions. Recently; human cord and fetal blood CD34(+)CD38(-)CXCR4(-) and CXCR4(+) cells; sorted with neutralizing anti-CXCR4 monoclonal antibody (mAb); were shown to have similar NOD/SCID repopulation potential. Herein we report that human cord blood CD34(+)CXCR4(+) (R4(+)) and CD34(+)CXCR4(-) (R4(-)) subsets; sorted with neutralizing anti-CXCR4 mAb; engrafted NOD/SCID mice with significantly lower levels of human cells compared with nonsorted and SDF-1-migrated CD34(+) cells. Coinjection of purified cells with 10 microg anti-CXCR4 mAb significantly reduced engraftment of all CD34(+) subsets; and 50 microg completely abrogated engraftment by R4(-) and CD34(+) cells. Importantly; R4(-) cells harbor intracellular CXCR4; which can be rapidly induced to cell surface expression within a few hours. Moreover; 48 hours of cytokine stimulation resulted in up-regulation of both cell surface and intracellular CXCR4; restoring migration capacities toward a gradient of SDF-1 and high-level NOD/SCID repopulation potential. In addition; homing of sorted R4(-) cells into the murine bone marrow and spleen was significantly slower and reduced compared to CD34(+) cells but yet CXCR4 dependent. In conclusion; R4(-) cells express intracellular CXCR4; which can be functionally expressed on the cell membrane to mediate SDF-1-dependent homing and repopulation. Our results suggest dynamic CXCR4 expression on CD34(+) stem and progenitor cells; regulating their motility and repopulation capacities.