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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.

A major limitation to clinical stem cell–mediated gene therapy protocols is the low levels of engraftment by transduced progenitors. We report that CXCR4 overexpression on human CD34+ progenitors using a lentiviral gene transfer technique helped navigate these cells to the murine bone marrow and spleen in response to stromal-derived factor 1 (SDF-1) signaling. Cells overexpressing CXCR4 exhibited significant increases in SDF-1–mediated chemotaxis and actin polymerization compared with control cells. A major advantage of CXCR4 overexpression was demonstrated by the ability of transduced CD34+ cells to respond to lower, physiologic levels of SDF-1 when compared to control cells, leading to improved SDF-1–induced migration and proliferation/survival, and finally resulting in significantly higher levels of in vivo repopulation of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice including primitive CD34+/CD38-/low cells. Importantly, no cellular transformation was observed following transduction with the CXCR4 vector. Unexpectedly, we documented lack of receptor internalization in response to high levels of SDF-1, which can also contribute to increased migration and proliferation by the transduced CD34+ cells. Our results suggest CXCR4 overexpression for improved definitive human stem cell motility, retention, and multilineage repopulation, which could be beneficial for in vivo navigation and expansion of hematopoietic progenitors. (Blood. 2004;103:2942-2949)

Stromal cell-derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 are implicated in the pathogenesis and prognosis of AML. Cellular microparticles (MPs), the submicron vesicles shed from the plasma membrane of various circulating cells, are associated with numerous human disorders. In the present study, we studied the putative relationships between CXCR4/SDF-1 axis and MPs in AML. We detected CXCR4 expressing MPs (CXCR4+MPs) in the peripheral blood and bone marrow plasma samples of normal donors (n=24) and newly diagnosed adult AML patients (n=26). The majority of CXCR4+MPs in AML patients were CD45+ whereas in normal individuals they were mostly CD41+. In samples from AML patients, the levels of CXCR4+MPs and total SDF-1 were significantly elevated as compared to normal individuals. Importantly, we found a strong correlation between the levels of CXCR4+MP and white blood cell (WBC) counts in the peripheral blood and bone marrow plasma obtained from the AML patients. Of interest, functional, non-cleaved SDF-1 levels were reduced in these patients compared to normal individuals, and also strongly correlated with the WBC counts. Furthermore, our data indicate N-terminal truncation of the CXCR4 molecule in the MPs of AML patients. This was found also in MPs obtained from the conditioned media of normal human CD34+ progenitors lentiviarlly transduced with CXCR4 vector in vitro. Appearance of MPs possessing N-terminally truncated CXCR4 in AML patients is likely to be dependent on proteolytic enzymes, such as elastase, which was elevated. However, MPs isolated from AML patients were capable of transferring functional CXCR4 molecule to the AML-derived HL-60 cells, enhancing their migration to SDF-1 in vitro and increasing their homing to the bone marrow of irradiated NOD/SCID mice. The CXCR4 antagonist AMD3100 reduced the increased migration and homing of MP treated HL-60 cells. Taken together, these findings suggest that functional CXCR4+MPs and SDF-1 are involved in the progression of AML. We propose that their levels are potentially valuable as an additional diagnostic AML parameter. Moreover, our findings suggest also the need for CXCR4- and SDF-1-target therapeutic approaches, clinically relevant in AML in the near future.