Stem cell homing and repopulation are not well understood. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 were found to be critical for murine bone marrow engraftment by human severe combined immunodeficient (SCID) repopulating stem cells. Treatment of human cells with antibodies to CXCR4 prevented engraftment. In vitro CXCR4-dependent migration to SDF-1 of CD34+CD38-/low cells correlated with in vivo engraftment and stem cell function. Stem cell factor and interleukin-6 induced CXCR4 expression on CD34+ cells, which potentiated migration to SDF-1 and engraftment in primary and secondary transplanted mice. Thus, up-regulation of CXCR4 expression may be useful for improving engraftment of repopulating stem cells in clinical transplantation.

Stem cell homing into the bone microenvironment is the first step in the initiation of marrow-derived blood cells. It is reported that human severe combined immunodeficient (SCID) repopulating cells home and accumulate rapidly, within a few hours, in the bone marrow and spleen of immunodeficient mice previously conditioned with total body irradiation. Primitive CD34 ؉ CD38 ؊/low CXCR4 ؉ cells capable of engrafting primary and secondary recipient mice selectively homed to the bone marrow and spleen, whereas CD34 ؊ CD38 ؊/low Lin ؊ cells were not detected. Moreover, whereas freshly isolated CD34 ؉ CD38 ؉/high cells did not home, in vivo stimulation with granulocyte colony-stimulating factor as part of the mobilization process, or in vitro stem cell factor stimulation for 2 to 4 days, potentiated the homing capabilities of cytokinestimulated CD34 ؉ CD38 ؉ cells. Homing of enriched human CD34 ؉ cells was inhibited by pretreatment with anti-CXCR4 antibodies. Moreover, primitive CD34 ؉ -CD38 ؊/low CXCR4 ؉ cells also homed in response to a gradient of human stromal cell-derived factor 1 (SDF-1), directly injected into the bone marrow or spleen of nonirradiated NOD/SCID mice. IntroductionDuring development hematopoietic stem cells migrate from the fetal liver into the bone marrow (BM) and continuously produce maturing hematopoietic cells that are released into the blood circulation. Hematopoietic stem cells are functionally defined, based on their ability to home to the BM microenvironment and to durably repopulate transplanted recipients with both myeloid and lymphoid cells. [1][2][3] In vivo repopulating assays for human stem cells have been developed by several groups, including ours. [4][5][6][7] In these assays human severe combined immunodeficient (SCID) repopulating cells (SRCs), characterized as CD34 ϩ CD38 Ϫ or as CD34 ϩ CD38 Ϫ/low cells, engraft the BM of sublethally irradiated immune-deficient NOD/ SCID and NOD/SCID/B2m null mice with high levels of myeloid and lymphoid cells. 7,8 Other primitive cells such as CD34 ϩ CD38 ϩ or CD34 Ϫ CD38 Ϫ cells were also found to have limited repopulation potential. 9,10 The chemokine stromal cell-derived factor 1 (SDF-1), which is also secreted by stromal cells in the BM microenvironment, was shown to attract human CD34 ϩ cells in vitro. [11][12][13] Recently we reported that engraftment of human SRC is dependent on the expression of SDF-1 and its receptor CXCR4, recharacterizing SRCs as CD34 ϩ CD38 Ϫ/low CXCR4 ϩ cells with major stem cell properties. 14 To home from the blood circulation into the BM microenvironment, hematopoietic stem and progenitor cells must first roll on E and P selectins, which are expressed on the BM vascular endothelial cells. [15][16][17] Following firm arrest and adhesion to the vessel wall under shear flow, a process mediated by the major integrins (VLA-4, VLA-5, and LFA-1) and their vascular ligands (VCAM-1 and ICAM-1), the cells extravasate through the endothelium into the hematopoietic compartment. 18-22 SDF-1 is constitutively expressed by ...

A major limitation to clinical stem cellmediated 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. ( IntroductionGene transfer into human hematopoietic stem cells (HSCs) may be a promising tool in the correction of a wide variety of hematopoietic and genetic disorders. HSC transplantation can be used to durably deliver these genetically modified cells to the bone marrow (BM), which in turn will release mature cells with the corrected gene into the circulation throughout life. Clinical and experimental HSC transplantation procedures mimic the physiologic process of HSC migration from the circulation into the BM occurring during late embryonic development and steady-state hematopoiesis in adults throughout life. [1][2][3] One of the disadvantages of BM transplantation is the long-lasting reduced levels of immature progenitors, including long-term culture-initiating cells (LTCICs; 1 log reduction), in the BM of patients who have received transplants compared with healthy individuals. [4][5][6] Furthermore, emerging evidence exists for impaired homing 7 and low engraftment 8 of retrovirally transduced human CD34 ϩ cells. Enhanced efficacy of HSC engraftment could improve the outcome of clinical transplantations as well as gene therapy protocols and might be achieved by modulating the ability of stem cells to home to and repopulate the recipient BM.Interactions between the chemokine stromal-derived factor 1 (SDF-1), also referred to as CXCL12, and its receptor CXCR4 play an essential role in stem cell seeding of the BM during murine embryonic development. 9,10 Moreover, we have previously demonstrated in a functional preclinical model, using nonobese diabetic/ severe ...