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

Abstract

IntroductionHematopoietic stem cells migrate during embryonic development from the fetal liver through the blood circulation, home to the bone marrow (BM) microenvironment, and repopulate it with immature and maturing blood cells of all lineages. Similarly, in clinical and experimental stem cell transplantation protocols, hematopoietic stem cells, which are infused into the blood circulation of patients and experimental animals, home and repopulate the BM. 1 The molecular mechanisms that regulate the homing and repopulation processes are crucial for stem cell function and development. [2][3][4][5] The CXC chemokine stromal cell-derived factor 1 (SDF-1) plays a major role in migration, proliferation, differentiation, and survival of many cell types including human and murine hematopoietic stem/progenitor cells. 6,7 SDF-1 is produced by multiple BM stromal cell types and by epithelial cells in many organs 8,9 and is highly expressed by human and murine BM endothelium. [10][11][12] CXCR4, the 7-transmembrane receptor of SDF-1, is widely expressed by a variety of hematopoietic cell types, neuronal cells, and different stromal cells. 13 SDF-1 is a chemotactic agent for human lymphoid, myeloid, and immature CD34 ϩ progenitor cells. 6,7,14,15 This chemokine induces integrin-dependent adhesion of CXCR4 ϩ human T lymphocytes 16 and immature CD34 ϩ CXCR4 ϩ cells 17 under shear flow and also mediates transendothelial migration of human progenitors. 18 In vivo cell migration and localization are also mediated by SDF-1/CXCR4 interactions. Murine T cells overexpressing human CXCR4 and CD4 accumulated in the BM of transgenic mice. 19 Prevention of CXCR4 expression by introducing SDF-1 intrakine blocked in vitro migration and in vivo dissemination of a T-cell hybridoma. 20 More important, mice reconstituted with progenitor cells expressing SDF-1-intrakine suffered impaired lymphoid and myeloid hematopoiesis, whereas transplantation of progenitors overexpressing SDF-1 led to increased myeloid and B-lymphoid hematopoiesis. 21 The key role of SDF-1 and CXCR4 in embryonic development was demonstrated by knockout studies in mice. The lack of either SDF-1 or its receptor in murine fetuses results in multiple lethal defects including impaired BM hematopoiesis. [22][23][24][25] Recently, Wright and colleagues have demonstrated that SDF-1 is the sole chemokine mediating in vitro migration of purified adult murine BM stem cells. 26 This important study suggests a major role for SDF-1/CXCR4 interactions also in adult murine stem cell migration and development.We demonstrated the essential role of SDF-1/CXCR4 interactions in both homing and high-level multilineage repopulation of nonobese diabetic/severe combined immunodeficient (NOD/SCID) 10,27,28 The antihuman CXCR4-neutralizing monoclonal antibody (mAb; clone 12G5) binds CXCR4 on the first and second extracellular domain as its ligand SDF-1, interfering with SDF-1 binding and signaling. 29,30 Coinjecting enriched human CD34 ϩ cells with neutralizing anti-CXCR4 mAb blocked homing and re...

The role of G-protein signaling in hematopoietic stem/progenitor cell mobilization

“…[24][25][26] Whether in vivo treatments with polyclonal antibodies versus different CXCR4 antagonistic molecules with potentially different modes of action could account for these differences is difficult to discern at this point and further experimentation is warranted. On a relevant vein, data with in vitro anti-CXCR4 antibody treatments are inconsistent [61][62][63][64] and at odds with studies using Ptx-treated cells in homing experiments of murine BM, 60 murine fetal liver cells, 65 or human CD34 ϩ cells. 62 Collectively, the weight of the evidence suggests that CXCR4/SDF-1 signaling may not be crucial for the initial anchoring of transplanted cells to endothelial cells within BM, but their subsequent retention within BM is greatly dependent on CXCR4/SDF-1 signaling.…”

“…On a relevant vein, data with in vitro anti-CXCR4 antibody treatments are inconsistent [61][62][63][64] and at odds with studies using Ptx-treated cells in homing experiments of murine BM, 60 murine fetal liver cells, 65 or human CD34 ϩ cells. 62 Collectively, the weight of the evidence suggests that CXCR4/SDF-1 signaling may not be crucial for the initial anchoring of transplanted cells to endothelial cells within BM, but their subsequent retention within BM is greatly dependent on CXCR4/SDF-1 signaling. Whether cells from Ptx-treated animals or hematopoietic cells with genetically modified G i protein signaling 35 display a different behavior in homing assays will be addressed in future experiments.…”

Hematopoietic Progenitor Cells (HPC) from Mobilized Peripheral Blood Display Enhanced Migration and Marrow Homing Compared to Steady-State Bone Marrow HPC

“…The seemingly counterintuitive increase of SDF-1-induced chemotaxis of MPB CFU-C despite down-regulated CXCR4 was previously reported, but not explained. [24,27,47,52,53] Since CXCR4 is reexpressed on HPC after brief cytokine exposure [54,55] or early after transplantation [47], the physiologic relevance of down-regulated CXCR4 on MPB HPC found in vitro is not clear, as it may not represent the status of the cells exposed to the in vivo environment in the conditioned host. Furthermore, the correlation between CXCR4 expression and in vitro migration may not be straight forward.…”

How do stem cells find their way home?

“…110,114 These studies demonstrate that proteolytic enzymes are involved in both turning on and off the locomotion machinery of human CD34 ϩ cells, thus mediating stem cell recruitment and organ localization. CD26, a cell-surface peptidase, is expressed on the surface of different hematopoietic subsets, including normal BM CD34 ϩ cells 115 41,63,79 These progenitors acquire in vivo expression of the CD34 ϩ marker, demonstrating slower differentiation and reconstitution kinetics but higher absolute numbers of CD45 ϩ and immature human CD34 ϩ cells in comparison with human cells initially expressing CD34. 41,65 Applying intrafemoral injection of CB CD34 ϩ CD38 Ϫ/low CD36 Ϫ purified cells, Dick's group could identify a new short-term SRC subset that can migrate to other bones, colonizing them with myeloid and erythroid cells.…”