Extracellular UTP Affects In Vitro Migration and In Vivo Homing and Engraftment of CD34⁺ Hematopoietic Stem Cells.

Homing and engraftment of hematopoietic stem cells (HSCs) depend on the complex interplay between chemokines, cytokines, growth factors and adhesion molecules in the intricate architecture of bone marrow (BM) microenvironment. We have recently reported that CD34⁺ HSCs express P2Y and P2X receptors (P2Rs) for extracellular nucleotides. P2Rs activation by ATP and UTP produced stimulatory effects, in synergy with other hematopoietic cytokines, on clonogenic CD34⁺ and lineage-negative CD34⁻ progenitor cells. ATP and UTP also expanded the more primitive CD34⁺- derived long-term culture-initiating cells. Extracellular nucleotides are also emerging as chemotactic factors for different cell types, including vascular endothelial cells, arterial smooth muscle cells and neutrophils, and regulate the trafficking of specific dendritic cell populations. In this study we asked if the extracellular nucleotide UTP modulates the migration of HSCs in response to the chemotactic peptide CXCL12/SDF-1α. Very low concentrations of UTP (10 μM) significantly improved stem cell migration, assayed with the dual-chamber migration system and evaluated by cell counting and clonogenic assays. Phenotypical analysis of CXCR4 antigen showed that SDF-1α gradients mainly attract HSCs expressing high levels of this receptor. Conversely, UTP induced the migration of HSCs expressing low levels of CXCR4. Furthermore, we evaluated actin polymerization as index of HSCs capacity to respond to chemotactic stimulation and to home to the BM microenvironment. The combined stimulation of CD34⁺ cells with UTP and SDF-1α produced a synergistic increase in actin polymerization, as indicated by the enhanced fluorescence intensity of FITC-phalloidin. Adhesion assays, performed in fibronectin-coated wells, also showed that UTP can stimulate the adhesion of HSCs to fibronectin, a key component of the extracelluar bone marrow microenvironment, without any substantial change in VLA-4 and VLA-5 expression levels. Moreover, xenogenic transplant studies showed that short-term pre-incubation with UTP significantly increased the engraftment efficiency of CD34⁺ HSC in nonobese diabetic / severe combined immunodeficiency mice. Taken together, our data suggest that UTP may affect HSC migration and homing to the BM, in synergy with the chemotactic peptide SDF-1α, perhaps activating a CXCR4-indipendent pathway.