CXCR2 modulates bone marrow vascular repair and haematopoietic recovery post-transplant.

Murine models of bone marrow transplantation show that pre-conditioning regimens affect the integrity of the bone marrow endothelium and that the repair of this vascular niche is an essential pre-requisite for successful haematopoietic stem and progenitor cell engraftment. Little is known about the angiogenic pathways that play a role in the repair of the human bone marrow vascular niche. We therefore established an in vitro humanized model, composed of bone marrow stromal and endothelial cells and have identified several pro-angiogenic factors, VEGFA, ANGPT1, CXCL8 and CXCL16, produced by the stromal component of this niche. We demonstrate for the first time that addition of CXCL8 or inhibition of its receptor, CXCR2, modulates blood vessel formation in our bone marrow endothelial niche model. Compared to wild type, Cxcr2(-/-) mice displayed a reduction in bone marrow cellularity and delayed platelet and leucocyte recovery following myeloablation and bone marrow transplantation. The delay in bone marrow recovery correlated with impaired bone marrow vascular repair. Taken together, our data demonstrate that CXCR2 regulates bone marrow blood vessel repair/regeneration and haematopoietic recovery, and clinically may be a therapeutic target for improving bone marrow transplantation.

A novel function for the haemopoietic supportive murine bone marrow MS-5 mesenchymal stromal cell line in promoting human vasculogenesis and angiogenesis.

The bone marrow contains specific microenvironmental stem cell niches that maintain haemopoiesis. CXCL12-expressing mesenchymal stromal cells are closely associated with the bone marrow sinusoidal endothelia, forming key elements of the haemopoietic stem cell niche, yet their ability to regulate endothelial function is not clearly defined. Given that the murine nestin(+) cell line, MS-5, provides a clonal surrogate bone marrow stromal niche capable of regulating both murine and human primitive haemopoietic stem/progenitor cell (HSC/HPC) fate in vitro, we hypothesized that MS-5 cells might also support new blood vessel formation and function. Here, for the first time, we demonstrate that this is indeed the case. Using proteome arrays, we identified HSC/HPC active angiogenic factors that are preferentially secreted by haemopoietic supportive nestin(+) MS-5 cells, including CXCL12 (SDF-1), NOV (CCN3), HGF, Angiopoietin-1 and CCL2 (MCP-1). Concentrating on CXCL12, we confirmed its presence in MS-5 conditioned media and demonstrated that its antagonist in receptor binding, AMD-3100, which mobilizes HSC/HPCs and endothelial progenitors from bone marrow, could significantly reduce MS-5 mediated human vasculogenesis in vitro, principally by regulating human endothelial cell migration. Thus, the clonal nestin(+) MS-5 murine bone marrow stromal cell line not only promotes human haemopoiesis but also induces human vasculogenesis, with CXCL12 playing important roles in both processes.

A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133(+) stem/progenitor cells.

Efficient homing/mobilization of human hematopoietic stem/progenitor cells to/from bone marrow niches enhances their therapeutic efficacy. Additionally, homing is dependent on cell source and may be modulated by prior ex vivo cell expansion. Here, we describe a novel application of a 3-dimensional time-lapse method for assessing trafficking of individual human cord blood CD133(+) hematopoietic stem/progenitor cells in vitro, using the key chemokine CXCL12 as a paradigm. This new methodology allows distinction between chemotactic responses (displacement of center of mass and the forward migration index of the cells), and chemokinetic responses such as total cell path traveled in any direction (accumulated distance) and cell velocity in a 3-dimensional matrix. Other key advantages of this novel assay over existing assays include the ability to assess individual cell migration over times comparable to in vivo homing and rapid mobilization assays (18-24h) and to directly compare the strength or response of individual hematopoietic progenitor cells to different or competing stimuli and small molecule inhibitors in a single assay prior to analyses in vivo. Importantly, using this method, our results demonstrate definitively that CXCL12 regulates the chemotactic responses of human cord blood CD133(+) cells, but not their random migration or chemokinesis.