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

Abstract

Summary 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.

The Hematopoietic Chemokine CXCL12 Promotes Integration of Human Endothelial Colony Forming Cell–Derived Cells into Immature Vessel Networks

“…ECFC-derived cells from CB and human umbilical vein were prepared and cultured in complete EGM2 medium [35][36][37][38]. hDFs, human umbilical cord vein endothelial cells (HUVECs), and hBM MSCs were also sourced from Lonza Biologics and cultured as described [35][36][37][38].…”

“…ECFC-derived cells from CB and human umbilical vein were prepared and cultured in complete EGM2 medium [35][36][37][38]. hDFs, human umbilical cord vein endothelial cells (HUVECs), and hBM MSCs were also sourced from Lonza Biologics and cultured as described [35][36][37][38]. Typical immunophenotypes of these CB and HUVEC ECFC-derived cells and hBM MSCs have been described previously [35][36][37][38] and in Supplementary Fig.…”

“…hDFs, human umbilical cord vein endothelial cells (HUVECs), and hBM MSCs were also sourced from Lonza Biologics and cultured as described [35][36][37][38]. Typical immunophenotypes of these CB and HUVEC ECFC-derived cells and hBM MSCs have been described previously [35][36][37][38] and in Supplementary Fig. S1 (Supplementary Data are available online at www.liebertpub .com/scd).…”

“…Ex vivo assays have been developed to mimic human vessel formation either in fibrin or collagen gels [28][29][30][31][32][33][34][35][36][37]. These have combined endothelial cells or ECFCs with mural cells (eg, lung fibroblasts, dermal fibroblasts, retinal pericytes, or mesenchymal stromal cells) and specialized endothelial growth medium containing cytokines [eg, VEGF and fibroblast growth factor-2 (FGF-2)].…”

“…We have adapted our version of these in vitro models, in which human umbilical cord blood (CB) or umbilical cord (UC) ECFC-derived cells are cocultured with human dermal fibroblasts (hDFs) or human bone marrow mesenchymal stromal cells (hBM MSCs) in a 3D collagen type I sandwich assay in media containing VEGF and FGF-2 [34][35][36][37], by further developing a method to assess integration of exogenous human ECFC-derived cells into immature vascular tubule networks. Here, using this model together with timelapse microscopy and fluorescently tagged cells, we analyzed the effects of the AMD3100 antagonist on human vasculogenesis and angiogenesis and showed that AMD3100 not only inhibits the dynamic refinement of the vascular network in vitro by enhancing endothelial cell retraction but also modulates the incorporation of ECFC-derived cells into extant vascular networks.…”