Abstract
Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) represent promising cell sources for angiogenic therapies. There are, however, conflicting reports regarding the ability of MSCs to support network formation of endothelial cells. The goal of this study was to assess the ability of human bone marrow-derived MSCs to support network formation of endothelial outgrowth cells (EOCs) derived from umbilical cord blood EPCs. We hypothesized that upon in vitro coculture, MSCs and EOCs promote a microenvironment conducive for EOC network formation without the addition of angiogenic growth supplements. EOC networks formed by coculture with MSCs underwent regression and cell loss by day 10 with a near 4-fold and 2-fold reduction in branch points and mean segment length, respectively, in comparison with networks formed by coculture vascular smooth muscle cell (SMC) cocultures. EOC network regression in MSC cocultures was not caused by lack of vascular endothelial growth factor (VEGF)-A or changes in TGF-β1 or Ang-2 supernatant concentrations in comparison with SMC cocultures. Removal of CD45+ cells from MSCs improved EOC network formation through a 2-fold increase in total segment length and number of branch points in comparison to unsorted MSCs by day 6. These improvements, however, were not sustained by day 10. CD45 expression in MSC cocultures correlated with EOC network regression with a 5-fold increase between day 6 and day 10 of culture. The addition of supplemental growth factors VEGF, fibroblastic growth factor-2, EGF, hydrocortisone, insulin growth factor-1, ascorbic acid, and heparin to MSC cocultures promoted stable EOC network formation over 2 weeks in vitro, without affecting CD45 expression, as evidenced by a lack of significant differences in total segment length (p=0.96). These findings demonstrate the ability of MSCs to support EOC network formation correlates with removal of CD45+ cells and improves upon the addition of soluble growth factors.
Highlights
Microvessels are critical for the healthy function of all organs in our body through the delivery of oxygen to tissues and removal of metabolic waste.[1]
In the absence of supplemental growth factors, endothelial outgrowth cells (EOCs) undergo regression and cell loss in mesenchymal stem cells (MSCs) cocultures To determine whether MSCs are able to function as mural cells by supporting stable, robust EOC network formation in vitro, we cocultured MSCs and EOCs using conditions previously determined to support EOC network formation while being cocultured with
Both the smooth muscle cell (SMC) and MSCs were dispersed throughout the coculture system at day 10 of culture, excluding the possibility of MSC loss contributing to EOC regression (Supplementary Fig. S3)
Summary
Microvessels are critical for the healthy function of all organs in our body through the delivery of oxygen to tissues and removal of metabolic waste.[1] In addition, regulation of microvessel development is essential for the management of several disease types.[2] Recent studies have demonstrated the ability of mesenchymal stem cells (MSCs) to serve as pericyte-progenitor cells, revealing novel opportunities for the use of MSCs in vascular therapies.[3,4,5,6] For example, human bone marrow– derived MSCs enhance early stages of angiogenesis in vitro through upregulation of angiogenesis-associated genes, such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases, allowing endothelial cells (ECs) to migrate and elongate.[7,8,9,10,11,12] These in vitro observations of MSCs’ function as mural cells are extended in vivo where MSCs combined with endothelial outgrowth cells (EOCs) derived from umbilical. Departments of 1Biomedical Engineering, 3Mechanical Engineering and Materials Science, 4Cell Biology, and 5Chemistry, Duke University, Durham, North Carolina.
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