Abstract
BackgroundSome studies demonstrated therapeutic angiogenesis attributable to the effects of endothelial progenitor cells (EPC), others have reported disappointing results. This may be due to the fact that EPC populations used in these contradictory studies were selected and defined by highly variable and differing experimental protocols. Indeed, the isolation and reliable characterization of ex vivo differentiated EPC raises considerable problems due to the fact there is no biomarker currently available to specifically identify EPC exclusively. On the other hand traditional differentiation of primary immature bone marrow cells towards the endothelial lineage is a time-consuming process of up to 5 weeks. To circumvent these shortcomings, we herein describe a facile method to isolate and enrich a primary cell population from rat bone marrow, combining differential attachment methodology with cell sorting technology.ResultsThe combination of these techniques enabled us to obtain a pure population of early endothelial precursor cells that show homogenous upregulation of CD31 and VEGF-R2 and that are positive for CD146. These cells exhibited typical sprouting on Matrigel™. Additionally, this population displayed endothelial tube formation when resuspended in Matrigel™ as well as in fibrin glue, demonstrating its functional angiogenic capacity. Moreover, these cells stained positive for DiI-ac-LDL and FITC-UEA, two markers that are commonly considered to stain differentiating EPCs. Based upon these observations in this study we describe a novel and time-saving method for obtaining a pure endothelial precursor cell population as early as 2–3 weeks post isolation that exhibits endothelial abilities in vitro and which still might have retained its early endothelial lineage properties.ConclusionThe rapid isolation and the high angiogenic potential of these syngeneic cells might facilitate and accelerate the pre-vascularization of transplanted tissues and organs also in a human setting in the future.
Highlights
Some studies demonstrated therapeutic angiogenesis attributable to the effects of endothelial progenitor cells (EPC), others have reported disappointing results
Since there is no unique cell surface marker that clearly defines the properties of EPC, a population separated via magnetic sorting does not necessarily reflect progenitor cells
Our findings demonstrate that mononuclear cells (MNC) from rat bone marrow show angiogenic characteristics which are commonly associated with EPC, including cobblestone-like morphology, lectin-binding, ac-LDLuptake, tube formation on and within MatrigelTM or Fibrin and expression of endothelial cell surface markers
Summary
Some studies demonstrated therapeutic angiogenesis attributable to the effects of endothelial progenitor cells (EPC), others have reported disappointing results This may be due to the fact that EPC populations used in these contradictory studies were selected and defined by highly variable and differing experimental protocols. Given that specific cell surface markers to identify endothelial progenitor cells are unknown, it is impossible to separate the EPC population at an early state of differentiation by using methodology based on magnetic activated cell sorting (MACS). Other approaches such as the differential attachment method [10,11,12] have been used to isolate EPC. Awaiting outgrowth of a pure cell population from the pluripotent heterogenous bone marrow fraction of mononuclear cells (MNC) is very time consuming
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