Culture and Characterization of Endothelial Colony Forming Cells from Peripheral Blood, Bone Marrow, and Umbilical Cord Blood of Horses

Abstract

Endothelial progenitors cells (EPCs), are a type of progenitor cell originating in the bone marrow and can be found circulating in peripheral blood, bone marrow, and umbilical cord blood. EPCs play an essential role in the formation of new blood vessels as well as maintaining vascular repair and homeostasis. These cells have become an intense area of interest in regenerative therapy due to the fact that they augment the formation of new blood vessels and promote endothelialization. Recent research has also shown that in humans with cardiovascular disease and metabolic disease there is a reduction in the number and function of EPCs. Since endothelial progenitor cells have yet to be investigated in horses and given the prevalence of diseases that can result in vasculature damage to horses, studying equine EPCs is an advantageous endeavor. Utilizing established protocols for human EPC isolation, we proposed that a specific subtype of EPCs, endothelial colony forming cells (ECFCs), could be isolated and cultured from peripheral, bone marrow, and umbilical cord blood samples from horses. Upon successful isolation and expansion, cells were characterized as true ECFCs using functional assays of acetylated low density lipoprotein (Ac-Di-LDL) uptake assay and vascular tube formation during culture in a Matrigel Basement Membrane Matrix. Cells from peripheral blood samples were analyzed for expression of specific cell markers including CD34, CD105, vascular endothelial growth factor 2 (VEGFR-2), and Von Willebrand factor (vWF). These markers were assessed via indirect immunofluorescence. Flow cytometry analysis was performed using the cell markers of CD14 and CD105. The number of cell colonies were recorded as well as cell performance in characterization assays including percent positive cells for the uptake of Di-Ac-LDL and vascular tube scoring of the Matrigel tubule formation assay. Cell performance and maximum number of passages before cell senescence were assessed on cells from peripheral blood samples through assessment of uptake of Ac-Di-LDL and vascular tube formation. Investigating equine endothelial progenitor cells and establishing culture and characterization methods is the initial step in gaining knowledge as to how these cells could be beneficial in equine regenerative medicine. Further investigation of the ECFCs will be carried out in future studies upon the establishment of a successful isolation protocol.