Isolation, Identification, Labelling Of The Canine Bone Marrow Endothelial Progenitor Cells And Tissue-engineered Urethra Construction

Endothelial progenitor cells(EPCs) are proposed as a potential regenerative tool for treating human vascular diseases and a possible target to restrict vessel growth in tumor pathology, however, few investigations were conducted on canine EPCs, despite dogs as large animal systems providing important references for human clinic. Thus we used a novel method to isolate EPCs from canine bone marrow, and morphology observation, cell surface marker determination as well as in vitro functional analysis were performed.Long segment circumferential urethra defect remains a challenge to urologists, therefore,we seeded canine bone marrow derived mesenchymal stem cells(BMSCs) and/or EPCs onto decellularized human amniotic scaffolds(d HAS) to construct tissue engineered urethra,hematoxylin-eosin(HE) staining and scanning electron microscopy scanning electron microscope(SEM) were used to make evaluations. The results were as follows:1. A facile endothelial progenitor cell isolation method was developed which is a combination of the whole bone marrow culture and the differential time attachment.2. This protocol was used to isolate two types of endothelial progenitor cells, early EPCs and late EPCs as short time as within1-2 weeks.3. Biological characteristics and functional analysis showed that isolated cells appeared typical colony and cobblestone morphology respectively and were positive for CD31,CD34, CD133 and VEGFR-2, whereas significant differences were observed on their expression level for some biomarkers between early and late EPCs, suggesting their different roles during angiogenesis and vasculogenesis.4. BMSCs and/or EPCs proliferated rapidly on d HAS, and distributed evenly.In summary, this method proved to be a novel, effective and time-saving way which could yield a population of enriched EPCs. In addition, d HAS seeded with BMSCs and EPCs proved to be a potential tissue engineered urethra and can be applied to preclinical study.