Study of Bone Marrow Derived Stem Cells and Gene Therapy for Engineering of Cardiovascular Grafts

Abstract

Recent study shows that endothelial progenitor cells (EPCs) and gene therapy technologies are effective strategies in the inhibition of stenosis and thrombus formation and improving the patency rate of the vascular graft in vivo. In this study, rat EPCs were cultured from bone marrow, and plated in fibronectin-coated plates with EBM-2 medium. Bone marrow mesenchymal stem cells (MSCs) were cultured with alpha minimum essential medium ( -MEM). After two weeks, EPCs were immunohistochemically characterized using antibodies specific for endothelial cells. Retroviral vectors pMSCV-eNOS, pMSCV-tPA, pMSCV-LacZ and pMCSV-GFP were constructed. Retroviral particles were produced using packaging cell line 293T cells. Gene transfer was carried out by exposing cells to virus solution for 6 hours in the presence of 8µg/ml polybrene. For constructing vessels, MSCs and EPCs were seeded on fibronectin coated ePTFE graft in tissue culture condition for 2-4 weeks. The attachment and growth of cells were analyzed with scanning electron microscopy (SEM). Our data showed that the EPCs expressed VEGF, Lectin BS-1, RECA-1, indicating they are endothelial lineage. The concentrated retroviral particles showed many folds higher transduction efficiency to NIH 3T3 cells than the commercial reagent Fugene. SEM data showed dense attachment of MSCs on the graft surface. MSCs/EPCs co-culture gave much better cell coverage on the graft than culture of EPCs alone.