Identifying Surface Markers of Smooth Muscle Progenitor Cells for Purification and Future Clinical Applications

Atherosclerosis and its complications represent the most common cause of death in the developing and developed countries. More than 1,500,000 patients require intervention every year because of ischemic symptoms refractory to medical treatment for coronary or peripheral artery disease. To fight with these problems, one of the best ways is to identify the high risk group subject to atherosclerosis by new risk stratification biomarkers far before the happening of diseases. Another way is to develop novel tissue engineering technology to treat patients at advanced stage of diseases. Vascular progenitor cells contain a few different cell types, at least smooth muscle progenitor cells (SMPCs) and endothelial progenitor cells (EPCs). The counterbalance between them decides part of the fate of a vessel to develop atherosclerosis after injury. These progenitor cells have unlimited potentials in both risk stratification and tissue engineering. Although the studies on EPCs and the phenotype expression of EPCs are well established, little is known for SMPCs, especially the cell surface markers of SMPCs. Current method of culturing SMPCs is by culture medium-selection. However, there is still a substantial amount of contamination with other cell types. Only after figuring out the surface markers of SMPCs can we purify SMPCs, followed by any vascular biological studies on SMPCs and any tissue engineering applications by SMPCs. In this 3-year project, we will use updated technologies to find out potential gene targets for separating SMPCs from EPCs. To use these targets to identify SMPCs, development of antibodies appropriate for flow cytometer use is mandatory to positively select SMPCs from other contaminated or mixed cell populations. Finally, combined with MACS system, we will test whether our work can estimate the number of circulating SMPCs for clinical risk stratification and separate pure SMPCs from whole blood for tissue engineering purposes. The central theme of our 3-year proposal includes a few parts: 1. To perform Microarray to identify the different RNA transcription patterns between smooth muscle and endothelial progenitor cells 2. To confirm the findings of RNA Microarray by Q-PCR and to estimate the protein expression levels of the candidate genes by Western blotting 3. To estimate the expression levels of these targeted proteins on human smooth muscle progenitor cells by immunostaining in an in vitro experiments (immunocytostaining) and in two animal models (bone marrow-transplantation model and transplanting human smooth muscle progenitor cells to SCID mice model) 4. To innovate new antibodies of these target proteins specific for flow cytometer use (for future cell separation) 5. To test whether the new innovated antibodies can specifically differentiate smooth muscle progenitor cells from endothelial progenitor cells 6. To separate smooth muscle progenitor cells from endothelial progenitor cells by MACS system 7. To estimate the percentage of circulating smooth muscle progenitor cells in patients with coronary artery disease and controls (by flow cytometer) and to separate smooth muscle progenitor cells from human whole blood Given the novelty of the project, we have developed a working group. Our group has experts in progenitor cell culture, flow cytometer, confocal microscope, vascular biology, molecular biology, atherosclerosis, interventional cardiology, and hematology. Currently, all the techniques and methods have been tested, and are up and running in our laboratory. We believe that this line of inquiry will identify the panel of target genes and proteins as the surface markers of SMPCs and for SMPCs separation. The work has a tremendous benefit in the future either in clinical risk stratification or in setting up a platform of vascular tissue engineering.

Project ID:PC10101-1552
External Project ID:NSC99-2628-B182-029-MY3