Cardiovascular disease is the leading cause of death in the United States. Millions of patients suffer each year from endothelial dysfunction and/or debilitating myocardial damage resulting in decreased quality of life and increased risk of death or disablement. Current pharmacological approaches are only partly effective at treating cardiovascular disease, and hence, better strategies are needed to provide significant improvements in treatment options. Cardiac stem/progenitor cells have the potential to regenerate myocardial tissue and repair damaged heart muscle. There are many different types of cardiac progenitor cells, and each may have certain unique properties and characteristics that would likely be useful for particular clinical applications. A current challenge in the field is to identify, isolate, and test specific cardiac stem/progenitor cell populations for their ability to repair/regenerate myocardial tissue. Our laboratory has discovered a new type of cardiac progenitor cell that expresses the enzyme, Phenylethanolamine-n-methyltransferase (Pnmt). My initial studies focused on identification of Pnmt+ cells based on knock-in of a nuclear-localized Enhanced Green Fluorescent Protein (nEGFP) reporter gene into exon 1 of the Pnmt gene in a stable recombinant Pnmt-nEGFP mouse embryonic stem cell (mESC) line. These cells were differentiated into cardiomyocytes, and I identified nEGFP+ cells using fluorescence, immunofluorescence, and phase-contrast microscopy techniques. Our results showed that only about 0.025% ( 1 per 4000) of the cardiac-differentiating stem cells expressed the nEGFP+ marker. Because of the relative rarity of these cells, optimization of isolation methods proved initially challenging. To overcome this technical barrier, I used a surrogate cell culture system to establish the methods of isolation based on expression of either a fluorescent cell marker (EGFP), or a unique cell surface receptor represented by an inactivated (truncated) version of the human low-affinity nerve growth factor receptor (LNGFR). Plasmid DNA containing these reporter genes was transiently transfected into a permissive cell line (RS1), and reporter gene expression was used to identify and isolate transfected from non-transfected cells using either Fluorescence-Activated Cell Sorting (FACS) or Magnetic-Activated Cell Sorting (MACS) methods. The main objective of the study was to establish the isolation techniques based on the expression of reporter genes (EGFP and LNGFR) in RS1 cells. Following transfection, EGFP+ cells were successfully isolated via FACS as verified by flow cytometric and microscopic analyses, which showed that approximately 96% of the isolated cells were indeed EGFP+. Despite the relative purity of the isolated cell population, however, their viability in culture following FACS was substantially compromised ( 50% attrition). In contrast, MACS enabled efficient isolation of LNGFR+ cells, and the vast majority of these ( 90%) retained viability in culture following MACS. The LNGFR expression was verified using RT-PCR. Further, MACS methods enabled isolation of marked cells in about 5-7 mins, whereas it took 2-4 hours to using FACS to perform similar isolations from the same amount of starting material (10^6 cells). In addition, MACS is a more economical method in that it does not require the use of an expensive laser-based instrument to perform the sorting. These results suggest that MACS was a more efficient, gentle, and feasible technique than FACS for isolation of reporter-tagged mammalian cells. Consequently, future studies aimed at isolation of Pnmt+ cardiac progenitor cells will thus primarily focus on MACS methods.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd-5648 |
| Date | 01 January 2014 |
| Creators | Varudkar, Namita |
| Publisher | University of Central Florida |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations |
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