abstract for my research proposal

i'm sure anyone who's reading this is thinking "just what is lilly going to be working on for the next X years (assuming she passes her comps)?" well, here for your reading pleasure is the abstract for my proposal. it's a little long and wordy (like i tend to be), and i really had to edit the hell out of it to fit it into one page in 11 point (or larger) font, but here it is:


Optimization of Differentiation of Mouse Embryonic Stem Cells into Cardiomyocytes for Use in Repair of Myocardial Infarction

While embryonic stem cells possess the ability to become virtually any cell type, many of the specific events involved in differentiation are unknown. The ability to direct the differentiation of stem cells into specific lineages would provide scientists with a powerful tool to generate cells for the treatment of many diseases and injuries. Stem cell-based treatment for myocardial infarction is particularly attractive due to the irreversible nature of the injury and the limited inherent regenerative capability of the heart. However transplant of undifferentiated stem cells is risky due to the possibility of teratoma formation, and the ability of these cells to engraft and participate in heart function is unknown. Researchers are beginning to understand the vital steps in cardiomyogenesis, and some of the earliest surface receptors and transcription factors expressed in cardiomyocyte progenitors have recently been identified. We hypothesize that by sorting murine embryonic stem (mES) cells differentiated via embryoid body (EB) formation based on expression of markers specific to the cardiac lineage, and ectopically expressing known cardiac transcription factors in these sorted cells using HSV vectors, we can produce a population of cells highly enriched for cardiomyocytes.

GATA4 and Nkx2.5 are two of the earliest transcription factors expressed in cardiac progenitors. They act together to regulate expression of cardiac specific genes in the developing heart, and exogenous expression of these two factors in mesenchymal stem cells has been shown to drive them towards a cardiac fate. We plan to construct HSV-1 vectors to express these genes in mES and embryoid body cells. HSV vectors are well suited for this work because they provide a pulse of gene expression which mimics the transient expression of developmentally regulated genes. The replication defective HSV vector JDββ has been shown to be capable of transgene expression in these cell types, and has a very low toxicity due to deletion of the immediate early genes ICP4 and ICP22, and reduced expression of ICP0 and ICP27, which are under control of an early (β) promoter. Previous work has also shown that this vector does not interfere with self-renewal or EB formation of mES cells, and does not alter expression of the mesoderm marker Brachyury (Bry). The latter is particularly important for our current work as cardiomyocytes are derived from this Bry positive mesoderm population.

Researchers have engineered a mES cell line which expresses EGFP under the control of the Bry promoter. They used FACS to sort cells based on expression of Bry along with Flk-1 to enrich for cardiac progenitors. A separate group used bioinformatic dissection to identify overexpression of the chemokine receptor CXCR4 during cardiogenesis, and used FACS for the combined expression of CXCR4 and Flk-1 to select for cells of a cardiopoietic lineage from EB cells. The advantage of their approach is that the Bry-GFP cell line is not required. We are awaiting the approval of an MTA to obtain this cell line, but if we are unable to do so, we plan to use the alternate approach of sorting for CXCR4+/Flk-1+ cells to enrich for cardiac progenitors from EB cells. We will infect both unsorted and sorted EB cells with our GATA4 and Nkx2-5 vectors, and predict that we will get high transgene expression and enhanced induction of cardiogenesis. We will use RT-PCR to examine cells for the expression of cardiac-specific genes such as α-MHC, myocardin, and MEF2C. Observation of spontaneously beating cells will also be used to confirm the enrichment of cardiomyocytes.

Upon optimization of the differentiation of mES cells into cardiomyocytes, including MOI and timing of infection with GATA4 and Nkx2-5 vectors, and optimization of sorting conditions, we plan to collaborate with another laboratory to evaluate the ability of these cells to repair myocardial infarction in mice. Ultimately we wish to compare their ability to survive and form large grafts to undifferentiated mES cells or non-cardiac adult stem cells like MDSCs following intramyocardial transplantation, and predict that these mES cell- derived cardiomyocytes will contribute better to heart contractility and function.