Laemmle, Lillian
(2015)
POLYCISTRONIC HSV VECTORS FOR THE DIFFERENTIATION OF EMBRYONIC STEM CELLS TOWARD A CARDIAC LINEAGE.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Cardiovascular disease is the leading cause of death in developed countries, but we lack the ability to regenerate cardiac tissue. Cell-based therapy holds promise to repopulate a damaged heart with functional cardiomyocytes. Developing technologies to produce cells for transplantation is key to the success of this approach. Pluripotent stem cells (PSC) are an ideal starting material for cell-based therapies because they can be expanded indefinitely in culture and their plasticity gives them the potential to regenerate any tissue or organ. The issue of teratoma formation in the host may be avoided by devising methods to differentiate PSC toward a desired lineage before transplantation. Because the heart is vital for life, and the biggest source of human morbidity and mortality, in vitro differentiation of PSC into cardiomyocytes for cell-based treatment of heart disease is an area of intense research.
Exogenous expression of vital cardiogenic genes in PSC can be a powerful tool. Transduction of PSC with recombinant viral vectors can deliver genes to activate cardiac programming and drive differentiation toward a cardiac lineage. Replication defective HSV vectors efficiently transduce PSC and can be engineered to express genes that alter the cellular differentiation program. The goal of this research was to develop highly defective HSV vectors to express multiple cardiac transcription factors in embryonic stem cells to increase their cardiogenic potential. Vectors vββG4Nk, vG4Nk, and vGTM were engineered to express GATA4 and NKX2.5 or GATA4, TBX5, and MEF2c in PSC with high efficiency and low toxicity. Transduction of mESC with these vectors induced the expression of endogenous genes that are vital for cardiogenesis. Differentiation of mESC transduced with cardiogenic HSV vectors had a positive impact on terminal cardiomyocyte differentiation, producing many more embryoid bodies with beating cardiomyocytes than those transduced with control vectors. In addition, we found that delaying the drastic dilution of viral genomes that occurs over the time interval to terminal differentiation could enhance the outcome. Our results indicate that infection of mESC with cardiogenic HSV vectors has long reaching effects on mESC differentiation, supporting the suggestion that HSV vectors can be a useful tool for producing lineage related changes in differentiating PSC to generate specialized cell types.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
1 May 2015 |
| Date Type: |
Publication |
| Defense Date: |
20 April 2015 |
| Approval Date: |
1 May 2015 |
| Submission Date: |
30 April 2015 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
179 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Virology and Microbiology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
GATA4 cardiomyocytes cell-based therapy embryonic stem cells cardiac differentiation HSV vectors |
| Date Deposited: |
01 May 2015 20:28 |
| Last Modified: |
15 Nov 2016 14:28 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/25091 |
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