Embryonic stem cells have the potential to differentiate into ectodermal, mesodermal and endodermal derivatives. This property makes them a valuable source of tissue specific progenitor and differentiated cells that can be used for cell replacement therapy. The first goal of this thesis is to test if cardiogenesis from embryonic stem (ES) cells can be influenced by the overexpression of different proteins. We find that cardiogenesis is significantly enhanced by the overexpression of the c isoform of the transcription factor 'Paired-Like Homeodomain Transcription Factor (Pitx2c). This effect was verified morphologically by differentiating ES cells with Pitx2c and immunostaining for cardiomyocyte-specific proteins. Similarly, changes in RNA transcripts specific to cardiomyocytes during the process of differentiation were also recorded by means of Quantitative RT-PCR (QRT PCR). Finally, the contractile properties of these cells were measured by means of calcium imaging and their action potential characteristics were measured. We transplanted these cells into a mouse model of myocardial infarction and found significant improvement of cardiac function. We subsequently, subjected Pitx2-ES cells to hypoxic conditions and observed that the supernatant also caused significant functional recovery when injected into infarcted mice. The second goal of this thesis is to study the development of cardiomyocytes from ES cells by utilizing a series of stage specific promoters to drive different fluorophores. We designed a system where three different constructs carrying the promoters of cardiac specific genes would express proteins which would heterodimerize and drive a response element expressing antibiotic resistance. This interdependence ensures the presence of the constructs in the non-silenced loci of the genome. We used this system to insert the promoters of NK2 transcription factor related, locus 5 (Nkx2.5), Myocyte-specific enhancer factor 2c (Mef2c), Alpha myosin Heavy Chain (-MHC) and Myosin light chain2 - ventricular isoform (MLC2v) in murine ES cells and such that the cardiac differentiation of these cells could be visualized by the various fluorophores driven by these promotes. This system will be a powerful tool to study the effects of various morphogens on cardiac differentiation of ES cells and will also be useful for cell-transplantation studies.

Last modified

• 10/08/2018

Creator

• Achuta K Guddati

DOI

• https://doi.org/10.21985/N29J16

Subject

• Integrated Graduate Program in the Life Sciences

Keyword

• Cell Biology

Date created

• 2008-05-09

Resource type

• Dissertation

Rights statement

• In Copyright