To achieve the goals of this PPG we will need to grow large quantities of cardiomyocytes (CMs) from mouse embryonic stem (ES) cell sources, for affinity-purification mass spectrometry (AP-MS). CM production will be scaled up from 6-well plates, to large tissue flasks and then suspension culture in a bioreactor. Although large-scale cell production is inefficient in a single lab, combining our efforts into a core laboratory will increase cell production so that the AP-MS can be achieved efficiently.
SPECIFIC AIMS SUMMARY Specific Aim 1: To provide high-quality cell production capabilities for program research projects. The cell production core will enable research projects to produce large quantities of cardiac progenitors and CMs derived from engineered mouse ES cells. By expanding cells in suspension, we can attain cell production levels that are at least two orders of magnitude greater than on petri dishes. These cells will be provided to the Proteomics Core to analyze biochemical complexes that underlie cardiac differentiation.
Specific Aim 2 : To develop new protocols for affordable high volume CM production. Bioreactors have successfully been used to produce large amounts of mouse and human CMs in academic and industrial laboratories, but the main limitation is the cost of reagents. Typical bioreactor runs will use 100 liters of media and state-of-the-art protocols can cost >$80,000 per run. We will adapt our current protocols to bring the cost down by over 70%, allowing for the proposed experiments in a large numbers of cell lines.
The networks we uncover in this study will inform our understanding of the molecular instructions that enable the embryo to make a heart, providing the underlying knowledge necessary to identify targets for therapeutic approaches to heart failure or severe heart damage.
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