16. A GENETIC SELECTION-BASED COCULTURE METHOD EXPLORING THE EFFECTS OF ENDOTHELIAL-CARDIOMYOCYTE INTERACTIONS ON THE CARDIOMYOGENESIS OF HUMAN EMBRYONIC STEM CELLS

Department: Bioengineering
Faculty Advisor(s): Andrew McCulloch | Mark Mercola

Primary Student
Name: Karen April Wei
Email: kwei@ucsd.edu
Phone: 858-534-5796
Grad Year: 2010

Abstract
Human embryonic stem cells (hESCs) represent a self-renewing, pluripotent cell source that possess the ability to generate functional cardiomyocytes. Using a dual-selectable marker system, we can purify cardiomyocytes from highly heterogeneous hESC cultures and improve differentiation into 3D beating cardiac cell clusters, termed cardiospheres. However, functional maturation and survival are currently inefficient and likely to require interactions with other cell types. For instance, when co-cultured in vitro with endothelial cells, the proliferation, survival, maturation, and spatial organization appears to improve. In addition, preliminary studies i our laboratory have shown that hESC-derived cardiomyocytes purified from other cell types in stem cell cultures at different differentiation stages exhibit different electrophysiological properties. Specifically, when puromycin selection of hESC-derived cardiomyocytes is performed at later stages of embryoid body culture, these cells exhibit action potential properties that more closely resemble mature cardiomyocytes as well as an increased dependence of sodium and HCN channels further suggesting cardiomyocyte maturation. Our hypothesis is that endothelial cells are major contributors to the development of immature hESC-derived cardiac tissue towards a more robust myocardial analog. To permit concurrent purification of both cell types in developing cardiospheres, we have designed a lentiviral vector system using the vascular endothelial-specific Tie2 promoter driving puromycin resistance. We will use these engineered cells to explore how cardiomyocytes and endothelial cells co-develop to create myocardial tissue and to investigate effects on maturation, proliferation, survival, and organization at different temporal developmental stages in both in vitro and in vivo within the infarcted heart.

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