VENTRICULAR TISSUE CULTURE MODEL FOR CARDIOVASCULAR RESEARCH

Abstract

IN THE PRESENT STUDY, A NOVEL REPORTER LINE WAS SUCCESSFULLY GENERATED FROM HESCS BACKGROUND. TWO REPORTERS (MCHERRY AND GAUSSIA LUCIFERASE) AND ONE SELECTABLE MARKER (NEOMYCIN RESISTANCE) WERE INSERTED VIA TALEN-MEDIATED HOMOLOGOUS RECOMBINATION (HR) INTO THE ENDOGENOUS MYL2 GENE LOCUS, WHICH CODES FOR MYOSIN LIGHT CHAIN SPECIFICALLY EXPRESSED IN VENTRICULAR CARDIOMYOCYTES (VCMS). FUNCTIONAL VALIDATION EXPERIMENTS FOLLOWING SUCCESSFUL DIFFERENTIATION USING NEWLY OPTIMIZED CARDIAC DIFFERENTIATION PROTOCOL DEMONSTRATED THAT THE REPORTER LINE WAS FUNCTIONAL AS DESIGNED AND CAN BE USED TO ENRICH FOR VCMS. CMS DERIVED FROM THIS REPORTER LINE WERE THEN USED TO GENERATE BIOMIMETIC CARDIAC TISSUE CONSTRUCTS: ENGINEERED HEART MUSCLES (EHMS), TO STUDY CM MATURATION IN A THREE-DIMENSIONAL TISSUE CONSTRUCT. A NUMBER OF MODALITIES WERE ATTEMPTED TO ENHANCE CM MATURATION IN VITRO. EMPIRICALLY, ELECTRICAL STIMULATION WAS FOUND IN THIS STUDY TO ENHANCE EHM MATURATION THE MOST. THESE NEW TOOLS AND INS