MATURATION OF VASCULARIZED CARDIAC ORGANOIDS THROUGH THE MODULATION OF GLUCOSE AND MECHANICAL MICROENVIRONMENT - PROJECT SUMMARY/ABSTRACT: HUMAN-INDUCED PLURIPOTENT STEM CELL-DERIVED VASCULARIZED CARDIAC ORGANOIDS (VCOS) REPRESENT A PROMISING IN VITRO MODEL FOR STUDYING HUMAN HEART DEVELOPMENT, DISEASE, AND DRUG RESPONSES. A ROBUST DIFFERENTIATION PROTOCOL HAS BEEN ESTABLISHED FOR GENERATING VCOS CONTAINING CARDIOMYOCYTES (CMS), ENDOTHELIAL CELLS (ECS), AND CARDIAC FIBROBLASTS (CFS). HOWEVER, VCOS REMAIN STRUCTURALLY AND FUNCTIONALLY IMMATURE, LIMITING THEIR UTILITY FOR MODELING LATE-ONSET CARDIOMYOPATHIES AND PRECLINICAL DRUG TESTING. CURRENT MATURATION STRATEGIES ENHANCE CONTRACTILITY AND METABOLIC ACTIVITY BUT OFTEN DISRUPT MULTICELLULAR COMPOSITION AND FAIL TO ACHIEVE KEY HALLMARKS OF ADULT CARDIAC MATURITY, SUCH AS A COMPACT MYOCARDIUM-LIKE STRUCTURE, ALIGNED SARCOMERES AND LONGER SARCOMERE LENGTH, FATTY ACID-DEPENDENT METABOLISM, HALLMARKS OF MARKER GENE EXPRESSIONS, AND CARDIAC FUNCTIONS. TO ADDRESS THESE CHALLENGES, THIS STUDY AIMS TO ENHANCE VCO MATURITY WHILE PRESERVING CELLULAR HETEROGENEITY BY IMPLEMENTING A DUAL-MODULATION STRATEGY TARGETING METABOLISM AND MECHANICAL STIFFNESS. THE FIRST AIM FOCUSES ON DEVELOPING A GLUCOSE ADMINISTRATION PROTOCOL THAT FACILITATES THE METABOLIC TRANSITION FROM GLYCOLYSIS TO FATTY ACID OXIDATION WHILE MAINTAINING ECS AND CFS WITHIN VCOS. OPTIMIZING GLUCOSE CONCENTRATION, TIMING, AND DURATION OF ADMINISTRATION IS EXPECTED TO ENHANCE CONTRACTILITY, CALCIUM KINETICS, AND MITOCHONDRIAL FUNCTION WITHOUT COMPROMISING VCO STRUCTURAL INTEGRITY. THE SECOND AIM INTRODUCES A STIFFNESS-TUNABLE BIOCOMPATIBLE HYDROGEL SYSTEM THAT MIMICS THE MECHANICAL TRANSITION FROM FETAL TO ADULT MYOCARDIUM. GRADUALLY INCREASING EXTRACELLULAR MATRIX STIFFNESS DURING MATURATION IS EXPECTED TO IMPROVE SARCOMERE ORGANIZATION, ENHANCE MYOCARDIAL COMPACTION, AND UPREGULATE KEY MATURATION GENETIC MARKERS. SUCCESSFUL COMPLETION OF THIS PROJECT WILL ESTABLISH A PHYSIOLOGICALLY RELEVANT MATURATION FRAMEWORK FOR VCOS, ENABLING ADVANCED CARDIAC DISEASE MODELING AND DRUG SCREENING. THIS RESEARCH ALIGNS WITH THE NIH'S MISSION TO DEVELOP INNOVATIVE IN VITRO HUMAN HEART MODELS FOR UNDERSTANDING CARDIOVASCULAR DISEASES AND IMPROVING THERAPEUTIC DEVELOPMENT.