THE LONG-TERM GOAL OF THIS PROPOSAL IS TO STUDY THE EFFECTS OF MODELED MICROGRAVITY AND PARTIAL GRAVITY ON AGGREGATE (ORGANOID) CULTURES OF INDUCED PLURIPOTENT STEM CELLS (IPSCS). OUR WORKING HYPOTHESIS, BASED ON SOME PRELIMINARY DATA, IS THAT HYPOGRAVITY, I.E., MODELED MICROGRAVITY AND/OR PARTIAL GRAVITY, WILL ENHANCE IPSC DIFFERENTIATION INTO THE ENDODERMAL LINEAGES. SPECIFICALLY, WE WILL COMPARE THE EFFICIENCY OF TWO DISTINCT MICROGRAVITY GRAVITY / PARTIAL GRAVITY SIMULATING DEVICES, I.E. THE ROTATING WALL VESSEL (RWV) BIOREACTOR AND RANDOM POSITIONING MACHINE (RPM) TO MODULATE THE MAINTENANCE OF IPSCS STEMNESS AND THE DIRECTED DIFFERENTIATION OF IPSC INTO DEFINITIVE ENDODERM. IN TESTING OUR HYPOTHESIS, WE DEVELOPED THE FOLLOWING SPECIFIC AIMS: SPECIFIC AIM 1. TO CHARACTERIZE THE DIFFERENTIATION OF IPSC INTO DEFINITIVE ENDODERM IN 2D MONOLAYER CULTURE IN NORMAL, AND SIMULATED PARTIAL- (MOON, MARS) AND MICROGRAVITY; AND IN 3D AGGREGATE SUSPENSION CULTURE IN PARTIAL- (RPM) AND MICROGRAVITY (RPM AND RWV). SPECIFIC AIM 2. EXAMINE THE UNDERLYING GENETIC, EPIGENETIC, MOLECULAR, AND BIOCHEMICAL MECHANISMS OF STEM CELL DIFFERENTIATION AND BEHAVIOR THAT ARE AFFECTED BY AGGREGATE CULTURE, HYPOXIA, AND HYPOGRAVITY; AND IDENTIFY SPECIFIC MOLECULAR AND SIGNALING FACTORS IMPORTANT IN MODULATION OF DIFFERENTIATION. IN THESE STUDIES, WE WILL USE BIOCHEMICAL, IMMUNOLOGICAL, AND MOLECULAR BIOLOGICAL APPROACHES TO ANALYZE THE DIFFERENTIAL EXPRESSION OF A SET OF LINEAGE-SPECIFIC SPECIFIC MARKER GENES AND GENE PRODUCTS UNDER ALTERED GRAVITY CONDITIONS. SUBSEQUENTLY, WE WILL IDENTIFY SPECIFIC GENETIC, EPIGENETIC, AND MOLECULAR MECHANISMS THAT UNDERLIE AND ARE INFLUENCED BY PARTIAL- AND MICROGRAVITY AND COMPARE THESE BIOLOGIC FACTORS WITH THOSE FROM INDUCED IPSC DIFFERENTIATION, IN BOTH AGGREGATE AND MONOLAYER CULTURE, IN STANDARD GRAVITY. THE LATTER DATA WILL BE ANALYZED BY SYSTEMS- / BIOINFORMATICS-BASED ANALYTICAL APPROACHES, SUCH AS PRINCIPAL COMPONENT ANALYSIS AND NETWORK ANALYSIS, WHICH WILL HELP TO IDENTIFY SPECIFIC HYPOGRAVITY-SENSITIVE CELLULAR SIGNALING MOLECULES AND PATHWAYS THAT MAY BE DIFFERENTIALLY AFFECTED BY VARYING LEVELS OF PARTIAL/MICRO-GRAVITY, AND WHICH COULD BE FURTHER EXPLORED IN ACTUAL SPACE EXPERIMENTS. IN ADDITION TO HAVING AVAILABLE BOTH HYPOGRAVITY SIMULATING DEVICES, WE HAVE INTRODUCED SEVERAL TECHNICAL INNOVATIONS, SUCH AS MODIFICATIONS IN THE RWV BIOREACTORS, ENHANCED OXYGENATION CAPABILITIES IN RPM CULTURE, AND A HIGH-RESOLUTION OPTICAL MONITORING SYSTEM THAT ALLOWS REAL-TIME VISUALIZATION OF INTRACELLULAR SIGNALING EVENTS. WE ANTICIPATE THAT WE WILL DEMONSTRATE A SUPERIOR ENVIRONMENT FOR IPSC DIFFERENTIATION IN SIMULATED AND/OR PARTIAL GRAVITY CONDITIONS AND THAT KNOWLEDGE GAINED FROM THESE STUDIES WILL CONTRIBUTE TO TECHNOLOGICAL AND MEDICAL ADVANCEMENTS BOTH ON EARTH AND IN SPACE AND ENHANCE OUR UNDERSTANDING OF THE ADAPTATION AND FUNCTION OF BIOLOGICAL PROCESSES IN ALTERED GRAVITY.