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.