The Stem Cell-Derived Muscle Function Assay: A High Throughput Screening Platform Utilizing Kinetic Image Cytometry to Discover Therapeutics for Duchenne Muscular Dystrophy

Duchenne Muscular DystrophyDMDis an early onsetprogressiveand inevitably fatal disease affectingmalescaused by mutations in the DMD geneX chromosomethat prevent expression of dystrophinDysThere is no cure for DMDand effective therapeutics are desperately neededThe overall goal of the proposed research is to develop an in vitro assay systemtheStem Cell derived Muscle Function AssaySCMFAthat will enable high throughput testing of chemical compounds for potential therapeutic effects against DMDThe SCMFA will feature skeletal muscleSkMdifferentiated from human pluripotent stem cellshPSCseither induced pluripotenthiPSCsor NIH approved human embryonic stem cellhESClinesrepresenting subjects that are unaffectedDMDor Becker Muscular Dystrophywhich is closely related to DMD but milderThe project will be a collaboration between Vala Sciences Inc that specializes in development of cell based assays using automated microscopyand Genea Biocells USa company that specializes in human stem cells and skeletal muscle diseasesThe Specific Aims for Phase I areDevelop hiPSC lines for the projectwhile we already have certain hESC linesthe assay will ultimately feature only hiPSC linesand methods to seed and differentiate the cells to SkM on patterned substrates and flexible membranesall inwell dishesDevelopment of Kinetic Image Cytometry methods to quantify muscle functioncalcium and voltage transientscontractile motionand generation of reactive oxygen speciesROSand Structured Illumination MicrosopySIMsimilar to confocalquantify the contractile apparatus and biomarker expressionandDevelopment of methods to stress the cells relevant to DMDpacing of contractionand stretch of the cellsExperiments will also be conducted to test the hypothesis that DMD SkM is more susceptible to stress induced activation ROS formation and activation of TRPCstretchchannelsIn Phase IIadditional hiPSC lines will be developedthe assay will be multiplexed and miniaturized towell dishesand pilot chemical libraries will be screenedVala Sciences Inc and Genea Biocells US will market the SCMFA to pharmaceutical companiesworld wideinterested in developing therapeutics for DMDThe methods developed will also be applicable to other inherited muscular dystrophies and related afflictions Duchenne Muscular DystrophyDMDis an inherited disease affectingout ofboysThe boys become weak because their muscle degenerateslosing their ability to walk by the time they are a teenagerand die before the age ofbecause they can t breath anymoreDMD is always fataland there is no curecertain medications prolong lifebut not by very longWe are proposing to develop a new way to test drugs for their ability to improve the health of muscle of DMD patientsWe will start with human stem cells that represent subjects with DMDwe will use stem cells that are approved for research use by the US governmentWe will then differentiate the stem cells to muscle cells which we will put into multi well dishesorwells per dishso that we can test many drugs on them in the same experimentWe will use automated digital microscopes to determine if any of the drugs increase the strength and health of the muscleOnce these test methods are developedwe will be able to test hundreds of thousands of different chemicals to identify drugs that will help boys with DMD