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 Dystrophy (DMD) is an early onset, progressive, and inevitably fatal disease affecting
1/5000 males, caused by mutations in the DMD gene (X-chromosome) that prevent expression of dystrophin
(Dys). There is no cure for DMD, and effective therapeutics are desperately needed. The overall goal of the
proposed research is to develop an in vitro assay system (the “Stem Cell-derived Muscle Function Assay”
[SCMFA]) that will enable high throughput testing of chemical compounds for potential therapeutic effects
against DMD. The SCMFA will feature skeletal muscle (SkM) differentiated from human pluripotent stem cells
(hPSCs, either induced pluripotent [hiPSCs], or NIH-approved human embryonic stem cell [hESC] lines)
representing subjects that are unaffected, DMD, or Becker Muscular Dystrophy (which is closely related to
DMD but milder). The project will be a collaboration between Vala Sciences Inc that specializes in
development of cell-based assays using automated microscopy, and Genea Biocells US, a company that
specializes in human stem cells and skeletal muscle diseases. The Specific Aims for Phase I are: 1) Develop
hiPSC lines for the project (while we already have certain hESC lines, the assay will ultimately feature only
hiPSC lines) and methods to seed and differentiate the cells to SkM on patterned substrates and flexible
membranes (all in 96-well dishes); 2) Development of Kinetic Image Cytometry methods to quantify muscle
function (calcium and voltage transients, contractile motion, and generation of reactive oxygen species [ROS])
and Structured Illumination Microsopy (SIM – similar to confocal) quantify the contractile apparatus and
biomarker expression; and 3) Development of methods to stress the cells relevant to DMD (pacing of
contraction, and stretch of the cells). Experiments will also be conducted to test the hypothesis that DMD SkM
is more susceptible to stress-induced activation ROS formation and activation of TRPC (stretch) channels. In
Phase II, additional hiPSC lines will be developed, the assay will be multiplexed and miniaturized to 384 well
dishes, and pilot chemical libraries will be screened. Vala Sciences Inc and Genea Biocells US will market the
SCMFA to pharmaceutical companies, world-wide, interested in developing therapeutics for DMD. The
methods developed will also be applicable to other inherited muscular dystrophies and related afflictions.
