Charles River has developed considerable expertise in both in vitro and in vivo models of DMD. Our in vitro expertise in DMD and other muscular dystrophies focuses primarily on cell-based assays in human primary myotubes that are differentiated using an established protocol from myoblasts. Disease-relevant readouts already established and available for compound screening include utrophin quantification and myoblast differentiation.
We are currently working with key opinion leaders and clinical stage researchers in the muscular dystrophy field to expand our portfolio of in vitro assays to include analysis of other disease phenotypes, such as muscle breakdown (e.g., MMP secretion, integrin/membrane stabilization) and fibrosis in patient-derived cells and human stem cells. This portfolio of in vitro assays complements Charles River’ in vivo phenotyping and efficacy testing capabilities in the DMD MDX mouse model and other models of muscular dystrophy.
UTROPHIN QUANTIFICATION ASSAY
The upregulation of utrophin, the fetal form of dystrophin, is thought to be able to compensate for the loss of dystrophin in Duchenne’s muscular dystrophy and is one of the well-established strategies to restore muscle function.
The utrophin quantification assay is highly specific for utrophin compared to dystrophin as the dystrophin signal is minimal at high utrophin concentrations.
MYOBLAST TO MYOTUBE DIFFERENTIATION ASSAY
The differentiation assay uses high-content imaging to measure the modulation of myoblast differentiation by disease-relevant inflammatory triggers. Myosin immunofluorescent staining is used to visualize myoblast to myotube differentiation. The percent differentiation is quantitated using a high-content algorithm that measures myotube area and length as well as the number of nuclei per myotube.
There is a dose-dependent decrease in myotube differentiation in response to inflammatory cytokines. The below images show increasing cytokine concentrations.