Human iPSC-derived Duchenne muscular dystrophy model
ioSkeletal Myocytes DMD Exon 52 Deletion are opti‑ox™ precision reprogrammed skeletal myocytes carrying a genetically engineered hemizygous deletion in exon 52 of the DMD gene encoding the Dystrophin protein. These cells offer a rapidly maturing, consistent, and scalable isogenic system to study Duchenne muscular dystrophy in a physiologically relevant human cell model. Use the cells to study how the exon deletion impacts muscle cell function, and investigate methods for dystrophin restoration, such as ASO-mediated exon skipping.
ioSkeletal Myocytes DMD Exon 52 Deletion disease model cells express skeletal muscle cell specific markers and lack expression of Dystrophin, demonstrating a Duchenne muscular dystrophy phenotype
Immunocytochemistry staining at day 10 post revival demonstrates robust expression of Desmin, a component of the contractile apparatus, and no expression of Dystrophin in the ioSkeletal Myocytes DMD Del Ex52/Y disease model cells, whereas ioSkeletal Myocytes, the wild type isogenic control, express both markers (upper panel). Robust expression of Myosin Heavy Chain (MHC) and the muscle transcription factor Myogenin is observed in ioSkeletal Myocytes DMD Del Ex44/Y and ioSkeletal Myocytes (lower panel). Anti-dystrophin antibody clone 2C6 (MANDYS106).
ioSkeletal Myocytes DMD Exon 52 Deletion form elongated, multinucleated myocytes over 10 days, comparable to the wild-type ioSkeletal Myocytes isogenic control. Day 3 to 10 post-revival; 100X magnification.
ioSkeletal Myocytes DMD Exon 52 Deletion disease model cells demonstrate gene expression of key myogenic markers following reprogramming
Following reprogramming, ioSkeletal Myocytes DMD Exon 52 Deletion (DMD DelEx52/Y) and wild type ioSkeletal Myocytes (WT Control) downregulate expression of pluripotency genes (A), while demonstrating expected expression of key myogenic markers (B). Gene expression levels assessed by RT-qPCR. Data expressed relative to the parental human iPSC (hiPSC), normalised to HMBS. Data represents day 10 post-revival samples.
ioSkeletal Myocytes DMD Exon Deletion disease model cells show absence of Dystrophin protein by immunocytochemistry, demonstrating a Duchenne muscular dystrophy phenotype
ioSkeletal Myocytes DMD Exon 44 Deletion and DMD Exon 52 Deletion disease model cells, and ioSkeletal Myocytes wild type isogenic control, were cultured in 96-well plates at a density of 32,000 cells per well, according to the user manual. Immunocytochemistry staining for Dystrophin and Myosin Heavy Chain (MHC) was carried out at day 10 post-revival. The data show robust expression of MHC, but no expression of Dystrophin in the ioSkeletal Myocytes DMD Del Ex44/Y or DMD Del Ex52/Y disease model cells compared to the wild-type control, demonstrating a Duchenne muscular dystrophy phenotype. Data courtesy of Charles River Laboratories.
ioSkeletal Myocytes DMD Deletion disease model cells are amenable to ASO-mediated Dystrophin restoration, demonstrated with the DMD Exon 44 Deletion product
ioSkeletal Myocytes (wild type control) and ioSkeletal Myocytes DMD Exon 44 Deletion (DMD Del Ex44) were cultured according to the user manual in 96-well plates at a density of 32,000 cells per well. On day 4 post-revival the cells were treated by gymnosis with exon 45 skipping antisense oligonucleotide (ASO-1), concentration range 1-50 µM for immunocytochemistry and high content image (HCI) analysis, and 0.01-50 µM for ddPCR. Cells were cultured to day 7 then analysed.
A) Dystrophin mRNA restoration: RNA was extracted and cDNA synthesised. PCR primers and a fluorescent (FAM) labelled probe were designed to amplify the region coding exons 43-45 (non-skip, NSKP) or exons 43-46 (skip, SKP). PCR was carried out to quantify the non-skip and skip transcripts. The graph shows a concentration-dependent increase in the amount of SKP transcript (blue) and a decrease in the NSKP transcript (yellow), indicating that ASO-1 treatment has been successful in creating an in frame mRNA transcript for dystrophin. B) Dystrophin protein restoration: cells were stained for dystrophin, myosin heavy chain (not shown) and DAPI for cell nuclei quantification (not shown) and measured by high content analysis of images captured on Yokogawa Cell Voyager 8000 using a proprietary algorithm. The graph shows dystrophin restoration levels calculated as % of dystrophin/MHC area compared to wild type. ASO-1 treatment increased dystrophin protein expression in a concentration-dependent manner.
Each condition was tested in technical duplicate for ddPCR and technical triplicate for HCI analysis, and in biological duplicate (N=2). Data courtesy of Charles River Laboratories.
ioSkeletal Myocytes DMD Del Ex52/Y are compatible with plates ranging from 6 to 384 wells. The recommended seeding density is 100,000 cells/cm2. One small vial can plate a minimum of 0.5 x 24-well plate, 0.75 x 96-well plate, or 1 x 384-well plate.
Cells arrive ready to plate
ioSkeletal Myocytes DMD Exon 52 Deletion are delivered in a cryopreserved format and are programmed to mature rapidly upon revival in the recommended medium. The protocol for the generation of these cells is a two-phase process: Phase 1. Stabilisation for 3 days. Phase 2. Maintenance during which the skeletal myocytes mature.
Human iPSC line
Normal (46, XY)
6, 12, 24, 48, 96 & 384 well plates
Caucasian adult male (skin fibroblast)
Small: >2.5 x 106 viable cells Large: >5 x 106 viable cells
Sterility, protein expression (ICC), gene expression (RT-qPCR) and genotype validation (Sanger sequencing)