myotube differentiation media  (PromoCell)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Splicing correction by peptide-conjugated morpholinos as a novel treatment for late-onset Pompe disease

doi: 10.1016/j.omtn.2025.102524

Figure Lengend Snippet: LOPD patient iPSC-derived myotubes recapitulate splicing deficits observed in LOPD patients (A) LOPD patient fibroblasts were reprogrammed into iPSC lines and pluripotency was confirmed by immunostaining with OCT3/4. LOPD iPSCs were terminally differentiated into LOPD patient derived iPSC myoblasts and myotubes that express MYOD and MHC by immunostaining, respectively. PC, phase contrast. Scale bars: fibroblasts (500 μm), iPSC and myoblasts (100 μm), myotubes (200μm) (B) LOPD patient iPSC-derived myotubes faithfully recapitulate aberrant splicing reported in LOPD patients, including skipping of exon 2. C) GAA TV expression in LOPD patient iPSC-derived myotubes. TV1, NM_000152.5 ; TV2, NM_001079803.3 ; TV3, NM_001079804.3 . Results are expressed as mean with error bars (SD).

Article Snippet: Patient iPSC-derived myoblasts were seeded in a 96-well or 24-well collagen-coated plate (Corning) and expanded in iPSC-derived myoblast expansion medium (iXCells Biotechnologies) for 48 h. Media was changed to myotube differentiation media (iXCells Biotechnologies) and differentiation was continued for 48 h. Media was then changed to fresh differentiation media containing the indicated concentrations of PPMO.

Techniques: Derivative Assay, Immunostaining, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Splicing correction by peptide-conjugated morpholinos as a novel treatment for late-onset Pompe disease

doi: 10.1016/j.omtn.2025.102524

Figure Lengend Snippet: PPMO correct GAA splicing in LOPD patient iPSC-derived myotubes (A) PPMO treatment decreases pathogenic splicing and increases full-length GAA as measured by endpoint RT-PCR. Marker in basepairs. Full-length GAA , 1100 bp; exon 2 skipping, 450 bp, 550 bp. (B) PPMO compounds increase levels of GAA TV1 and TV2 upon treatment. (C) PPMO treatment precisely restores the LOPD-critical GAA exon 1–2 junction with no detectable undesired splicing variants as measured by RNA amplicon sequencing. Results are expressed as mean with error bars (SD).

Article Snippet: Patient iPSC-derived myoblasts were seeded in a 96-well or 24-well collagen-coated plate (Corning) and expanded in iPSC-derived myoblast expansion medium (iXCells Biotechnologies) for 48 h. Media was changed to myotube differentiation media (iXCells Biotechnologies) and differentiation was continued for 48 h. Media was then changed to fresh differentiation media containing the indicated concentrations of PPMO.

Techniques: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Marker, Amplification, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Splicing correction by peptide-conjugated morpholinos as a novel treatment for late-onset Pompe disease

doi: 10.1016/j.omtn.2025.102524

Figure Lengend Snippet: PPMO correct pathogenic GAA levels at transcript, protein, and lysosomal enzyme activity levels in LOPD patient iPSC-derived myotubes (A) PPMO treatment increases GAA TV1 and TV2 at the LOPD critical exon 1-2 junction and also increases. (B) Total GAA (all TVs) transcript levels as measured by qPCR. (C and D) PPMO treatment similarly increases GAA protein (one-way ANOVA with Dunnett’s multiple comparison test; ∗ p < 0.05; ∗∗ p < 0.01). Arrows indicate size of GAA primary translation (76 kDa) and post-translationally modified GAA (76 kDa). (E) Lysosomal GAA enzyme activity levels in patient iPSC-derived myotubes were increased after treatment with PPMO in a dose-dependent fashion. Results are expressed as mean with error bars (SD).

Article Snippet: Patient iPSC-derived myoblasts were seeded in a 96-well or 24-well collagen-coated plate (Corning) and expanded in iPSC-derived myoblast expansion medium (iXCells Biotechnologies) for 48 h. Media was changed to myotube differentiation media (iXCells Biotechnologies) and differentiation was continued for 48 h. Media was then changed to fresh differentiation media containing the indicated concentrations of PPMO.

Techniques: Activity Assay, Derivative Assay, Comparison, Modification