iPSC-derived skeletal muscle spheroids for Duchenne Muscular Dystrophy modeling

Area of Science:

• Biomedical Engineering
• Stem Cell Biology
• Regenerative Medicine

Background:

• Duchenne Muscular Dystrophy (DMD) involves progressive skeletal muscle degeneration, requiring satellite cell (SC) activation for regeneration.
• Dystrophic SCs have intrinsic defects, and the fibrotic niche impairs muscle recovery.
• Traditional 2D cell cultures struggle to accurately model the DMD muscle niche and SC behavior.

Purpose of the Study:

• To validate a 3D culture of induced skeletal muscle spheroids (iSMS) for modeling DMD.
• To compare the iSMS model with traditional 2D cultures for DMD studies.
• To investigate dystrophin deficiency mechanisms in vitro using these models.

Main Methods:

• Differentiated wild-type (WT) and DMD-derived induced pluripotent stem cells (iPSCs) in both iSMS and 2D cultures.
• Analyzed myogenic marker expression, proliferation, and differentiation using RT-qPCR, immunofluorescence, and flow cytometry.
• Compared iSMS and 2D responses to terminal differentiation medium and characterized DMD iSMS-derived progenitor behavior.

Main Results:

• iSMS cultures maintained satellite-like cells in a less activated state compared to 2D cultures, indicated by altered PAX7 and reduced MYOD1, MYOG, MYF5, and MYH3 expression.
• Notch signaling pathway genes (JAG1, NOTCH1) were upregulated in iSMS compared to 2D.
• DMD iSMS-derived progenitors exhibited reduced proliferation compared to WT, a defect not seen in 2D cultures.

Conclusions:

• The iSMS platform offers significant advantages over 2D cultures for in vitro DMD modeling.
• 3D iSMS systems provide deeper insights into SC behavior, crucial for developing novel DMD treatments.
• This 3D modeling approach may also benefit research into other muscular disorders.