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Cell Biology JoVE (Open Access) Citable · DOI

Generation of Myospheres From hESCs by Epigenetic Reprogramming

DOI: 10.3791/51243-v
What you'll learn
  • ✓ Apply epigenetic reprogramming to differentiate hESCs into skeletal muscle progenitors
  • ✓ Culture infected cells and induce myogenic differentiation via EB-like formation
  • ✓ Generate and characterize three-dimensional myospheres with contractile properties
Protocol

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Difficulty
advanced
Total time
~7–10 days
Model organism
Human embryonic stem cells (hESCs)
Biosafety
BSL-1

Steps

1
Infect hESCs with epigenetic reprogramming vector

Introduce viral or transgenic factors to initiate epigenetic modifications in human embryonic stem cells, directing them toward myogenic lineage commitment.

▶ 01:23
2
Culture infected cells under myogenic conditions

Maintain infected hESCs in culture medium optimized to support expansion and early differentiation of myogenic progenitor populations.

▶ 03:46
3
Induce embryoid body formation and differentiation

Promote three-dimensional aggregate formation (EB-like structures) and apply differentiation signals to drive myogenic maturation and myofiber formation.

▶ 05:35
4
Assess myogenic differentiation and myosphere characteristics

Evaluate contractile myofiber organization, three-dimensional cluster morphology, and biological features recapitulating human skeletal muscle tissue.

▶ 07:33
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