Table 1.
Main items of myotubes differentiation methods from ESCs and iPSCs.
| Reference | Darabi35 | Goudenege37 | Abujarour38 | Shoji39 | Choi43 |
|---|---|---|---|---|---|
| Myogenic precursor isolation | |||||
| Starting cells | ESCs and iPSCs | ESCs and iPSCs | iPSCs | iPSCs | ESCs and iPSCs |
| EB acquisition | by dissociation | not used | not used | not used | not used |
| Lentiviral vector | Pax7 | MyoD | MyoD | not used | not used |
| Transposon vector | not used | not used | not used | MyoD | not used |
| Proliferation media | mTeSR, IMDM | mTeSR, MB1 | DMEM / F12 | ESC medium | DMEM / F12 |
| Factors | dox, bFGF, 10% HS | 15% FBS | KSR, bFGF, SMC4 | bFGF | KSR, bFGF |
| FACS | Pax7+ | CD73+ | not used | not used | not used |
| Duration (days) | 14 | 28 | 7 | 14 | 7 |
| Myogenic cell differentiation | |||||
| Differentiation medium |
low glucose DMEM | DMEM |
aDMEM / F12; bDMEM; clow glucose DMEM |
bESC medium; cαMEM; dDMEM |
MEF conditioned N2 medium |
| Factors | dox-, bFGF-, 5% HS | 2% FBS |
abFGF-; b15% FBS, dox; c5% HS, dox |
bdox; c5% KSR, 2 ME, dox; d2% HS, IGF-1, 2-ME, dox |
CHIR99021; DAPT |
| Duration (days) | 7 | 7 | 7 | 14 | 30 |
| Myotubes protein expression | MyoD, MYOG, MHC, dystrophin | MyoD, MYOG, MHC, dystrophin |
MyoD, MYOG, MHC, dystrophin, DES, RYR1, TNNC1 | MHC, SMA, CKM | MyoD, MYOG, MHC, Dystrophin, TTN, ACTN1 |
| Final differentiation markers | CD56, CD63, CD146, CD105, CD90, CD13 | CD56, TBX1, TBX4 | CD56, CD44, CD29 | not analyzed | DES; αSARC; MYL1, |
| Advantages | Sorting to select pure myogenic population | Rapid and efficient differentiation by small molecules | Efficient cell differentiation by transposon | Efficient, serum and transfection-free cell differentiation by small molecules | |
| Disadvantages | Longer time to reach suitable number of myoblasts by FACS; possible mutagenesis due to transfection; exogenous growth factor and serum-based protocol | Possible mutagenesis due to transfection; serum-based protocol | Longer time to reach suitable number of myoblasts; exogenous growth factor-based protocol | ||
Pre-induction medium;
Induction medium;
Differentiation medium;
Maturation medium.
2-ME, 2-mercaptoethanol; αMEM, minimum essential medium; αSARC, αsarcoglycan; ACTN1, alpha-actinin-1; bFGF, basic fibroblast growth factor; CD, cluster of differentiation; CHIR99021, inhibitor of glycogen synthase kinase 3; DAPT, inhibitor of the gamma-secretase complex; DES, desmin; DMEM, Dulbecco’s modified Eagle medium; dox, doxycycline; EB, embryoid body; ESC, embryonic stem cell; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; HS, horse serum; IGF-1, hypertrophy-inducing factor insulin-like growth factor 1; IMDM, Iscove’s modified Dulbecco’s media; iPSC, induced pluripotent stem cell; MB1, basal medium 1; KSR, knockout serum replacement; MEF, mouse embryonic fibroblast; mTeSR, cGMP, feeder-free maintenance medium for human ESCs and iPSCs; MYL1, myosin light chain 1; N2, serum-free supplement; RYR1, ryanodine receptor 1; SMC4, small molecule cocktail 4; TNNC1, troponin C1; TTN, titin.