Keeping your strength up: induced pluripotent stem cell-based approaches for the treatment and investigation of skeletal muscle disorders

Figures & data

Figure 1. Highly organized structure of skeletal muscle. Individual muscles are surrounded by a layer of connective tissue called the perimysium. Each muscle is composed of bundles of contractile cells termed muscle fibers. These fibers contain sarcomeres, which are repeating units of myofibrils that give skeletal muscle its banding pattern. Two contractile proteins, myosin and actin, form the thick and thin filaments of the sarcomere, respectively. The interaction between myosin and actin produces muscle contraction.

Table 1. Culture strategies to enhance iPSC differentiation into myogenic precursor cells.

Myogenic progenitor induction	Terminal differentiation	Culture	Myogenic cell selection	Ref.
MYOD1 (tetracycline-inducible system) StemFit AK02N, 0.5% pen/strep (1 d); primate ES Cell media, 0.5% pen/strep (1 d); same medium as before, 1 μg/mL dox (1-2 days); α-MEM, 0.5% pen/strep, 5% KSR, 200 μM 2-ME, 1 μg/mL dox (6–7 days) Sorting markers GE: MYOD1, MYOG, MYH PE: MYH2, MYOG	DMEM, 0.5% pen/strep, 2 mM L-glutamine, 200 μM 2-ME, 5% HS, 10 ng/mL IGF-1, 5 μM SB431542 (2-3 days); DMEM, 0.5% pen/strep, 2 mM L-glutamine, 200 μM 2-ME, 2% HS, 10 ng/mL IGF-1, 5 μM SB431542 (7 days) Sorting markers GE: n/a PE: MYH2	Adherent	n/a	(Uchimura et al. 2017)
PAX7 (dox-inducible lentiviral system) Matrigel® coating, with E6 media, 10 µM CHIR99021 (2 days); E6 media, 1 µg/mL dox (18 days) Sorting markers GE: MYHs PE: MYOD1, PAX3, MYF5, MYOG, sarcomeric α-actinin, PAX7	logDMEM, 10% FBS, Fetuin (500X), hEGF (1000X), DE (1000X), pen (100 unit/mL), strep (50 µg/mL) until 80% confluence (for 2D culture) or 4 days (for 3D culture); logDMEM, N-2 Supplement (100X), pen G (100 unit/mL) (∼ 2 weeks) Sorting markers GE: n/a PE: sarcomeric α-actinin, ACh receptor, MYH, Ca^2+ handling genes	Adherent vs 3D	Myogenic progenitors: FACS for iPAX7+ (GFP+)	(Rao et al. 2018)
Collagen I coating with SKM-01, 5% HS, 3 µM CHIR99021, 2 µM Alk5 Inhibitor, 10 ng/mL EGF, 10 µg/mL insulin, 0.4 µg/mL DE, 200 µM AsAc (10 days); SKM-02, 5% HS, 10 µg/mL insulin, 10 µg/mL EGF, 20 ng/mL HGF, 10 ng/mL PDGF, 20 ng/mL FGF2, 20 µg/mL oncostatin, 10 ng/mL IGF-1, 2 mM SB431542, 200 µM AsAc (8 days) Sorting markers GE: n/a PE: PAX7, DES	SKM-03, 10 µg/mL insulin, 20 µg/mL oncostatin, 50 nM necrosulfonamide, 200 µM AsAc (7 days) Sorting markers GE: n/a PE: MYH	3D	n/a	(Uchimura et al. 2020)
iMatrix coating, DMEM/F12, 1% ITS, 3 μM CHIR99021, 0.5 μM LDN193189 (3 days); same medium as before with 20 ng/mL FGF2 (3 days); DMEM/F12, 10 ng/mL HGF, 2 ng/mL IGF-1, 20 ng/mL FGF2, 0.5 μM LDN-193189 (2 days); DMEM/F12, 15% KOSR, 2 ng/mL IGF-1 (4 days). Induction of EZ sphere culture in Stemline, 100 ng/mL FGF2, 100 ng/mL EGF, 5 µg/mL heparin sodium salt for at different time points (6–10 weeks) (Lim et al. 2018) Sorting markers GE: T, TBX6, PAX3, PAX7 PE: MYOD, PAX7, MYOG	Collagen coating, DMEM, 10% FBS (4 weeks); DMEM, 10% FBS, 10 μM SB431542, and/or 10 μM DAPT Sorting markers GE: MRF4, MYH2, MSTN, MYH8, PAX7 PE: MYH2, MYOG, PAX7	Combination of adherent 2D culture and EZ spheres	Myogenic progenitors: FACS for CD57−/CD108− /CD271+/ERBB3+	(Sakai-Takemura et al. 2018)
DMEM/F12, 1% ITS, 1% pen/strep/L-glutamine, 3.5 µM CHIR99021 (5 days); same medium as before with 20 ng/mL FGF2 (14 days) Sorting markers GE: n/a PE: PAX7	DMEM/F12, 1% ITS, 1% pen/strep/L-glutamine (16 days) Sorting markers GE: RNA seq PE: MYH, TTN, α-ACTN	Adherent	Myogenic progenitors: FACS for c-MET+ /HNK-1-	(van der Wal et al. 2018)
PAX7 (dox-inducible lentiviral system)	KO DMEM, 20% KOSR, 1% NEAA, 1% GlutaMAX^TM, 1% pen/strep, 10 µM SB431542, 10 µM DAPT, 10 µM DE, 10 µM PD0325901, 10 µM Forskolin (5 days)	EBs then adherent	Myogenic progenitors: FACS for iPAX7+ (GFP+)	(Selvaraj et al. 2019)
IMDM basal medium, 15% FBS, 10% HS, 1% pen/strep, 1% GlutaMAX^TM, 1% KOSR, 50 µg/mL AsAc, 4.5 mM MTG (altogether defined ‘myogenic medium’), 10 µM CHIR99021 (2 days); myogenic medium, 200 nM LDN193189, 10 µM SB431542 (1 d); myogenic medium, 1 µg/mL dox (3 days); gelatin coating, myogenic medium, 1 µg/mL dox, 5 ng/mL FGF2 (4 days) Sorting markers GE: MEOX1, TCF15, PAX3, FOXC2 PE: n/a	Sorting markers GE: MYOG, MYOD1, MYH2, MYH3, MYH8, MYH7 PE: MYH8, TTN, DES
Matrigel® coating, DMEM/F12, 20 IU/mL pen, 0.02 mg/mL strep (2%), 1% NEAA, 1% ITS, 3 μM CHIR99021, 0.5 μM LDN193189 (3 days); same medium as before with 20 ng/mL FGF2 (3 days); DMEM/F12, 15% KOSR, 1% NEAA, 2% pen/strep, 0.1 mM 2-ME, 10 ng/mL HGF, 2 ng/mL IGF-1, 20 ng/mL FGF2, 0.5 μM LDN193189 (2 days); DMEM/F12, 15% KOSR, 1% NEAA, 2% pen/strep, 0.1 mM 2-ME, 2 ng/mL IGF-1 (4 days); same medium as before with 10 ng/mL HGF (9 days); DMEM/F12, 1% ITS, 2% pen/strep, 1% N-2 Supplement, 1% L-glutamine Sorting markers GE: n/a PE: TBX6, PAX3, MYOD1, MYOG, PAX7, MYH2, TTN, DMD	Skeletal muscle growth medium (SkGM-2, Lonza), 10 µM ROCK inhibitor (1 d); SkGM-2 (1-2 days); DMEM/F12, 2% KSR, 1 µM Chiron, 0.2% pen/strep, 1× ITS (1-2 weeks) Sorting markers GE: n/a PE: PAX7, MYH2	Adherent	Myogenic progenitors (dissociated after 21 days of differentiation): FACS for PAX7^Venus	(Al Tanoury et al. 2020)
Geltrex matrix, E6 medium, 1% ITS, 10 µM CHIR99021 (2 days); E6 medium, 1% ITS, 5 mM LiCl, 10 ng FGF2, 10 ng IGF-1, 50 µg/mL EVs from MT (4 days); cell splitting; collagen I coating, E6 medium, 1% ITS, 10 ng FGF2, 10 ng IGF-1, 50 µg/mL EVs from MT (10 days) Sorting markers GE: MSGN1, PAX3, PAX7, MYOD1, MYOG PE: n/a	E6 medium, 1% ITS (10 days) Sorting markers GE: MYOG, MYH8, CKM, MYH PE: MYH2, NCAM1, MYOD1, MYOG, MYH2	Adherent	n/a	(Baci et al. 2020)
STEM Diff Apel medium (STEMCELL Technologies), 10 ng/mL FGF2, 0.5 mM BIO, 20 mM forskolin (7 days) Sorting markers GE: n/a PE: PAX7	Matrigel® coating, DMEM, 2% HS (26 days) Sorting markers GE: n/a PE: PAX7, MYF5, MYH4, DES, DMD	EBs and then adherent	n/a	(Uchimura et al. 2020)

Figure 2. Schematic of iPSC-based cell therapy for DMD patients. iPSCs are generated from a tissue biopsy, and the genetic mutation in dystrophin corrected. After expansion in culture to generate sufficient cell numbers for transplantation, the cells are then induced to enter the myogenic pathway to yield cultures of muscle precursor cells. These cells are then transplanted into the patient’s skeletal muscle and produce dystrophin positive myofibers and satellite cells.

Figure 3. Schematic of iPSC-based cell therapy for VML patients. Similar to DMD patients, a tissue biopsy is used to generate iPSCs, which are then expanded in culture and induced to enter the myogenic pathway to produce muscle precursor cells. Alternatively, culture conditions may be modified to yield cultures containing multiple cells types found in skeletal muscle, such as motor neurons and immature myotubes. Due to the loss of the connective tissue scaffold in VML, a biomaterials-based matrix is transplanted with the iPSC-derived cells to support skeletal muscle regeneration. The transplanted cells utilize the artificial matrix enter myogenesis and effectively replace the loss muscle tissue.

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Data availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.