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| Reference | PSC type | PSC culture | Myogenic progenitor derivation and proliferation | Progenitor purification | Terminal differentiation | Efficiency of myogenic differentiation | In vivo engraftment | Use of disease-specific iPSCs |
| Culture condition | Culture condition |
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| Barberi et al. 2007 [66] | ESCs | Feeder-dependent protocol using MEF | Monolayer cells were plated at 1 × 103 cells/cm2 on MEF, or 3 × 103 cells/cm2 on feeder-freegelatin-coated plates, for 3-4 days under standard ESC culture conditions. The cells were then switched in DMEM/F12 supplemented with ITS for 20 days, and in αMEM, 10% FBS for an additional 14 days. | FACS based on CD73+/NCAM+. Sorted NCAM+ cells were grown in αMEM, 10% FBS until confluence. | The cells were differentiated in serum-free N2 medium. | 60–80% of sorted NCAM+ cells were MyoD+. At 24 hours after exposure to N2 medium, approximately 7% and 46% of the total cells expressed Pax7+ and MyoG+, respectively. Upon terminal differentiation, MyoG, desmin, skeletal muscle actin, and myosin (MHC) were identified. Spontaneous twitching of myotubes was confirmed. | ESC-derived cells (5 × 105 cells, CD73+/NCAM+ cells) were transplanted into a muscle injury model in SCID/Beige mice. The expression of reporter proteins (luciferase and GFP), human cell-specific nuclei, and laminin-positive myofibers were identified in the grafted muscles. | |
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| Awaya et al. 2012 [53] | ESCs and iPSCs | Feeder-dependent protocol, using human ES cell maintenance medium (hESM) | EBs were formed by suspension in hESM for 7 days and then plated onto gelatin-coated tissue culture plates in ITS medium for an additional 14 days. | | EBs were differentiated in skeletal muscle induction medium containing 10% FCS and 5% HS until day 112 of differentiation. In some experiments, dissociated EB cells (3 × 103 cells/cm2) were seeded on collagen type I-coated plates. On day 49, the medium was changed to ITS medium. | xIn the cells migrating out of the EBs, the clusters Pax3+ and Pax7+ cells were randomly distributed at day 21. Skeletal myosin-positive multinucleated myofibers had appeared within most of the attached EBs at day 63. | Progenitors (1–5 × 105 cells) were transplanted into the muscle of immunodeficient NOG (NOD/Shiscid/IL-2Rγnull) mice following cardiotoxin injury. Four weeks after transplant, human cell-specific laminin A/C-positive nuclei were detected in the TA muscles. Further, the detection of human-specific laminin a2 proved that the transplanted cells produced human protein around the muscle fibers into which they had integrated. | |
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| Xu et al. 2013 [20] | iPSCs | Feeder-free protocol using mTeSR on Matrigel-coated plates | EB culture in STEMdiff APEL Medium supplemented with 10 ng/ml FGF2, 0.5 μM GSK3β inhibitor BIO, 20 μM forskolin (“triple cocktail”) for 7 days. | | EB cells were then cultured on Matrigel-coatedplates in DMEM, 2% HS for an additional 29 days. | Under terminal differentiation procedures (day 36), most of the cells expressed desmin (72%) and MyoG (92%), forming multinucleated myofibers. Sarcomere structures were also confirmed by electron microscopy. | iPSC-derived myogenic progenitors (1 × 105 cells at day 14 of differentiation) were transplanted into cardiotoxin-injured muscles in NSG (NOD/SCID/IL-2Rγnull) mice. Human δ-sarcoglycan expression in myofibers and colocalization of human-specific histone H2A and Pax7 were characterized in the grafted muscles. | |
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| Borchin et al. 2013 [55] | ESCs and iPSCs | Feeder-free protocol using mTeSR1 on Matrigel-coated plates | PSC colonies were cultured in ITS medium (DMEM/F12 supplemented with ITS) in the presence of 3 μM GSK3β inhibitor CHIR99021 for 4 days, and then in ITS medium containing 20 ng/ml FGF2 for an additional 14 days. The cells were then maintained in ITS medium alone for a further 17 days of culture in ITS medium alone. | FACS based on the expression of HNK, AChR, CXCR4, C-MET, following the differentiation for 35 days. | FACS-sorted AChR+ myocytes and CXCR4−/C-MET+ and CXCR4+/C-MET+ precursors were plated onto fibronectin/laminin-coated tissue culture wells in ITS medium supplemented with 10 μM ROCK inhibitor Y-27632. The myocytes were maintained in ITS medium with 50 ng/ml IGF-I. The progenitor cells were differentiated in ITS medium. | In presorting cultures of CXCR4−/C-MET+ and CXCR4+/C-MET+ cells isolated at day 35 of differentiation, >18% Pax3+/Pax7+ and >8% MF20+ muscle cells were identified. In postsorting cultures at day 35, 97% in CXCR4−/C-MET+ and 98% in CXCR4+/C-MET+ were PAX3+; 84% in CXCR4−/C-MET+ and 96% in CXCR4+/C-MET+ were PAX7+. After 3 days of culture, few cells retained PAX7 expression, whereas all cells expressed MYH5. In postsorting cultures of AChR+ myocytes, all AChR+ cells were MyoG+ and MHC+ at 24 hours after plating. | | |
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| Hwang et al. 2013 [58] | ESCs (OCT4-GFP reporter line) | Feeder-dependent protocol using MEF | Single cells were cultured in suspension on ultra-low attachment plates to form EBs for 9 days, in high glucose DMEM containing 5% FBS, 2 mM L-glutamine, 100 nM dexamethasone, 100 μM hydrocortisone, 1% penicillin/streptomycin, 1 mM transferrin, 86.1 μM recombinant insulin, 2 mM progesterone, 10.01 mM putrescine, and 3.01 mM selenite. The EBs were then spilt, transferred, and cultured on a Matrigel-coated dish for an additional 8 days. | The cells growing out of the EBs were concentrated by FACS based on PDGFRA and OCT4-GFP.PDGFRA+ and PDGFRA− cells were expanded in growth medium, containing high glucose DMEM, 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin. | PDGFRA+ and PDGFRA− cells (1 × 104 cells/cm2) were plated on gelatin-coated culture plates and differentiated in high glucose DMEM containing 2 mM L-glutamine, 100 nM dexamethasone, 100 mM hydrocortisone, 1% penicillin/streptomycin, 1 mM transferrin, 86.1 mM insulin, 2 mM progesterone, 10.01 mM putrescine, and 3.01 mM selenite with 10% FBS or without FBS. | The morphology of PDGFRA+ cells progressively became more spindle-like and fused and formed multinucleated myotubes (approximately 30% MHC+) after 14 days of terminal differentiation. In contrast, little or no myogenic differentiation was observed in PDGFRA− cells population. | ESC-derived PDGFRA+ cells were transplanted into the muscle of NOD/SCID mice following cardiotoxin injury. Human laminin+ myofibers were identified after 14 days posttransplantation. | |
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| Hosoyama et al. 2014 [59] | ESC and iPSCs | Feeder-dependent with MEF (ESCs) or feeder-independent protocols (iPSCs) | Sphere-based culture (EZ sphere) was maintained in Stemline medium containing 100 ng/ml FGF2, 100 ng/ml EGF, 5 ng/ml heparin sulfate for 6–12 weeks (42–84 days). The spheres were passaged by mechanical chopping every week. | | Monolayer culture in high glucose DMEM, 2% B27 serum-free supplement on poly-L-lysine/laminin-coated coverslips. | Before terminal differentiation, progenitors were approximately 40% and 56% Pax7+, respectively. After 14 days of differentiation, the prevalence of Pax7+, MyoD+, 36–61% MyoG+, 24-25% MHC+ after 14 days of differentiation. Spontaneous contraction and AChR+ in myotubes were confirmed after 25 days of terminal differentiation. | | The protocol was applied to patient-derivediPSCs (ALS with SOD1 or VAPB mutation, BMD, and SMA iPSC lines) for myogenic differentiation. |
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| Shelton et al. 2014 [54] and 2016 [22] | ESCs | Feeder-free protocol in E8 medium | Monolayer cells (1.5 × 105 cells per well) were plated on Matrigel-coated dishes in E8 medium supplemented with 10 μM Y-27632 overnight. Cells were grown in E6 medium supplemented with 0.1% CHIR99021, BMP4, or activin-A for 2 days and then switched in unsupplemented E6 medium until day 12. From day 12 to 20, the medium was replaced with StemPro-34 media. Cells were then returned to E6 medium from day 20 to 35. | | The cells were differentiated in N2 medium until the endpoint of the experiment. | Skeletal myocytes were prominent approximately 37% Pax7+ and 14% MHC+ by day 40 following 5 days of growth in N2 medium. Skeletal muscle contractions could be observed at this time point. When the cells were left in N2 media until day 50, 43% Pax7+ and 47% MHC+ were identified. | | |
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| Chal et al. 2015 [56] and 2016 [64] | ESCs and iPSCs | Feeder-free protocol in mTeSR1 media | Single cells from PSC colonies were seeded on Matrigel-coated plates (15,000–18,000 cells/cm2) in mTeSR medium supplemented with Y-27632 for 1 day. The medium was changed to a DMEM-based medium supplement with ITS, 3 μM CHIR99021, and 0.5 μM LDN193189 for 2 days. At day 3, 20 ng/ml FGF2 was added for an additional 3 days. At day 6, the cells were changed to a DMEM-based medium supplemented with 10 ng/ml HGF, 2 ng/ml IGF-I, 20 ng/ml FGF-2, and 0.5 mM LDN193189 for 2 days. | | At day 8 in culture, the medium was changed to DMEM, 15% KSR, supplemented with 2 ng/ml IGF-I for 4 days and then supplemented with both 10 ng/ml HGF and 2 ng/ml IGF-I after day 12. | After 20 days, the cultures contained large fields comprising MHC+ and MyoG+ fibers and PAX7+ cells. By 4 weeks, ~22% nuclei were MyoG+ and 23% of nuclei were Pax7+. The muscle fibers showed sarcomeres, as demonstrated by titin and fast MHC staining. These striated fibers exhibited spontaneous twitching. The diameter of the muscle fibers was ~3.5 μm. | | |
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| Caron et al. 2016 [57] | ESCs and iPSCs | Feeder-free protocol in serum-free M2 medium | Monolayer culture at 2500 cells/cm2 on collagen-coated plates and maintained in skeletal muscle induction medium containing 5% HS, 3 μM CHIR99021, 2 μM Alk5 inhibitor, 10 ng/ml EGF, 10 μg/ml insulin, 0.4 μg/ml dexamethasone, and 200 μM ascorbic acid for 10 days. | | At day 10, cells were dissociated with trypsin and replated at 2500 cells/cm2 onto collagen-coated plates and maintained for 8 days in skeletal myoblast medium containing 5% HS, 10 mg/ml insulin, 10 ng/ml EGF, 20 ng/ml HGF, 10 ng/ml PDGF, 20 ng/ml FGF2, 20 mg/ml oncostatin, 10 ng/ml IGF-I, 2 μM SB431542, and 200 μM ascorbic acid. After 18 days of differentiation, cells were maintained in myotube medium, containing 10 μg/ml insulin, 20 μg/ml oncostatin, 50 mM necrosulfonamide, and 200 μM ascorbic acid, for 7 days. | After 10 days in skeletal muscle induction medium, 80% Pax3+, 20% Pax7+, and 30–40% CD56+ cells were identified. At day 18 (after the second step of differentiation), 50–60% of the cells were MyoD1+ and 20% desmin+. At day 26 (after the third and final stage of the process), 50–80% of the cells formed elongated and multinucleated myotubes that stained positive for MyoG, MHC, dystrophin, and α-actinin. | | FSHD1- or BMD-affected ESCs were differentiated into MHC+ myotubes expressing MyoG. Patient-derived iPSCs with FSHD1 were also tested. |
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| Choi et al. 2016 [65] | ESCs and iPSCs | Feeder-dependent protocol using MEF | At day 0, nonadherent cells were plated on a gelatin-coated dish (at 1.5 × 105 cells per well of a 24-well plate), in MEF-conditioned N2 media containing 10 ng/ml FGF2 and 10 μM Y-27632. At day 1, N2 media with 3 μM GSK3β inhibitor CHIR99021 was added. At day 4, N2 media with 10 mM γ-secretase inhibitor DAPT were added until day 12. The resulting cells (“CHIR99021-DAPT culture”) were maintained in defined N2 media until day 30. | | To determine the presence of fusion component myoblasts, the dissociated cells from the CHIR99021-DAPT culture (days 25–30) were also replated. | At day 30 in the CHIR99021-DAPT culture, approximately 63% of cells were MHC+ and 61% were MyoG+. The culture resulted in differentiation of myoblasts into multinucleated and spontaneously contractile myotubes with sarcomere structures. When the cells from the CHIR99021-DAPT culture were replated, the attached and surviving cells were mono-nucleated at day 2 after replating and then formed multinucleated myotubes at day 10 after replating with typical striations and expression of 35% dystrophin+, 37% titin+, and 40% α-actinin+. | The dissociated CHIR99021-DAPT culture cells (1–3 × 106 cells) were transplanted into the injured TA muscle of NRG mice. At 6 weeks after transplantation, human nuclei (human-specific lamin A/C+) and human-specific laminin+ myofibers were detected in the grafted muscles. | Disease-specific cellular characteristics were characterized in the myotubes from patient-derived iPSC lines (FSHD, ALS with C9orf72 repeats, and DMD). |
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| Swartz et al. 2016 [60] | iPSCs | Feeder-free culture on vitronectin-coated plates in TeSR-E8. | When iPSC colonies were ~250–400 μm in diameter (day −1), 1.5% DMSO in TeSR-E8 medium was added. On day 0, cells were cultured in chemically defined medium (CDM) supplemented with 20 ng/ml FGF2, 10 μM LY294002, 10 ng/ml BMP4, 10 μM CHIR99021 (FLyBC) for 36 hours. Cells were then cultured in CDM supplemented with 20 ng/ml FGF-2 and 10 μM LY294002 (FLy) for an additional 5.5 days. On day 7, cells were cultured in MB-1 and 15% FBS for 6 days. On day 12, the cells were cultured in fusion medium (2% HS in DMEM). | | After 10 days of fusion medium (22 days total from the start of differentiation), the cells were changed to N2 medium (DMEM/F12 supplemented with 1% N2 supplement and 1% ITS). | At day 5, <5% of the total cells were Pax3+ mesodermal progenitors. At day 36, up to 64% (median 44.8%) of nuclei were MyoG+. A mix of intermediate- and late-stage muscle cells as demonstrated by desmin+ and MHC+. After 63 total days in fusion medium, brief and spontaneous contractions in a small set of myotubes were observed. Seven to 10 days after the addition of N2 medium, robust spontaneous contractions throughout the cell cultures were observed. Titin+ striation was displayed. | | |
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| Xi et al. 2017 [61] | ESCs and iPSCs | Feeder-free culture on Matrigel-coated plates in mTeSR1 medium. | On day −1, single cells from PSC colonies (25,000 cells/cm2) and seeded on Matrigel-coated plates in mTeSR1 medium containing 10 μM Y-27632. On day 0, cells were switched to basal differentiation medium (BDM; DMEM/F12, 1% ITS and 0.5% penicillin–streptomycin) supplemented with 3 μM CHIR99021 for 2 days. On day 2, cells were switched to BDM supplemented with 200 nM LDN193189 and 10 μM SB431542 for another 2 days. On day 4, culture medium was changed to BDM supplemented with 3 μM CHIR99021 and 20 ng/ml FGF2 for 2 days. On day 6, medium was switched to a KSR/HGF/IGF-I-based differentiation medium (DMEM, 0.5% penicillin–streptomycin and 15% KSR, 10 ng/ml HGF, 2 ng/ml IGF-I) for 14–21 days. | | At day 29, cell suspension was filtered through cell strainers to exclude cell aggregates. Filtered cells were resuspended in SkGM2 medium supplemented with 20 ng/ml FGF-2 and replated at 15,000–20,000 cells onto Matrigel-coated plates. Cells were cultured for 7–10 days until reaching >70% confluency, and then medium was switched to N2 medium (BDM containing 1% N2 supplement) for 5 days. | At day 2, ~80% cells were Pax3+. Expression of myogenic markers was gradually increased toward day 20. At day 27, large areas of MHC+ cells emerged throughout the culture, and the majority also expressed titin. A high proportion of Pax7+, MyoD+, and MyoG+ was also identified. At day 44, approximately 58% MHC+ myocytes and myotubes were identified, as well as cells outside MHC+ area (6.5% Pax7+/MyoD−, 9.1% Pax7−/MyoD+, and 4.9% Pax7+/MyoD+). | | |
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| Hicks et al. 2017 [79] | ESCs and iPSCs | Feeder-free culture on Matrigel-coated plates in mTeSR1 medium. | For direct differentiation from PSCs, two published protocols (Shelton et al. 2014 [54] and Chal et al. 2015 [56], listed in Table 1) were used. Myogenic progenitors from day 50 culture were dissociated and filtered through 100 mm meshes. | FACS based on HNK−/NCAM+ or ERBB3+/NGFR+ were performed. Sorted cells could be grown in SkBM-2 media. | Myogenic progenitors were induced to differentiate in N2 media for 7 days. In some experiments, TGF-β inhibitors (SB431542 or A83-01) were added in differentiation media. | HNK−/NCAM+ enrichment increased PAX7 and MYF5 expression by ~1.7-fold in comparison to unsorted SMPCs. When differentiated in culture, the number of MHC+ cells was increased in HNK−/NCAM+ cells compared to replated/unsorted cells. ERBB3+/NGFR+ progenitors were enriched for PAX7 and MYF5 by 20-fold in comparison to ERBB3−/NGFR− cells. ERBB3+/NGFR+ progenitors could form homogenous myotubes following terminal differentiation. TGF-β inhibitors (SB431542 or A83-01) significantly facilitated myotube fusion and maturation, as demonstrated by MHC protein levels (MYH1 and MYH8) and sarcomere formation. | The enriched cells (1 × 106 cells per 5 μl, injected 5–10 μl of cells) were transplanted into the injured or irradiated TA muscle of mdx-NRG mice. At 30 days after transplantation, human-specific lamin A/C+, spectrin+, and dystrophin+ myofibers were detected in the grafted muscle. HNK−/NCAM+ sorted cells survived in the grafted muscle but did not improve engraftment in comparison to unsorted cells. ERBB3+/NGFR+ progenitors significantly increased the number of engrafted myofibers in comparison to NCAM+ sorted cells. | Disease-specific iPSCs from DMD patients were used in this study. The mutation in DMD-iPSC lines was corrected by CRISPR-Cas9 gene editing, which could restore dystrophin expression. |
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