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This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.
Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.
Cellular models obtained directly from human tissues are useful to model many human genetic disorders, with the advantage of having the original genomic context and, in many cases, reproducing the same molecular and cellular hallmarks observed in the patients. In the field of neuromuscular disorders, muscle biopsies have been a great source of human myoblasts and have helped in the elucidation of pathological mechanisms. Additionally, they are an important tool for in vivo testing of drugs and gene therapies. On one hand, the derivation of myoblasts from muscle fragments is relatively easy. On the other hand, the culture and maintenance of primary myoblasts a....
All experiments and biopsies were carried out following the ethical rules of the institutions involved under the approval of the Nationwide Children's Hospital Institutional Review Board.
1. Initiation of dermal fibroblasts culture
This protocol shows how to establish human skin-derived fibroblast cultures and convert them into myoblasts and then into differentiated myotubes. This type of cell line is extremely useful for the study of neuromuscular disorders and in vitro testing of potential therapies.
A schematic representation of the fibroblast conversion is shown in Figure 1. Figure 2A shows a fragment of skin and the fibroblasts emerging from it. Th.......
To obtain FM cell lines with good quality, some steps are critical. The sooner the skin biopsy is processed, the greater the chances are to obtain healthy fibroblasts. The passage number of fibroblasts cultures is also important. Primary cells have limited proliferative capacity and after many passages, they enter in replicative senescence. Thus, it is better to have a stock of fibroblasts at a low passage number and transform cells at the earliest passage as possible.
Another important step .......
We would like to thank Dr. Vincent Mouly for sharing his knowledge in the past regarding the model. This work has been supported by the US National Institutes of Health National Institute of Neurological Disorders and Stroke (R01 NS043264 (K.M.F., and R.B.W.)), the US National Institutes of Health National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) (P50 AR070604-01 (K.M.F., K.M., R.N., and N.W.). N.W. has received fellowship support from the Ohio State University/Nationwide Children's Hospital Muscle Group and the Philippe Foundation. This work was also supported by internal discretionary funds and part of the exon 2 skipping work has bee....
Name | Company | Catalog Number | Comments |
100 mm dish | Corning | 430167 | |
0.25% Trypsin-EDTA, phenol red | Thermo Fisher | 2500056 | |
10X Phosphate buffered saline (PBS) | Fisher Scientific | BP3994 | |
12-well plate | Corning | 3513 | |
20X Transfer buffer | Thermo Fisher | NP00061 | |
20X Tris-acetate SDS running buffer | Thermo Fisher | LA0041 | |
3-8% Tris-Acetate gel | Thermo Fisher | EA0378BOX | |
75 cm2 flask | Corning | 430641U | |
Antibiotic-Antimicotic 100X | Thermo Fisher | 15240062 | |
Anti-myosin heavy chain, sarcomere antibody | Developmental Studies Hybridome Bank | MF20 supernatant | Dilution 1:50 |
Antioxidant | Thermo Fisher | NP0005 | |
BCA Protein Assay | Thermo Fisher | 23227 | |
Chloroform | Sigma-Aldrich | C2432 | |
DAPI | Thermo Fisher | D3571 | Dilution 1:1000 |
Digitonin | Millipore Sigma | 300410250MG | |
Dimethyl sulfoxide | Sigma-Aldrich | D2438 | |
DMEM, High glucose, GlutaMAX supplement, Pyruvate | Thermo Fisher | 10569044 | |
DNAse I set (250U) | Zymo Research Corporation | E1010 | |
Doxycycline Hydrochloride | Fisher Scientific | BP2653-5 | |
Dup2 human primers | Fw_5' GCTGCTGAAGTTTGTTGG TTTCTC 3' | Rv_5' CTTTTGGCAGTTTTTGCC CTGT 3' | |
Dystrophin antibody | Abcam | ab15277 | Dilution 1:200 |
Fetal bovine serum | Thermo Fisher | 16000 | |
Glycine | Sigma-Aldrich | G8898 | |
Goat anti-mouse, Alexa Fluor 488 | Thermo Fisher | A11001 | Dilution 1:1000 |
Halt Protease inhibitor cocktail 100X | Thermo Fisher | 78430 | |
Hemocytometer | Hausser Scientific | 3100 | |
Hygromycin B | Thermo Fisher | 10687010 | |
IRDye 680RD goat anti-Rabbit IgG (H+L) | Li-Cor | 926-68071 | Dilution 1:5000 |
Lab-Tek II CC2 chamber slide system | Thermo Fisher | 15852 | |
Laemmli | Bioworld | 105700201 | |
Lipofectamine 3000 Transfection Reagent | Thermo Fisher | L3000008 | |
Matrigel GFR membrane matrix | Corning | 354230 | |
Methanol | Fisher Scientific | A412P-4 | |
Mr. Frosty Freezing Container | Thermo Fisher | 51000001 | |
Nitrocellulose membrane 0.45 µm | GE Healthcare Life Sciences | 10600002 | |
Normal Goat serum control | Thermo Fisher | 10000C | |
Odyssey Blocking Buffer (PBS) | Li-Cor | 927-40003 | Blocking buffer for Western blot |
Opti-MEM I Reduced Serum Medium | Thermo Fisher | 11058021 | |
Paraformaldehyde | Sigma-Aldrich | 158127 | |
PCR master mix | Thermo Fisher | K0172 | |
Phosphatase inhibitor | Thermo Fisher | A32957 | |
Precision Plus Protein Dual Color Standards | Bio Rad | 1610374 | |
Puromycin | Thermo Fisher | A1113803 | |
Revert 700 Total Protein Stain for Western Blot Normalization | Li-Cor | 926-11021 | |
RevertAid kit | Thermo Fisher | K1691 | |
RNA Clean & Concentrator-25 | Zymo Research Corporation | R1018 | |
Scalpels | Aspen Surgical | 372611 | |
Skeletal Muscle Cell Differentiation medium | Promocell | C23061 | |
Skeletal Muscle Cell Growth medium | Promocell | C23060 | |
Triton X-100 | Acros Organics | 215682500 | |
TRIzol reagent | Thermo Fisher | 15596026 | |
Tween 20 | Fisher Scientific | BP337500 | |
Ultra low temperature freezer | Thermo Scientific | 7402 | |
UltraPure 0.5M EDTA, pH 8.0 | Thermo Fisher | 15575020 | |
Vectashield antifade mounting medium | Vector Labs | H1000 |
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