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Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders
Chapters
Summary April 3rd, 2021
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.
Transcript
Hello everyone. My name is Nicolas Wein. I'm a principal investigator at Nationwide Children's Hospital in Columbus, Ohio.
And today we're gonna show you a video about how to take a skin biopsy and convert it into myoblasts and myotube. This is a very useful tool to study neuromuscular disorder. In addition, it's a way faster technology compared to IPSC because we are using two different type of virus to directly combat this type of cell.
This video demonstrates the protocol for establishing human skin derived fibroblast cultures, the conversion into myoblasts, and then differentiated myotubes. A skin biopsy is transferred to cell culture to derive dermal fibroblasts. Lentiviruses carrying hTERT and MYOD genes are used to transduce these fibroblasts.
Upon addition of myoblast growth medium supplemented with Doxycycline, the MYOD gene is expressed, inducing the conversion of fibroblasts into myoblasts. Next, the medium is switched to a differentiation medium, also supplemented with Doxycycline. Then the myoblasts fuse with each other, forming multinucleated myotubes.
Aspirate the media from the tube and rinse the biopsy with 10 milliliters, one x PBS at room temperature, three times. After the third wash, leave the PBS in the tube. Pour out the PBS and the skin biopsy onto a 10 centimeter squared dish.
Using sterile scalpels, cut the biopsy into pieces that are as small as possible. Using a pipette, transfer an individual skin piece onto a sterile 10 centimeter squared dish. Aspirate the excess of PBS from around each piece.
Be careful not to aspirate the piece itself. Cover the dishes with the lid partially and allow the skin biopsy pieces to dry for five to 20 minutes. Do not allow the pieces to dry for too long.
Once the pieces are dry, tilt the dish at a 45 degree angle and slowly add 12 milliliters of fibroblast medium to the corner of the dish. Lower down the dish, carefully distributing the media so that the pieces are not lifted up by the media. Place the dishes into the incubator.
Replace the media in five to seven days, and once a week thereafter. Seed primary fibroblasts at 30%of confluency and two wells of a 12 well plate, approximately 20, 000 cells per well, in order to have about 50%of confluency the next day. For lentiviral transduction, add two to 5 billion viral genomes of hTERP Puromycin Lentivirus and 400 microliters of fibroblast medium.
Add the lentivirus mix to one well. To the second well, add just 400 microliters of fibroblast medium. The following day, add one milliliter of media.
One or two days later, transfer the cells into a six well plate and grow them until reaching 60 to 70%confluency. Supplement the fibroblast medium with one microgram per milliliter of Puromycin and add two milliliters to each well. Keep the cells under selection until all cells in the control well are dead for at least 12 days, changing the media every two to three days.
Seed H chart expressing fibroblasts at 30%confluence in three wells of the 12 well plate to have about 50%confluence the next day. For lentiviral transduction, mix two to five billion viral genomes of MYOD Hygromycin B lentivirus and 400 microliters of fibroblast medium and add to two wells. Add one milliliter of medium the next day.
One or two days later transfer the cells into a six well plate and grow until 60 to 70%confluent. Supplement growth media with 400 micrograms per milliliter of Hygromycin B.Add two milliliters to each well. Keep the cells under selection until all cells in the control wells have died, typically taking at least 12 days, changing the media every two to three days.
For induction of myoblasts, grow fibroblasts until they're 70%confluent. Add eight micrograms per milliliter of Doxycycline to the myoblast growth medium. Always use fresh aliquots of Doxycycline and medium.
The medium should be prepared right before any media changes. Additionally, the Doxycycline should not be refrozen. Aspirate the fibroblast medium and rinse the cells with PBS.
Add 10 milliliters of supplemented myoblast media. Two to three days later, cells are 90 to 95%confluent and their morphology will have changed. Aspirate the myoblast growth media and rinse the cell surface with PBS.
Add 10 milliliters of differentiation media that has been supplemented with eight micrograms per milliliter of fresh Doxycycline. Continue to change the media every two to three days until myotubes are formed. Seven to 10 days after initiation of myotube differentiation, cells should be fully differentiated, but this varies from one cell line to another.
The first fibroblasts may emerge five days after the skin pieces have been in culture. In picture B, fibroblasts are confluent and ready to be transferred to 75 centimeter square flasks for further proliferation. It's important to freeze several vials of fibroblasts at low passage numbers as primary cells enter the replicative senescence.
Once the FM cell lines are established for the conversion of fibroblasts into myotubes, the fibroblasts must proliferate until 70%confluent as shown in picture A.It's important not to exceed 80%confluency, as a high density may impair the differentiation. After addition of Doxycycline to the medium, within two to four days the cells become myoblasts, characterized by an elongated morphology and parallel orientation as seen in picture B.Differentiated myotubes are shown in picture C.The time for fusion and differentiation of myotubes may vary from seven to 21 days depending on cell genotype. The cells must be collected or fixed before they detach.
The beginning of the detachment can be observed by color and brightness of the myotube's edges, as pointed out by the arrows in this picture. By immunofluorescence, the expression of mature muscle specific proteins confirms the successful conversion of fibroblasts into myotubes. In these pictures, three different cells are shown.
The degree of differentiation will vary from cell to cell, and may or may not be effected by the primary mutation as it's the case of cells in figures B and C.Further validation of the model was confirmed by RNAseq. As shown in this plot there are more than 12, 000 differentially expressed genes detected in FM myotubes compared to that of skeletal muscle. The significant correlation between both transcriptomes supports that FM cells have a muscle specific expression profile demonstrating that they're a useful and reliable surrogate for muscle-derived cell lines.
To illustrate the usefulness of this in vitro model, we have used one of the most common exonic duplications in the DMD gene. Duplication of Exon two leads to the disruption of the DMD reading frame, and its skipping can either lead to restoration of that reading frame or utilization of the internal ribosome entry site. This therapy has already been validated with in vivo studies Figure A, we demonstrated proof of concept by RTPCR analysis of this particular mutated cell line, with and without treatment by exon skipping.
The western blot in figure B demonstrates that functional Dystrophin protein is expressed in treated cells. When comparing the efficiency of this therapy to in vivo models, results support the reliability of the cell differentiation technique and support its use for testing various mutation specific gene therapies. Before clinical studies required to test the efficacy of official therapy support on neuromuscular disorders, depending on the ability of animal and cellular models.
The development of animal models, although being easier nowadays, its not a feasible option for each specific mutation. The generation of myoblasts through IPS cells derived from dermal fibroblasts is an alternative, but its a time consuming technique. Thus, this procedure is a simple alternative to IPS generation that allows for the direct conversion of fibroblasts into myoblasts.
It facilitates the access to human cell models by avoiding the more invasive application of muscle biopsies In conclusion, this direct conversion of fibroblasts into myoblasts is a powerful tool for testing therapies for neuro muscular disorders in limitation specific context.
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