Exon Skipping in Directly Reprogrammed Myotubes Obtained from Human Urine-Derived Cells

We have established a new protocol for the direct reprogramming of human urine-derived cells into myotubes. This in vitro modeling enables us to evaluate exon skipping in the myotubes derived from patient with Duchenne muscular dystrophy. We discovered that 3-deazaneplanocin A hydrochloride, DZnep, are histone methyltransferase inhibitor could significantly promote direct screening of UDCs into myotube in a short amount of time.

This new autologous UDC based disease modeling could lead to the application precision medicine for various muscular diseases. To begin, centrifuge the entire urine sample at 400 x g for 10 minutes at room temperature. Next, aspirate the supernatant leaving one milliliter in the tube.

Resuspend the pellets individually in the remaining one milliliter of urine. Collect those in a single 50 milliliter tube and add 10 milliliters of washing buffer to the tube. Centrifuge the samples at 200 x g for 10 minutes at room temperature.

Aspirate the supernatant leaving 0.2 milliliters in the tube. Resuspend the cell pellets in 4.5 milliliters of primary medium. Seed the cells in three wells of gelatine-coated six-well plate, 1.5 milliliters of cell suspension per well.

Culture at 37 degree Celsius and 5%carbon dioxide. For three days add 1.5 milliliters of the primary medium each day. On day four, replace the medium with 1.5 milliliters of growth medium supplemented as described in the manuscript.

After day four, replace the supplemented growth medium every other day until the UDC culture is 80 to 90%confluent. Then remove the medium and wash the cells with PBS. Split the cells using 0.25%trypsin-EDTA.

Seed the cells at a density of 3, 000 to 5, 000 cells per squared centimeter onto a new gelatine-coated 60 millimeter dish. Seed the UDCs at 3, 000 to 5, 000 cells per square centimeter on a gelatin-coated 60 millimeter dish and place the dish in the incubator. 24 hours after seeding, infect the cells with thawed retrovirus at a multiplicity of infection of 200.

Use fresh medium with a hexadimethrine bromide concentration of 8 micrograms per milliliter. Incubate for 24 hours at 37 degrees Celsius and 5%carbon dioxide. To select the MYOD1-transduced cells, replace the culture medium with fresh growth medium containing one microgram per milliliter of puromycin.

Continue to change the medium every other day for seven to 10 days. Plate the MYOD1-transduced UDCs in a collagen coated multi-well plate. After incubating the cells for 24 hours, change the growth medium to differentiation medium described in the manuscript.

Then after three days, change the differentiation medium to fresh differentiation medium without DZNep. Continue to change the medium every three days. To transfect the antisense oligonucleotide into the MYOD1-transduced UDCs, mix ASO transfection reagent and differentiation medium to a final concentration of one to 10 micromolar.

And use this mixture to replace the medium in the wells. Incubate the plate at 37 degree Celsius and 5%humidity. After 72 hours incubation with ASO, change the medium to fresh differentiation medium without ASO.

Three to seven days after ASO transfection, remove the differentiation medium. Wash the UDCs once with PBS and add cell lysis buffer. Harvest the total RNA using an RNA extraction kit.

Measure the RNA concentration with a spectrophotometer. As described in the manuscript, combine the reagents for one-step RT-PCR in PCR tubes. Place the PCR tubes in the thermocycler and run the thermocycler.

Perform microchip electrophoresis and calculate the exon skipping efficiency. UDCs were collected easily and noninvasively and formed colonies within a week of primary culture. MYOD1 transfected UDCs in differentiation medium with DZNep could fuse to each other and formed multinucleated myotubes efficiently.

RT-PCR clearly detected exon skipping in MYOD1-UDCs derived from patients with Duchenne muscular dystrophy. Skipping efficiency was dependent on the dose of ASO. Dose-dependent exon skipping was also detected by Western blot.

Intensities of dystrophin were measured with fluorescent microscope one week after ASO transfection. Markedly higher fluorescent signals were observed in MYOD1-UDCs treated with ASO than in the control. We can model any muscular disease leading to the understanding of pathophysiology of those diseases.