Duchenne is a muscular dystrophy causing premature exhausting of muscle progenitor cells. It is one of the most severe Muscular Dystrophies. To promote a functional muscle regeneration, we use CRISPR/Cas9-mediated gene editing to restore dystrophin expression in Induced Pluripotent Stem Cell-derived muscle progenitor cells.
The CRISPR-Cas9 gene editing has the potential to restore dystrophin gene expression. Autologous iPSC-derived muscle progenitor cells can replenish the stem cell pore, repair damage, and prevent further complications in DMD without causing an immune response. Different agents of iPS cells into Myogenic Progenitor Cells reduced the risk of tumor re-genesis in iPS cells.
Several therapies that combine autologous Induced Pluripotent Stem Cell-derived muscle progenitor cells with CRISPR-Cas9-mediated genetic correction are promising Duchenne Muscular Dystrophy treatment strategies. This technology can be used to treat many congenital diseases by genetically editing autologous stem cells, including heart, brain, and blood disease. We showed demonstration of this approach can provide the readers with experience in handling cell reprogramming and gene editing, which remains a challenge.
Under an inverted microscope, examine the colonies of infected mouse embryonic stem cells. Mark the colonies at the bottom of the dish with the self-inking object marker. Apply greased cloning rings to cover the marked cell colonies.
Add 100 microliters of 0.05%Trypsin-EDTA to each cloning ring and place the plate in an incubator at 37 degrees Celsius for five minutes. Then, transfer the digested cells with 100-microliter pipette tips to 48-well culture plates containing 250 milliliters of MES growth medium. Incubate the cells in the 37 Degree Celsius incubator with 5%Carbon Dioxide and 85%humidity.
When they read 70%confluence, select the wells with well-grown cells and add 250 milliliters of TVP solution to digest for 30 minutes. Then, transfer the cell culture from each well to a six-centimeter dish. Repeat applying cloning rings and incubation for several times until uniform, dorm-shaped clones are obtained.
Now, digest the prepared mouse iPSCs by adding one milliliter of TVP solution. Transfer the cell culture into a 24-well plate coated with Fibronectin and Gelatin and incubate at 37 degrees Celsius with 5%Carbon Dioxide. After the cells reach 50%confluence, switch to 500 microliters of fresh culture medium containing eight micrograms per milliliter Polybrene.
Then, add 100 microliters of the lentiviral particle solution to the mouse iPSCs. Incubate the cells for three days at 37 degrees Celsius with 5%Carbon Dioxide. Next, determine the minimum concentration of Blasticidin and Hygromycin B which is at half kill rate within 24 hours.
Add the media of 2.5 micrograms per milliliter Blasticidin and 100 micrograms per milliliter Hygromycin B into the cells, wait for three days, and select the cells that remain adherent as stabling infected cells. Digest the selected mouse iPSCs with 0.5 milliliters of TVP solution, each well in a new 24-well plate, and incubate cells for 30 minutes at 37 degrees Celsius with 5%Carbon Dioxide. Then, pipette the iPSCs into single cells and count the cells with a cell counting chamber.
Select about 150 digested single cells and dilute with MES medium into 10 centimeter dish, culture at 37 degrees Celsius with 5%Carbon Dioxide. After about 10 days, under an inverted microscope, use 10 microliter sterile pipette tips to pick single colonies and transfer each colony into 50 microliters of TVP solution in a 96-well plate. Digest at 37 degrees Celsius for 30 minutes.
Reliable manual cloning picking is critical for the success of harvesting CRISPR/Cas9-edited Induced Pluripotent Stem Cells. Then, transfer and divide the digested cells from each well into another two 96-well culture plates. Incubate at 37 degrees Celsius until 70%confluent.
With the cell colonies at 70%confluence, remove the medium in the 96-well plate. Add 25 microliters of Lysis reagent containing 1%Proteinase K solution to each well and transfer the lysate to another 96-well PCR plate. Seal the PCR plate with adhesive seals and incubate the plate in a thermal cycler at 55 degrees Celsius for 30 minutes and then at 95 degrees Celsius for 45 minutes to lyse the cells and denature the Proteinase K.Then, in a PCR tube, add two microliters of the lysate, 10 microliters of 2x DNA Polymerase Premix, 7 microliters of DNase-free water, and one microliter of DMD Exon23 primers.
Start the PCR reaction according to the manuscript. Then, load the PCR reaction supplied with 2.2 microliters of loading buffer onto a 2%agarose gel. Run the gel at 100 to 150 volts for 30 minutes and inspect the gel under UV light.
In this protocol, two guide RNAs that flank the mutant Exon23 were designed. After Cas9-mediated double-stranded breaks and non-homologous end-joining, mutant Exon23 was deleted, allowing for truncated but functional Dystrophin production. To avoid the leakage of Trypsin solution, we need to apply grease evenly to the bottom of the rings.
The combination of CRISPR/Cas9 genome editing with a tacked-on MyoD activation system provide a safe, feasible, and effective strategy for autologous transplantation in Duchenne Muscular Dystrophy patients with gene recovery basal progenitor cells.
