This method can help answer key questions in the cardiac re-programming field, such as identifying what pathways promote or repair cardio-myogenesis. The main advantage of this technique, is that we can generate functional induced cardiomyocytes, with higher efficiency than previously published methods. These chemic heart disease results in loss of cardiomyocytes and impaired heart function.
Therefore, this technique may aid in high repair, by converting fibroblasts into functional induced cardiomyocytes. To begin the production of retrovirus, grow Platinum E, or PE cells in PE medium. Supplement it with selection markers in a 10CM dish, in a bio-safety level two cabinet.
Every two days passage the cells, at one to four, or one to fifth dilusion. One day before the planned transfection, seed PE cells in 10ML of growth medium, in a 10CM culture dish, and then gently rock the dishes back and forth. Carefully place the dishes in an incubator, to allow even distribution of the cells, and incubate for 24 hours.
On the day of transfection, add the GHMT2M cocktail, to a 1.5ML tube. Next add 360 microliters of reduced serum media, to a separate 1.5ML tube. Subsequently, add 36 microliters of transfection reagent to the reduced serum media, and gently flick the tube to mix.
Incubate a room temperature for five minutes. The transfection reagent used in this step binds to plastic. To avoid transfection efficiency, take care to pipet the transfection reagent, directly into the reduced serum media, and avoid touching the pipet tip to the tube wall.
Then add the reduced serum media transfection reagent mixture to the tube, containing the GHMT2M DNA cocktail. Gently flick the tube to mix, and incubate at room temperature for 15 minutes. Finally add the transfection reaction mixture drop wise to the PE cells.
Gently swirl the dish for 10 to 15 seconds. Incubate the transfected cells at 37 degrees celsius, for 16 to 20 hours. Now coat 60MM dishes with collagen, and incubate at 37 degrees celsius, for a minimum of two hours.
Then dispense freshly thawed mouse embryonic fibroblasts, or MEFs into the collagen coated dishes. And add growth medium into a final volume, of 4ML per 60MM dish. Gently rock the dishes, and place them in the incubator.
When plating MEFs it's important that cells are plated evenly. Avoid swirling the dishes, this can cause uneven plating. Instead, gently rock the dish back and forth, and carefully place it in the incubator.
24 hours after PE cell transfection, use a 30ML syringe to collect the retroviral medium generated by the cells. Pass the first collection of retroviral medium through a 45 micrometer pour size filter, into a 50ML conical tube. Then add 10ML of fresh growth medium onto the wall of the dish, taking care not to displace the PE cells.
Incubate the cells at 37 degrees celsius for another 24 hours. Next add 7.2 microliters of 10MG per milliliter hexidine methrine bromide transduction reagent, to the 50ML conical tube containing viral supernatant. Now aspirate the medium from the MEFs, and add 4ML of the freshly harvested retroviral medium to the dish.
Incubate cells at 37 degrees celsius for 24 hours. 48 hours post transfection of the PE cells, collect the retroviral medium, and perform the transduction exactly the same way, as for the first collection. Discard the PE cells after the second collection of retroviral medium and add 10%bleach, to all materials in contact with retroviral medium.
After the incubation aspirate the retroviral medium from the MEFs, and add 4ML of ICM medium, containing 05 micromolar, TGF Beta Type 1 receptor inhibitor. Every two days change the ICM medium, until further use. To begin the immunostaining, aspirate the ICM medium from day 14 reprogrammed MEFs.
Then wash the cells once with 1ML of 1X ice cold PBS, per well of the 12 well plate. Next, aspirate the PBS, add 500 microliters of 2%paraformaldehyde to fix the cells, and incubate for 10 minutes at room temperature. After aspirating the paraformaldehyde, wash the cells three times with 1ML of PBS.
After aspirating the PBS, add 500 microliters of. 02%Triton X 100, and incubate for 15 minutes at room temperature. Following aspiration of Triton X 100, block the cells with 500 microliters of 10%horse serum, for 30 minutes at room temperature.
Next, aspirate the horse serum, and add 500 microliters of diluted primary antibody. Incubate for one hour at room temperature. After aspirating the primary antibody, wash three times with 1ML of PBS.
After the last wash of PBS, add 500 microliters of diluted secondary antibody. Wrap the plate in foil and incubate in the dark, for one hour at room temperature. Finally, aspirate the secondary antibody, and wash three times with 1ML of PBS.
Wrap the plate in foil, and store at four degrees celsius until imaging is done. This method shows high efficiency conversion of MEFs into cardiomyocytes. A representative flow cytometry analysis, add day nine post transduction, shows activated cardiac Troponin T expression, in approximately 70%of the cells.
Similarly, cardiac Alpha-actinin expression, is observed in approximately 55%of the cells, when compared to the GFP transduction control. Additionally, immuno-staining for cardiac markers, performed two weeks post transduction, shows the majority of the cells express Troponin I, Alpha-actinin, and Troponin T.The gap junction protein Cx43, was also detected along the periphery of the ICMs. Furthermore, co-labeling of ICMs with Alpha-actinin, or Troponin T and Cx43, reveal formations of gap junctions between neighboring cells.
GHMT2M transduction, and treatment with A83O1, reprograms a fraction of MEFs, into beating cells by day eight. On day 11, approximately 10000 beating cells per square centimeter are observed. Spontaneous calcium transients, are detected in beating cardiomyocytes.
Addition of beta adrenergic agonist isoproterenol, significantly increases the frequency of spontaneous calcium transients. In contrast, sedition of L-type calcium channel blocker nifedipine, significantly decreases the frequency of calcium transients. After watching this video, one should have a good understanding of how to reprogram fibroblasts, into functional, induced cardiomyocytes with high efficiency.
Following this protocol, other methods, including RNA sequencing and chip sequencing, can be used to answer additional questions, such as global gene expression and chromatin status, are altered during cardiac reprogramming. Don't forget that working with retrovirus can be hazardous. Precautions, such as working in a bio-safety level two cabinet, wearing personal protective equipment, and proper disposal of all retroviral waste, should always be implemented while performing this protocol.
