Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

The overall goal of this experiment is to differentiate human pluripotent stem cells into cardiomyocytes with high efficiency and reproducibility. This protocol can be scaled up in a bioreactor or modified for differentiating cell lines, which are sensitive to single cell culture. This method can help answer key questions in cardiac disease modeling using human induced pluripotent stem cells.

The main advantages of this technique are that it is cost-effective and has high efficiency, reproducibility, and applicability for both human embryonic and induced pluripotent stem cells. Our demonstrator today is Hananeh Fonoudi, currently a PhD student at the Victor Chang Cardiac Research Institute, coming to us from the Royan Institute. Before coating, use a cleaning brush and distilled water to thoroughly remove any dust or culture residue from a 100 milliliter spinner flask with a glass pendulum.

Next, fill the flask with 70%ethanol. After 30 minutes, rinse the flask with distilled water, and fill it with five molar sodium hydroxide. The next morning, rinse the flash under running water for five minutes, and fill it with one molar hydrochloric acid.

After 15 minutes, rinse the flask under running water for another five minutes, followed by two rinses with double-distilled water. Place the flask in a biosafety cabinet to dry. Then, add 1.5 milliliters of siliconizing solution to the flask, and rotate the flask horizontally to cover all of the surfaces with silicon.

Heat the coated flask in a dry oven for one hour at 100 degrees Celsius. When the flask has cooled, rinse it three times for 15 minutes with deionized water. Then, autoclave sterilize the container at 121 degrees Celsius for 20 minutes.

After passaging undifferentiated spheroids, using the single cell passaging method at least three times, use a five milliliter pipette to transfer all of the undifferentiated spheroids into a 15 milliliter conical tube. Wash the wells with one milliliter of human embryonic stem cell medium to collect any remaining spheroids, and pool the wash with the harvested spheroids. Allow the spheroids to sediment for five minutes to form a loose pellet.

Then, aspirate the supernatant, taking care not remove any spheroids. Next, rinse the spheroids with one milliliter of PBS for five minutes, and incubate them in 5 milliliters of Trypsin EDTA at 37 degrees Celsius. After four to five minutes, stop the reaction with one milliliter of human embryonic stem cell medium, and use a P1000 pipette to dissociate the spheroids into a single cell suspension.

After counting by trypan blue exclusion, resuspend the cells at a 200, 000 cell per milliliter concentration, in conditioned medium supplemented with basic fibroblast growth factor and ROCK inhibitor, and transfer 100 milliliters of the cell suspension into the siliconized bioreactor flask. Place the flask on the agitator at 35 RPM. After 24 hours, increase the agitation to 40 RPM.

After 48 hours, stop the agitation for five to ten minutes until all of the reformed spheroids have settled to the bottom of the flask, and replace half of the medium with conditioned medium supplemented with basic fibroblast growth factor. Then, restart the agitator at 40 RPM for another 24 hours, replacing half of the medium by the same process every day for the next three days. On day five, stop the agitator for five to ten minutes.

Then, transfer all of the spheroids to a 50 milliliter tube in 50 milliliters of medium or less. Discard any of the remaining medium in the flask, taking care that no spheroids remain in the vessel. Then, carefully remove the supernatant from the settled spheroids, and wash cell clusters in 25 milliliters of PBS with calcium and magnesium.

After five to ten minutes, remove the wash, and resuspend the spheroids in 40 milliliters of RPMI-B27 medium supplemented with glycogen synthase kinase-3 inhibitor, ROCK inhibitor, and polyvinyl alcohol. Transfer all of the spheroids back to the bioreactor flask. Then, add fresh RPMI-B27 medium to a total volume of 100 milliliters, and restart the agitation at 40 RPM.

After 24 hours, follow the same process to change media from RPMI-B27 medium. After an additional 24 hours, follow the same process and change media with RPMI-B27 plus IWP2, purmorphamine, and SB43. Leave for 48 hours.

First beating is usually observed after seven days. For the fast differentiation of human pluripotent stem cell lines that have not been adapted to single cell culture, first treat to 70 to 80%confluent human pluripotent stem cell culture, grown on mouse embryonic fibroblast cells, with 5 milliliters of dissociation solution for 30 to 60 seconds. Dissociation solution should only be left until the MEFs round up and hPSC colony edges become clear.

If the cells are left too long, they will not differentiate correctly. At the appropriate time, quickly replace the dissociation solution with 75 milliliters of collagenase type IV, and incubate the cells for five to 15 minutes at 37 degrees Celsius, checking the culture under the microscope every few minutes. When whole colonies begin to detach, replace the collagenase with 1.5 milliliters of human embryonic stem cell medium, and use a P1000 pipette to gently detach whole cell colonies from the bottom of the plate.

Cells should be carefully detached so that the colonies do not break up into small pieces. Then, use a five milliliter pipette to transfer the cells into a 15 milliliter conical tube, and wash the wells with one milliliter of human embryonic stem cell medium, pooling any remaining cells in the 15 milliliter tube. After five minutes of sedimentation, carefully replace the supernatant with two milliliters of fresh human embryonic stem cell medium supplemented with basic fibroblast growth factor.

Using a new five milliliter pipette, transfer the cells into an ultra-low attachment plate, and incubate the colonies for six to 12 hours. At the end of the incubation, use a five milliliter pipette to carefully transfer the colony aggregates into a new 15 milliliter tube, and collect any remaining aggregates with an RPMI-B27 medium wash. When the colonies have settled, replace the supernatant with fresh RPMI-B27 medium supplemented with glycogen synthase kinase-3 inhibitor, and carefully transfer the colonies to laminin-coated plates for attachment at 37 degrees Celsius.

After 24 hours, change the medium with RPMI-B27 medium. After another 24 hours, change the medium with RPMI-B27 containing IWP2, purmorphamine, and SB43. First beating is usually observed after seven days.

To date, the static suspension differentiation protocol has been tested on five human embryonic stem cell and four human induced pluripotent stem cells lines resulting in more than 90%beating spheroids from each line, demonstrating the high reproducibility of this protocol among different human pluripotent stem cell lines. The differentiating spheroids exhibit a similar behavior in a stirred suspension bioreactor environment as in the static system, with approximately 100%of spheroids observed to beating at day 10. Indeed, beating spheroid sections collected at day 30 reveal the cytoplasmic and nuclear expression of cardiac troponin T and the cardiac transcription factor NKX2.5 respectively.

Further, when undifferentiated spheroids are replaced with undifferentiated cell aggregates, beating clusters appear seven days after differentiation. With 42 tested human induced pluripotent stem cell lines generating on average more than 60%cardiac troponin T positive cell populations by day 15. The beating clusters also demonstrate a similar cardiac troponin T cytoplasmic and NKX2.5 nuclear expression as observed for the stirred suspension bioreactor-generated spheroids.

While attempting this procedure, it is important to remember to use hPSCs which has been adapted to single cell culture for at least three passages prior to the differentiation in the bioreactor. This procedure can be combined with other methods for enriching cardiomyocyte to acquire a pure population of cells. This technique has paved the way for commercialization of hPSC-derived cardiomyocyte.

It has the potential for use in clinical, pharmaceutical, tissue engineering, and organ or organoid development applications. After watching this video, you should have a good understanding of how to efficiently differentiate human pluripotent stem cells into cardiomyocyte and to scale up the culture in bioreactor.