The overall goal of this differentiation protocol is to generate homogeneous populations of atrial cardiomyocytes in culture. So this method can help answer key questions in the study of cardiac development and cardiac diseases such as atrial arrhythmia. The main advantages of this technique are that it produces homogeneous cultures of atrial cardiomyocytes in vitro and that it can be done with minimal training.
Up to four days before setting up the mouse embryonic stem cell, or mESC cultures, code 10-centimeter tissue dishes with 5 milliliters of 0.2%gelatin solution each, in six well plates with 1 milliliter of gelatin per well in a sterilized tissue culture hood. On the day of the culture, remove the extra gelatin solution, and add 10 milliliters of medium to one of the gelatin-coated 10-centimeter dishes. Then, use cryotongs to gently swirl a vial of 1 times 10 to the 6 mESC's in a 37-degree Celcius water bath until only a small ice crystal remains in the center of the vial.
Dry the vial with a disposable, lint-free wipe and sterilize it with 70%ethanol. Then place the vial in the tissue culture hood and use a P1000 pipette to add the ESC's to the dish. Place the cells in a 37-degree Celcius humidified cell culture incubator with 5%carbon dioxide, and gently move the plate front to back and side to side to evenly distribute the cells.
At the end of the incubation, use a brightfield microscope to confirm the cell attachment. Some debris and dead cells will be observed. Replace the medium with 10 milliliters of fresh mESC medium, and return the cells to the incubator.
When the culture is 60 to 70%confluent, rinse the plate with 5 milliliters of sterile DPBS without magnesium and calcium, and incubate the cells in 2 milliliters of trypsin EDTA at 37 degrees Celcius for five minutes. While the cells are incubating, aspirate the excess gelatin solution from one new 10-centimeter dish per passaged culture, and add 10 milliliters of medium to each plate. Then collect the detached cells by centrifugation in a 15 milliliter tube, and re-suspend the pellet in 10 milliliters of mESC medium.
Depending on the desired split ratio, add 0.5 to 1 milliliter of cell suspension to each 10 centimeter dish and return the cells to the incubator, gently distributing the cells in each dish as just demonstrated. When the passaged cells are 70%confluent, detach them with trypsin EDTA as just demonstrated, then resuspend the single-cell suspension in 5 milliliters of embryoid body or EB medium. Count the mESC's and then dilute them in the appropriate volume of EB medium to a final concentration of 2.5 times 10 to the 3rd cells per milliliter.
Next, add 2 milliliters of PBS to the bottom of one sterile 10-centimeter bacterial petri dish per hanging drop culture, and transfer the EB suspension into a sterile solution basin. Now, remove the lid from one of the prepared dishes and use a multichannel pipette, equipped with six tips, to carefully deposit 60 20-microliter drops of EB suspension onto the inner surface. Gently place the lid back onto the dish, taking care not to dislodge the hanging drops and place the dishes into the cell culture incubator for two days.
To wash down the EB'ss, carefully remove the lid from the hanging drop culture, and tilt it at a 45-degree angle. Then, using a 5-milliliter serological pipette containing room-temperature EB medium, gently wash the EB's into a pool at the bottom of the lid. Carefully inspect the lid for EB's that are stuck to the surface, then transfer the EB's into an uncoated, 6-centimeter petri dish.
Return the EB's to the tissue culture incubator for another two days, checking the plates daily for EB aggregation and attachment. Before plating the EB's, replace the excess gelatin coating solution in each well of the prepared six-well plate, with 2 milliliters of freshly prepared, room temperature Grem2 treatment medium. Then use a P1000 pipette set to 100 microliters to transfer 30 EB's to each well, then return the plate to the incubator overnight, evenly dispersing the cells with gentle movement as demonstrated.
After the EB's have attached to the bottom of the wells, replace the Grem2 medium every two days until day 10. Then, feed the cells with fresh EB medium every two days to maintain a healthy cell culture. Prior to their hanging drop culture, the pluripotent stem cells should be compact and free of spontaneous differentiation.
If spontaneous differentiation is observed, the cells should not be used in the hanging drop cultures. Quality EB is generally formed best within evenly spaced, well rounded hanging drops. By differentiation day two, the EB's should be visible to the naked eye, as spherical arrangements of differentiating stem cells at the tips of the hanging drops.
At day four, well-formed EB's should be free floating and homogeneous in size and shape. Once plated, the EB's will flatten as the cells migrate out from the EB onto the surface of the tissue culture plate, as observed here on differentiation days eight and 10. In non-treated control wells, small, slowly contracting patches are observed as early as day six and as late as day nine.
While in Grem2-treated wells, large patches with a relatively fast contracting rate are common. RT-QPCR analysis of gene expression in the differentiated cells typically reveals high levels of cardiac structural and regulatory genes in Grem2-treated cultures. If the alpha-MHC DsRed nuke cell line is used, the increased yield of cardiomyocytes is observed as a higher number of DsRed-labeled nuclei in the Grem2-treated wells.
Grem2 treatment also biases the differentiation toward an atrial-like phenotype, as confirmed by RT-QPCR and patch clamp measurements of cellular action potential duration. Once mastered, the differentiating stem cells can be treated with Grem2 in as little as 60 minutes and beading cells are usually observed as early as day seven. While attempting this procedure, it's important to remember to consistently add Grem2 at day four after the peak expression of gastrulation markers such as brachyury.
Following this procedure, other methods like electrophysiology and QPCR can be performed to understand the functional and molecular characteristics of the cells that have been generated. After watching this video, you should have a good understanding of how to generate atrial cardiomyocytes from mouse ESC's, using Grem2.
