Reproducibility is major concern when inducing stem cell-derived cardiomyocytes. Using these methods, high purity and high quality cardiomyocytes can be generated from iPS for their use in functional studies. The main advantage of this technique is that is a simple, reliable, cost-effective, and extremely powerful method for obtaining purified, high quality preparations of iPSC-derived cardiomyocytes.
For cell passaging, rinse the cells with one milliliter of PBS without calcium and magnesium for seven to 10 minutes before replacing the PBS with one milliliter of passaging medium. Using a cell lifter, gently scrape the cells from the bottom of the wells, and use sterile two milliliter glass pipette to mechanically dissociate the detached cells. When the cells have lifted into small clusters as visible under the microscope add passaging medium to split the cells at a one to six ratio.
Next, aspirate the human embryonic stem cell qualified matrix from the six well plate. Then add one milliliter of passaging medium and one milliliter of dissociated cells to each well, and let the cells grow until they reach 70 to 80%confluency before starting a cardiac differentiation. At least 30 minutes before dissociating the iPSC-CMs, wipe individual glass cover slips with 70%ethanol, and place the cover slips into individual wells of a sterile six well plate.
When the cover slips have dried, add a 250 to 300 microliters of human embryonic stem cell qualified matrix solution directly onto the center of each cover slip, and place the six well plate in the tissue culture hood. To dissociate the metabolically selected iPSC-CM add one milliliter of sterile 0.25%trypsin with EDTA to each well for a five minute incubation at 35 degrees celsius, and use a 1000 microliter pipette to mechanically dissociate the cells until a single cell suspension has been achieved. Pull the cells in sterile 15 milliliter conical tube, and wash each well with two milliliters of RPMI 20 medium per well.
Then add the washes to the tube and pellet the cells by centrifugation. Resuspend the cells in a sufficient volume of medium to achieve a three times 10 to the fifth cells per 250 microliters of cells concentration. Use a 1000 milliliter pipette tip to dissociate the cells until the solution appears to be homogenous.
Then aspirate 250 microliters of cells into a 1000 microliter pipette tip, and slowly dispense the entire volume of solution onto one human embryonic stem cell qualified matrix coded cover slip. When all of the cells have been seeded, carefully place the plate into the cell culture incubator overnight. Gently adding two milliliters of D7 medium to each well the next morning.
When the selected iPSC-CMs reach at least three months old treat the cells with two microliters of Fura-2 AM for 10 minutes at 37 degrees Celsius, and power on the calcium analysis system. Initiate the ArcLamp and connect the tubes from the pump to the appropriate inlet and outlets and the electronic wire from the stimulator to the chamber. Place the chamber onto the system.
Fill the ProFusion tube that runs through the fluidic inlined heater with 37 degrees Celsius warmed Tyrode's solution, and adjust the camera and framing aperture dimensions to minimize the background area. Fasten the glass cover slip seeded with iPSC-CMs into the chamber, and gently add 500 microliters of Tyrode's solution directly on top of the cover slip. Begin profusing the chamber with Tyrode's solution at a 1.5 milliliter per minute rate, and use an electric stimulator to pace the iPC-CMs with 1 Hertz field stimulation at 10 volts every four milliseconds for three to five minutes.
When all of the dye has been washed out of the chamber adjust the viewing window to the upper left region of the cover slip, and begin the recording. After collecting a consistent stream of five to 10 peaks click pause to temporarily stop the recording and move the microscope stage to an adjacent area for the opposite end of the cover slip before resuming the recording. After scanning the calcium transience across the entire cover slip in the same manner over a period of 10 minutes analyze the data with an appropriate florescence trace analysis software according to the manufacturer's instructions.
This protocol generates highly pure cardiomyocytes that acquire a ventricular, adult-like phenotype in culture over time. As assessed by immuno fluorescent staining for the atrial and ventricular MLC2 isoforms, the majority of cells generated by this protocol are atrial MLC2 isoform positive at day 30 after the induction of cardiac differentiation. While ventricular MLC2 isoforms are expressed in much numbers at the same time point.
As the time in culture increases, a complete switch to the MLC2 isoforms is observed. These data were also confirmed by flow cytometric quantification of the percentage of atrial and ventricular MLC2 marker expressing cells. The calcium amplitude is significantly increased in the iPSC-CMs at day 90, similar to that observed for adult rat CMs.
It's important to note that contaminating non-cardiac cells may influence the functional maturation of the human iPSC-CMs during differentiation. Further, when record calcium transients in iPSC-CMs it's necessary to allow the cells to stabilize at 37 degrees Celsius under constant stimulation for at least 200 seconds, and to restrict the recordings to a specific time window to ensure reproducible results. Remember to confirm that the iPSCs exhibit a homogeneous morphology and to allow the cells to grow to 70 to 80%confluency before initiating a cardiac differentiation.
Pure quality in immature iPS-derived cardiomyocytes might show altered functional characteristics, which do not reflect the real phenotype, but could be misinterpreted as the diseased phenotype. Therefore, obtaining pure and high quality cardiomyocytes is important to provide consistent and reproducible results.
