It is difficult to assess sarcomere function in the pluripotent stem cell-derived cardiomyocytes due to their underdeveloped and disorganized sarcomere. Using fluorescent-tagged sarcomere proteins, we can now access sarcomere shortening. We can visualize sarcomeres and assess their function in vitro using stem cell-derived cardiomyocytes and fluorescent-tagged sarcomere proteins.
Using AAV-based transduction, we can expand the method to any patient-derived induced pluripotent stem cells. This method can be expanded to the development of cardiomyopathy therapy, as it can be used to directly assess disease-related sarcomere dysfunction in vitro. This method is useful in the cardiology field, including pediatric cardiology study.
However, it can also be applied to the study of skeletal muscle dysfunction, such as myopathy. On day minus four of differentiation induction, coat a six-well plate with 0.5 micrograms per square centimeter of laminin-511 E8 diluted in PBS for a 30-minute incubation at 37 degrees Celsius and 5%carbon dioxide. Nearing the end of the incubation, treat the human-induced pluripotent stem cells with recombinant trypsin for three minutes at 37 degrees Celsius.
When the cells have detached, harvest cells to medium supplemented with 10%FBS for counting, and resuspend the cells at a 1.25 times 10 to the fifth cells per two milliliters of AK02N medium supplemented with laminin-511 E8 and ROCK inhibitor after centrifugation. Next, aspirate the coating solution from each well of the six-well plate, and seed two milliliters of cells into each well. Return the plate to the cell culture incubator.
After four days, the cells reach to 80%confluency. Replace the supernatant in each well with two milliliters of RPMI plus B27 without insulin medium supplemented with GSK-3 inhibitor per well to induce the differentiation. After two days of differentiation, gently replace the medium with two milliliters of RPMI plus B27 without insulin medium supplemented with porcupine inhibitor per well.
The cells should have reached 100%confluency. On day four of differentiation, gently replace the supernatants with two milliliters of RPMI plus B27 without insulin per well. The cells should remain at a high density, and some may have begun to float.
On day seven and nine of differentiation, replace the supernatant in each well with two milliliters of RPMI plus B27 with insulin supplemented with puromycin for selective pluripotent stem cell-derived cardiomyocyte culture. At this point, beating cells should be observed within the cultures. To set up a patterned pluripotent stem cell-derived cardiomyocyte culture, first use mending tape to remove any dust from the surface of a custom-made PDMS stamp, and briefly submerge the stamp in 70%ethanol.
Use an air duster to remove the ethanol from the stamp surface, and treat the surface with five to 10 microliters of 0.5%MPC polymer and ethanol. When the polymer has completely dried, place the stamp onto a polymer coverslip in a 35-millimeter imaging dish, and place a weight onto the stamp. After 10 minutes, use a light microscope to confirm that the pattern has been transferred onto the coverslip, and wash the dish and coverslip two times with PBS.
After the second wash, coat the coverslip with 0.5 to one microgram per square centimeter of laminin-511 E8 diluted in 0.1%gelatin for a two-to four-hour incubation at room temperature. On day 10 of differentiation, wash the cells two times with PBS, followed by treatment with one milliliter of recombinant trypsin per well for three to five minutes at 37 degrees Celsius. After counting, resuspend the cells at a 2.5 to five times 10 to the fifth cells per milliliter of RPMI plus B27 with insulin supplemented with puromycin concentration, and seed one milliliter of cells onto the plate for an overnight incubation at 37 degrees Celsius.
The next morning, replace the supernatant with fresh RPMI plus B27 with insulin supplemented with puromycin, and return the cells to the cell culture incubator, refreshing the medium two to three times per week until days 21 to 28 for imaging. For time-lapse imaging of the sarcomere shortening in the PCCSM cultures, apply oil to the 100 times objective lens of a fluorescent confocal microscope, and select live imaging conditions in the associated software. To obtain good representative data, select the highest frame rate, set the shutter to open, and apply a four-by-four binning in a crop of the acquisition area to achieve the shortest intervals between images during the time-lapse imaging.
If the beating rate of the cells is low, evoke the cells by electrical field stimulation, and start the time-lapse recording, taking care that the imaged field remains in focus during the imaging. At the end of the imaging period, save the time-lapse images into an appropriate folder. To analyze the time-lapse images, open a series of time-lapse images into ImageJ, and adjust the brightness and contrast of the images until the sarcomere pattern can be clearly observed.
In the More Tools menu, select SarcOptiM, and press Control Shift P and the one micron button on the toolbar to use the instructions to calibrate the program. Then draw a line across the region of the sarcomere to be used to measure the sarcomere shortening, and click Single Cell AVI to begin the sarcomere shortening analysis. Time-lapse imaging of sarcomere-labeled, patterned pluripotent stem cell-derived cardiomyocytes can be used to measure sarcomere shortening at different time points.
To overcome the sarcomere disorganization typically observed in patterned pluripotent stem cell-derived cardiomyocyte cultures, specific PDMS stamps can be used to culture patterned pluripotent stem cell-derived cardiomyocytes in a stripe pattern, which promotes an elongated cell shape and a more organized sarcomere pattern compared to cells cultured in a non-pattern area. Patterned cultures also promote a better cell contraction and provide a smooth sarcomere length profile compared to non-pattern cultured cells. AAV-transduced pattern pluripotent stem cell-derived cardiomyocytes express sarcomeric GFP signals along the patterned pluripotent stem cell-derived cardiomyocytes as early as three days post-transduction and can also be used to measure sarcomere contraction profiles.
Further, the addition of blasticidin-resistant gene during transduction can also be used to facilitate patterned pluripotent stem cell-derived cardiomyocyte selection to a purity of over 90%To facilitate an easier and higher quality image analysis, it is essential to identify an area with sarcomeres that move linearly and to obtain images at the highest frame rate. Using this method, we can assess the role of maturation in sarcoma proteins to study sarcomere dysfunction in lab cardiomyocyte derived from disease-specific, patient-derived iPS cells.
