For neonatal diseases such as cardiomyopathy, regenerative therapy using induced pluripotent stem cell-derived cells has massive potential. However, in vitro accelerated cell growth leads to DNA damage by accumulated metabolites such as reactive oxygen species. Transplanting these DNA-damaged cells would result in poor engraftment and regeneration of the organ.
Before transplantation, the quality of the cells should be assessed. Here we provide two step-by-step protocols to assess the DNA damage in stem cells. Demonstrating the procedures will be Jessica Miller, a researcher, Nikhil Mardhekar, a researcher, and Vasanthi Rajasekaran, a lab technician.
To begin, place 500 milligrams of low-melting agarose in 100 milliliters of DNA-RNA free water. Heat the bottle in a microwave oven until the agarose dissolves. Then, place the bottle in a 37-degree-Celsius water bath until needed.
To prepare pre-coated comet slides, place a few drops of 0.5%agarose on a glass slide. Immediately spread the agarose to form a thin layer of agarose coating using a coverslip. Working under low-light conditions to avoid ultraviolet light-induced cell damage, mix 10 microliters of cell suspension and 90 microliters of agarose solution in a tube.
Place the tube in a 37-degree-Celsius water bath to prevent the agarose from solidifying. Mix the solution without introducing air bubbles, and pipette 70 microliters per spot onto the pre-coated comet slide. Using a pipette tip, spread the cell agarose mixture to form a thin layer.
Place the slide at four degrees Celsius for 15 minutes. In a container, completely submerge the slide in cold lysis buffer. Place the container in the dark at four degrees Celsius for one hour.
Replace the lysis buffer with cold alkaline solution. Make sure the slides are completely submerged. After leaving the container in the dark at four degrees Celsius for another 30 minutes, place the slide in a horizontal electrophoresis tank.
Fill the tank with cold alkaline electrophoresis buffer until the slides are completely submerged. Apply voltage at one volt per centimeter for 15 to 30 minutes. In a container, completely submerge the slide in cold deionized water.
After two minutes, remove the deionized water and add fresh deionized water, and then repeat this step a second time. Next, replace the deionized water with cold 70%ethanol. After five minutes, gently remove the slide without tilting it and let it air dry.
Once dried, add 100 microliters of diluted DNA dye to each spot, and wait 15 minutes at room temperature. Using a FITC filter in an epifluorescence microscope, take images of 50 to 100 comets in total per sample. Culture iPS-derived cardiomyocytes as described in the text protocol.
To see the iPS-derived cardiomyocytes in chamber slides, first discard the culture medium from the wells, and wash the cells three times with PBS. Add one milliliter of 0.25%Trypsin per well, and incubate at 37 degrees Celsius for five minutes. Check the plate under a microscope for cell detachment.
Then add one milliliter of CMM to stop the Trypsin. Place the cell suspension in a 15-milliliter tube and centrifuge for five minutes at four degrees Celsius and 200 times g. Discard the medium, add one milliliter of CMM, and resuspend the cells.
Count the cells and adjust the cell counts to obtain two million cells in 1.6 milliliters of CMM. Now seed 200 microliters of cell suspension per well of the eight-well chamber slide. Tap the slide gently to spread the cells around the well.
Incubate the cells at 37 degrees Celsius. For the iPS-derived cardiomyocyte doxorubicin treatment, discard the culture medium from the iPS-derived cardiomyocyte wells, and wash the cells with PBS. Treat the cells with one-micromolar doxorubicin for four hours at 37 degrees Celsius.
Aspirate the medium, and wash the cells with PBS. To perform immunolabeling, wash the cells three times with PBS and add 200 microliters of freshly-prepared 4%paraformaldehyde to each well. Fix the cells for 20 minutes in the dark at room temperature.
After washing the cells with PBS, add 200 microliters of blocking buffer per well, and block for 30 minutes in a humidified chamber at room temperature. Remove the blocking buffer and add 200 microliters of primary antibody solution. After incubating for 45 minutes in a dark humidified chamber at room temperature, wash the wells three times with PBS.
Remove the PBS and add 200 microliters of secondary antibody mix. Incubate for 45 minutes in a dark humidified chamber at room temperature. Then wash the wells three times with PBS.
Wash with deionized water before mounting the slides with antifade. Place a cover glass, and store the slides in the dark at four degrees Celsius. Using a confocal microscope, take three to five images of random fields per sample and proceed to image analysis.
To count the DNA damage response foci, download and install CellProfiler. Select file, import, pipeline from the file, and choose the pipeline file named puncti gamma-H2AX. Drag and drop the folder containing the acquired images of gamma-H2AX foci to the file list window in the input modules images section for analysis.
Then follow the instructions as shown in the text protocol. To set the parameters for the images, drag the desired image for analysis onto the file list. Under input modules, select images.
Also in input modules, select metadata, and then select names and types. For groups, select no. Working in analysis modules, select color to gray.
Then select smooth, select identify primary objects, and again select smooth. Next select enhance or suppress features, and then select identify primary objects. Still working in analysis modules, select relate objects, then classify objects, and finally select export to spreadsheet.
Click analyze images at the bottom left panel to perform image analysis. At the successful completion of the analysis, several images are generated. Note the CSV file that is generated and saved in the appropriate location on the computer.
This file contains the quantitative data for further analysis. In the spreadsheet called my experiment image, column B will have the number of nuclei that have up to five puncti, and column D will have the number of nuclei that have more than five puncti. Add columns B and D to get the total number of nuclei.
Repeat these steps for all images, changing the my experiment filename before every analysis. Representative micrographs of comets from doxo-treated and non-treated iPS cells are shown here. A basal amount of DNA damage was found in iPS cells, expressed as a fraction of DNA damage and tail moment.
However, the doxo treatment increased the DNA damage in iPS cells as expected, indicating that the comet assay can be used to assess DNA integrity in pluripotent stem cells. Representative micrographs of gamma-H2AX immunolabeling are shown here, at lower magnification and at higher magnification. In the control iPS-derived cardiomyocytes, more than 90%of the cells had less than five DDR foci per nuclei.
And a total of less than 10%of the cells had more than six DDR foci per nuclei. For iPS-derived cardiomyocytes cultured for six months, less than 90%of the cells had up to five DDR foci per nuclei, and a total of more than 13%of the cells had more than six DDR foci per nuclei. Whereas in the doxo-treated iPS-derived cardiomyocytes, less than 80%of the cells had up to five DDR foci per nuclei.
And a total of about 24%of the cells had more than six DDR foci per nuclei. This data indicates that prolonged iPS-derived cardiomyocyte cell culture and doxo treatment induced significant DNA damage, making them unsuitable for cell transplantation. It is very important to avoid variables while mixing the cell suspension, as this might affect the electrophoresis.
Following this procedure, you can perform the comet assays and the immunolabeling procedures with cells of various types. These techniques will allow for cells of various kinds to be assessed for DNA damage before being used in cell transplantation studies.
