Generating Single Positive CD8 T Cells from Induced Pluripotent Stem Cells

Prepare gelatinized OP9-delta-1 dishes one week prior to co-culture with human IPSCs. Add 4 milliliters of 0.1% gelatin to three new cell culture dishes, and incubate them for 30 minutes at 37 degrees Celsius. After the incubation, aspirate the gelatin and add 8 milliliters of OP9 media to each dish. Passage one confluent dish of OP9-delta-1 cells to three gelatin-coated dishes.

After four days, add 10 milliliters of OP9 media to each dish of cells for a total of 20 milliliters of media per dish. Begin human IPSC co-culture on OP9-delta-1 confluence dishes seven to eight days after passaging the cells. Aspirate the media from a confluent dish of human IPSCs, and add 10 milliliters of OP9 media.

Cut and detach cell colonies using a disposable cell passaging tool, and transfer 350 to 600 clumps of cut colonies onto a pregelatinized OP9-delta-1 dish with 10 milliliters of fresh OP9 media. Rock the culture dish side-to-side and front-to-back to ensure even distribution of colonies. The next day, aspirate the spent media, and replace it with 20 milliliters of fresh OP9 media.

The human IPSC clumps co-cultured on OP9-delta-1 for one day should appear as small, round monolayer colonies. On day 5, aspirate 10 milliliters of spent media and add 10 milliliters of fresh OP9 media. The colonies will begin to differentiate into primitive mesoderm, which is characterized by a multilayered dark center.

Repeat this process on day 9, at which point the multilayer center structures will evolve into dome-like shapes. Harvest the hematopoietic progenitor cells on day 13, at which point the structures are composed of a dark central organoid surrounded by a network of dome-like areas. Aspirate the media, and wash the cells once with 5 milliliters of 1x phenol red-free Hank's balanced salt solution with calcium and magnesium or HBSS.

After the wash, add 250 microliters of 5,000 units per milliliter collagenase-4 to 10 milliliters of HBSS. Add the mixture to the cells and incubate them at 37 degrees Celsius for 45 minutes. Aspirate the HBSS-collagenase mixture, and wash the cells once with 5 milliliters of PBS. After washing with PBS, add 5 milliliters of 0.25% trypsin-EDTA, and incubate the cells at 37 degrees Celsius for 20 minutes.

Then, add 4 milliliters of OP9 media, and associate the cell layer by pipetting to make a single-cell suspension. Transfer the cell suspension to a 50-milliliter conical tube through a 100-micrometer strainer, and centrifuge the tube at 300 times g and 4 degrees Celsius for 5 minutes. Aspirate the supernatant, and resuspend the cells in 10 milliliters of OP9 media.

Plate the cell suspension on a new gelatinized 10-centimeter cell culture dish, and incubate them at 37 degrees Celsius for 45 minutes. Then, collect any nonadherent cells by gentle pipetting. Transfer the cell suspension to a 50-milliliter conical tube through a strainer, and centrifuge as previously described. Then, aspirate the supernatant, and resuspend the cells in 10 milliliters of differentiation media.

Plate the cell suspension onto a new OP9-delta-1 confluent dish. Passage the cells on day 16 according to the manuscript directions, and continue passaging the nonadherent cells every five to seven days thereafter. After 35 days of differentiation, enrich the CD4-positive cell population by CD4 magnetic bead isolation according to the manufacturer's protocol.

Then, resuspend the cells in OP9 media and plate 1 milliliter of cell suspension into each well of a tissue culture flat-bottom 24-well plate of confluent OP9-delta-1. Stimulate cells by adding 100 international units of human IL-2, 5 nanograms of human IL-7, 500 nanograms of anti-human CD3 antibody, and 2 micrograms of anti-human CD28 antibody to each well. After four to seven days, cells can be collected for further analysis.