We present a method to generate human induced pluripotent stem cell-derived tumor antigen-specific CD8 alpha beta single-positive T cells by using OP9-Delta-1 co-culture system. This method is a powerful tool for in vitro T cell generation and will facilitate the development of in vitro-derived T cells for use in regenerative medicine and cell-based therapies. It can also be adapted for the generation of other lymphocytes, like NK cells.
When performing this technique, it is important to pre-evaluate the lot of FBS and passage OP9-Delta-1 cells consistently when cell-to-cell cytoplasmic contact begin to prevent cell differentiation and senescence. Demonstrating the procedure will be Dr.Meghan Good, a surgical oncology fellow from my team. Start by checking human iPSC colonies in a stereo-microscope, and remove any areas of differentiation from the culture with the plastic edge of a 200-microliter pipette tip.
When the colonies reach 0.8 to 1.2 millimeters in diameter, passage the human iPSCs. Aspirate the spent media, and add 10 milliliters of human iPSC media supplemented with 10-micromolar ROCK inhibitor. Roll a disposable cell passaging tool across the entire dish in one direction, making sure to maintain uniform pressure and that the entire roller blade touches the culture dish.
Rotate the culture dish 90 degrees, and repeat the rolling. Visually confirm the proper cutting of the colonies with a microscope. They should appear checkered.
Then, detach the cut colonies by gentle mechanical flushing with a 200-microliter pipette. Visually estimate the number of colony clumps, and transfer between 350 and 600 clumps to a new 10-centimeter dish of mouse embryonic fibroblasts with 10 milliliters of fresh human iPSC media supplemented with ROCK inhibitor. Incubate the dish overnight at 37 degrees Celsius.
The next day, aspirate the spent media, and add 10 milliliters of fresh human iPSC media. Change the media every one or two days, depending on the cell growth rate. Culture the OP9-Delta-1 cells in OP9 media at 37 degrees Celsius.
When they reach confluency, aspirate the media, and wash the cells once with five milliliters of 1x magnesium, calcium, and phenol red-free PBS. Aspirate the PBS, add two milliliters of 05%trypsin-EDTA, and incubate the cells for five minutes at 37 degrees Celsius. Then, add four milliliters of OP9 media, and mechanically dissociate the cell layer by pipetting.
Transfer the cell suspension to a 50-milliliter conical tube through a 100-micrometer cell strainer, and centrifuge at 300 times g at four degrees Celsius for five minutes. Aspirate the supernatant, and resuspend the cells in 12 milliliters of OP9 media. Add eight milliliters of OP9 media to each of six new cell culture dishes, and plate two milliliters of the OP9-delta-1 cell suspension onto each dish.
Rock the dish side to side and front to back to ensure even distribution of the cells. Incubate the cells at 37 degrees Celsius, and repeat passage when the cells reach confluency. Prepare gelatinized OP9-Delta-1 dishes one week prior to co-culture with human iPSCs.
Add four 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 eight 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 confluent 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 pre-gelatinized 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 five, 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 nine, 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 five milliliters of 1x phenol red-free Hanks'balanced salt solution with calcium and magnesium, or HBSS.
After the wash, add 250 microliters of 5, 000-units-per-milliliter collagenase IV 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 five milliliters of PBS.
After washing with PBS, add five milliliters of 0.25%trypsin-EDTA, and incubate the cells at 37 degrees Celsius for 20 minutes. Then, add four milliliters of OP9 media, and dissociate 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 four degrees Celsius for five 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 non-adherent 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 non-adherent 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 one 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, five nanograms of human IL-7, 500 nanograms of anti-human CD3 antibody, and two micrograms of anti-human CD28 antibody to each well. After four to seven days, cells can be collected for further analysis. After culture on non-gelatinized OP9-Delta-1 in the presence of human stem cell factor, human FLT3-ligand, and human interleukin-7, hematopoietic progenitors differentiated into CD3, CD7, CD4, and CD8 double-positive T lineage cells, the majority of which expressed T cell receptors specific to the MART1 epitope.
When the CD4, CD8 double-positive T cells were stimulated with anti-human CD3 and CD28 antibodies in the presence of human interleukin-7 and 2, the number of CD3-positive CD8 alpha beta single-positive cells increased and remained specific for the MART1 epitope, confirming the preservation of their inherited antigen specificity. An ELISpot assay revealed that when cultured in the presence of MART1 peptide, human iPSC-derived CD8 alpha beta single-positive T cells secrete higher amounts of interferon gamma compared with both bulk and CD8 alpha alpha single-positive T cells. No interferon gamma expression was detected for T cells and antigen-presenting cells alone, demonstrating that human T-iPSC-derived T cells are antigen-specific and functional.
An important step in this method is CD4 magnetic bead enrichment to eliminate CD4-negative, CD8-negative double-negative cells, which can cause direct killing of double-positive cells upon stimulation. This technique is applicable for use with several sources of human cells, including hematopoietic stem cells and embryonic stem cells. iPSC-derived immature T cells generated by this method could be further improved for the generation of mature human tumor antigen-specific naive-like T cells for future therapeutic use.
After watching this video, the viewer should understand how to generate human iPSC-derived CD8 alpha beta single-positive T cells using the OP9-Delta-1 co-culture system.
