Generating Single Positive CD8 T Cells from Induced Pluripotent Stem Cells

1. Culturing Human iPSCs (hiPSCs) on Mouse Embryonic Fibroblasts (MEF)

NOTE: Alternative methods for culturing hiPSCs can also be used, including but not limited to seeding onto a 6-well plate pre-coated with gelatin, a gelatinous protein mixture, recombinant laminin 511, or any other extracellular matrix used in hiPSC expansion, and cultured using defined media specially formulated for human pluripotent stem cell culture.

1. Culturing MEF
   1. Coat a 10 cm cell culture Petri dish with 4 mL of 0.1% gelatin and incubate for 30 min at 37 °C.
   2. Thaw a vial of 4 x 10⁶ irradiated MEF quickly into 10 mL of 37 °C MEF media (Dulbecco's Modified Eagle Medium [DMEM] + 10% fetal bovine serum (FBS) + 1x penicillin-streptomycin + 1x L-glutamine supplement). Centrifuge at 300 x g for 5 min at 4 °C. Aspirate the supernatant and resuspend the cell pellet in 9 mL of MEF media.
   3. Remove the gelatin-coated dish from the incubator. Aspirate gelatin and add 7 mL of MEF media. Plate 3 mL of MEF suspension (from step 1.1.2) onto the gelatin-coated dish. Rock the dish side-to-side and front-to-back to ensure even distribution of MEF over the dish. Incubate at 37 °C for 8-36 h.
2. Passaging hiPSC on MEF
   NOTE: The data was generated using melanoma antigen recognized by T cells 1 (MART-1) iPSC derived from long-term cultured melanoma tumor-infiltrating lymphocyte (TIL), which specifically recognizes MART-1 peptide in the context of human leukocyte antigen (HLA)-A*02:01.
   1. Passage hiPSCs when colonies are between 0.8 – 1.2 mm in diameter. Before passaging, check hiPSC colonies in a stereo-microscope and remove any areas of differentiation from the culture using the plastic edge of a 200 µL tip.
   2. Aspirate spent media and add 10 mL hiPSC media (human embryonic stem cell [ES] culture media [Table of Materials] + 10 ng/mL human basic fibroblast growth factor [hbFGF]) supplemented with 10 μM Rho-associated protein kinase (ROCK) inhibitor.
   3. Hold the cell culture dish in one hand and roll a disposable cell passaging tool across the entire dish in one direction. Apply enough pressure so that the entire roller blade touches the culture dish and maintains uniform pressure during the rolling action.
   4. Rotate the culture dish at 90° and repeat step 1.2.3. View the plate in the microscope to visually confirm the proper cutting of the colonies, which should appear checkered. Detach cut colonies by gentle mechanical flushing using a 200 μL pipette.
      NOTE: Detachment of cut colonies by mechanical flushing must be done immediately after cutting colonies with the roller because after 3 min, the cut colonies will start to reattach to the dish, and it will become difficult to detach colonies of homogeneous size by flushing.
   5. Transfer 350 – 600 clumps of cut colonies onto a new 10 cm dish of MEF (plated 8 – 36 h before hiPSC passaging) with 10 mL of fresh hiPSC media supplemented with 10 μM ROCK inhibitor. Incubate at 37 °C.
      NOTE: 600 clumps represent approximately 1.0 x 10⁶ MART-1 iPSC and will yield 0.5-1.0 x 10⁶ DP cells on day 35. However, expected numbers will vary depending on the potency of the starting cell line and culture conditions.
   6. The following day, aspirate spent media and add 10 mL of fresh hiPSC media. Change hiPSC media every 1-2 days, depending on the hiPSC growth rate.

2. Preparation of OP9/DLL1 Cells for Co-culture with hiPSCs

1. Culture OP9/DLL1 cells in OP9 media [α-minimum essential medium (α-MEM) + 20% fetal bovine serum (FBS) + 1x penicillin-streptomycin] at 37 °C. When OP9/DLL1 cells reach confluency, aspirate media and wash once with 5 mL of 1x magnesium, calcium, and phenol red-free phosphate-buffered saline (PBS).
2. Aspirate PBS and add 2 mL of 0.05% Trypsin-ethylenediaminetetraacetic acid (EDTA). Incubate for 5 min at 37 °C. Then, add 4 mL of OP9 media and mechanically dissociate the cell layer by pipetting to make a single-cell suspension.
3. Transfer the cell suspension into a 50 mL conical tube through a 100 μm cell strainer to avoid cell clumps. Centrifuge at 300 x g for 5 min at 4 °C. Aspirate the supernatant and resuspend in 12 mL of OP9 media.
4. Add 8 mL of OP9 media to six new 10 cm cell culture Petri dishes. Plate 2 mL of OP9/DLL1 cell suspension from step 2.3 onto each new 10 cm dish. Rock the dish side-to-side and then front-to-back to ensure even distribution of OP9/DLL1 over the dish.
5. Incubate at 37 °C. Repeat passage every 2 – 3 days when cells reach confluency.
   NOTE: It is important to make enough frozen stock of OP9/DLL1 cells and thaw a new stock every 4-6 weeks.

3. In Vitro Differentiation of hiPSCs into CD8αβ+ Single Positive (SP) T Cells

1. Prepare gelatinized OP9/DLL1 dishes one week before co-culture with hiPSCs. To prepare 0.1% gelatin solution, add 5 mL of room temperature (RT) tissue-grade stock gelatin solution to 500 mL of PBS.
   1. Coat 3 new 10 cm cell culture Petri dishes by adding 4 mL per dish of 0.1% gelatin. Incubate 30 min at 37 °C.
   2. Aspirate gelatin and add 8 mL of OP9 media to each dish. Passage one confluent dish of OP9/DLL1 (as done in section 2 above) to three gelatin pre-coated dishes.
2. After 4 days, add 10 mL of OP9 media to each 10 cm dish of OP9/DLL1 on gelatin for 20 mL of media per dish.
3. After 7 – 8 days, begin the hiPSC co-culture on OP9/DLL1 confluent dishes (differentiation day 0).
   1. Aspirate spent media from confluent 10 cm dish of hiPSCs on MEF. Add 10 mL of OP9 media. Cut and detach hiPSC colonies using a disposable cell passaging tool, as done in steps 1.2.3 and 1.2.4.
   2. Transfer 350 – 600 clumps of cut colonies onto a 10 cm pre-gelatinized OP9/DLL1 dish (step 3.1) with 10 mL of fresh OP9 media using a 200 μL pipette. Rock the culture dish side-to-side and then front-to-back to ensure an even distribution of colonies.
      NOTE: Alternatively, pre-formed hiPSC embryoid bodies (EBs) or small clump suspension may be used. However, using a disposable cell passaging tool or EB formation system is preferred to produce hiPSC clumps of uniform size.
4. On day 1, aspirate spent media and replace it with 20 mL of fresh OP9 media. hiPSC clumps co-cultured on OP9/DLL1 for 1 day will appear as small round monolayer colonies (Figure 1).
5. On day 5, aspirate 10 mL of spent media and add 10 mL of fresh OP9 media. hiPSC colonies will begin to differentiate into primitive mesoderm, characterized by a multilayered dark center.
6. On day 9, aspirate 10 mL of spent media and add 10 mL of fresh OP9 media. At this point, multilayered center structures will evolve into dome-like shapes, and a peripheral network-like area will become evident.
7. On day 13, harvest hematopoietic progenitor cells (HPCs) (Figure 1). hiPSC-derived structures on day 13 are characterized by a dark central organoid surrounded by a network of dome-like areas, representative of hematopoietic zones (HZs) previously reported to enclose human embryonic stem cell-derived hematopoietic progenitors.
   NOTE: The presence of the dome-like structures indicates a successful process even in the absence of dark centers. The inability to produce HPCs may be due to poor quality of OP9/DLL1, quality of FBS lot, confluency of iPSC clumps seeded onto OP9/DLL1 (350-600 clumps is optimal), and/or variations in potency of iPSC lines to produce hematopoietic precursors.
   1. Aspirate spent media and wash 1x with 5 mL of 1x phenol red-free Hanks' balanced salt solution modified with calcium and magnesium (HBSS).
   2. Aspirate HBSS and add 250 μL of 5000 Units/mL collagenase IV in 10 mL of HBSS. Incubate at 37 °C for 45 min. Aspirate HBSS with collagenase IV and wash once with 5 mL of PBS.
   3. Aspirate PBS and add 5 mL of 0.25% Trypsin-EDTA. Incubate at 37 °C for 20 min. Then, add 4 mL of OP9 media and dissociate the cell layer by pipetting to make a single-cell suspension.
   4. Transfer the cell suspension into a 50 mL conical tube through a 100 μm cell strainer. Centrifuge at 300 x g for 5 min at 4 °C. Aspirate the supernatant and resuspend in 10 mL of OP9 media.
   5. Plate cell suspension onto a new gelatinized 10 cm cell-culture Petri dish (see steps 3.1.1 and 3.1.2). Incubate at 37 °C for 45 min. Then, collect non-adherent cells by gentle pipetting.
   6. Transfer collected cell suspension into a 50 mL conical tube through a 100 μm cell strainer. Centrifuge at 300 x g for 5 min at 4 °C. Aspirate the supernatant and resuspend in 10 mL of differentiation media [OP9 media with 5 ng/mL human stem cell factor (hSCF), 5 ng/mL human Flt3 ligand (hFLT3L), and 5 ng/mL human interleukin 7 (hIL-7)].
   7. Plate the cell suspension onto a new 10 cm OP9/DLL1 confluent dish.
8. On day 16, passage the cells.
   1. Mechanically detach non-adherent cells by gentle pipetting and filter through a 100 μm cell strainer. Centrifuge at 300 x g for 5 min at 4 °C. Aspirate the supernatant and resuspend in 10 mL differentiation media.
   2. Plate the cell suspension onto a new 10 cm OP9/DLL1 confluent dish.
9. Continue passaging non-adherent cells every 5-7 days thereafter by repeating step 3.8.
10. On day 35, enrich CD4+CD8+ double positive (DP) population and stimulate to produce CD8αβ+ single positive (SP) T cells (Figure 2).
    1. Mechanically detach non-adherent cells by gently pipetting and filtering through a 100 μm cell strainer to remove clumps. Enrich the CD4+ cell population by CD4 magnetic bead isolation according to the manufacturer's protocol.
       NOTE: The rationale for using CD4 magnetic beads is to remove CD4-CD8- double negative (DN) cells from the culture, as these have been demonstrated to cause direct killing of CD4+CD8+ DP cells after stimulation.
    2. Count live CD4 enriched cells using a Neubauer hemocytometer and Trypan blue dye. Suspend in OP9 media at a total concentration of 0.5 x 10⁶ cells/mL. Aliquot 1 mL of the cell suspension (0.5 x 10⁶ cells) into each well of a tissue culture flat bottom 24 healthy plates of confluent OP9/DLL1.
    3. Add 100 IU human interleukin 2 (hIL-2), 5 ng/mL hIL-7, 500 ng/mL anti-human CD3 antibody, and 2 μg/mL anti-human CD28 antibody, then culture at 37 °C.