The protocol below outlines how to generate T cells from pluripotent stem cells (PSCs). First, CD34+ hematopoietic progenitor cells are differentiated from PSCs. Subsequently, CD4CD8 double-positive (DP) T cells are generated from PSC-derived CD34+ cells. An optional protocol to mature DP T cells to CD8 single-positive (SP) T cells can then be performed.

Materials

• STEMdiff™ T Cell Kit (Catalog #100-0194)
• Y-27632 (Catalog #72302)
• AggreWell™400 Plate (Catalog #34421)
• Anti-Adherence Rinsing Solution (Catalog #07010)
• DMEM/F-12 with 15 mM HEPES (Catalog #36254)
• ACCUTASE™ (Catalog #07922)
• 37 µm Reversible Strainer (Catalog #27250)
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• Collagenase Type II (Catalog #07418)
• TrypLE™ Express (Thermo Fisher #12605010)
• EasySep™ Human CD34 Positive Selection Kit II (Catalog #17856)
• UM729 (Catalog #72332)
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Protocol

This procedure has been optimized for use with multiple human embryonic stem (ES) cell and induced pluripotent stem (iPS) cell lines; refer to the Technical Manual (Document #10000007541) for complete instructions.

Part I: Differentiate CD34+ Cells from PSCs

1. Prepare EB Medium A (STEMdiff™ Hematopoietic - EB Basal Medium + STEMdiff™ Hematopoietic - EB Supplement A). Prepare EB Formation Medium by adding Y-27632 at 10 µM to EB Medium A.
2. Prepare an AggreWell™400 plate by rinsing with Anti-Adherence Rinsing Solution, washing with DMEM/F-12 with 15 mM HEPES, and adding a half-volume of EB Formation Medium.
3. Harvest PSCs and generate a single-cell suspension using ACCUTASE™.
4. Dilute PSCs to 1.4 x 10⁶ cells/mL in 2.5 mL of EB Formation Medium, then seed into the AggreWell™ plate that was prepared in step 2.
5. Perform a half-medium change with EB Medium A on day 2.
6. Prepare EB Medium B (STEMdiff™ Hematopoietic - EB Basal Medium + STEMdiff™ Hematopoietic - EB Supplement B).
7. Perform a half-medium change with EB Medium B on day 3.
8. Harvest EBs on day 5, then filter and elute these with EB Medium B, using a 37 µm reversible strainer.
9. Transfer eluted EBs to a non-tissue culture-treated plate.
10. Add EB Medium B on day 7.
11. Perform a half-medium change with EB Medium B on day 10.
12. Harvest EBs and dissociate into a single-cell suspension using Collagenase Type II and TrypLE™ Express. Isolate CD34⁺ cells using EasySep™ Human CD34 Positive Selection Kit II.
13. Proceed to the protocol below for T cell generation.

Part II: Differentiate DP T Cells from PSC-Derived CD34+ Cells

1. Coat non-tissue culture-treated plates with StemSpan™ Lymphoid Differentiation Coating Material; refer to the Technical Manual #10000007541) for complete instructions.
2. Prepare StemSpan™ Lymphoid Progenitor Expansion Medium (StemSpan™ SFEM II + StemSpan™ Lymphoid Progenitor Expansion Supplement).
3. Dilute CD34⁺ cells to 5 x 10⁴ cells/mL in StemSpan™ Lymphoid Progenitor Expansion Medium and seed onto the coated plate.
4. Incubate at 37°C for 7 days, following instructions in the Technical Manual (Document #10000007541) for required half-medium changes and plate transfer on day 7. On day 14, harvest lymphoid progenitor cells for further differentiation to DP T cells.
5. Prepare StemSpan™ T Cell Progenitor Maturation Medium (StemSpan™ SFEM II + StemSpan™ T Cell Progenitor Maturation Supplement).
6. Dilute lymphoid progenitor cells to 0.5 - 1 x 10⁶ cells/mL in StemSpan™ T Cell Progenitor Maturation Medium. Seed onto a freshly coated plate (see step 1), incubate at 37°C, and follow instructions in the manual for required half-medium changes (removing dead cells using fluorescence-activated cell sorting [FACS] at this stage may improve frequency and yield of DP T cells).
7. On day 28, harvest cells containing DP T cells (see Figure 1) for use in downstream assays, or follow the optional protocol extension for further maturation to CD8 SP T cells.

Part III (Optional): Mature DP T Cells to CD8 SP T Cells

1. Prepare a freshly coated plate (See step 1 of Part II: Differentiate DP T Cells from PSC-Derived CD34+ Cells).
2. Prepare complete CD8 SP T Cell Maturation Medium (StemSpan™ SFEM II + StemSpan™ T Cell Progenitor Maturation Supplement + IL-15). See Technical Manual for details.
3. Add ImmunoCult™ T Cell Activator at half of the recommended concentration.
4. Dilute cells to 1 x 10⁶ cells/mL in complete CD8 SP T Cell Maturation Medium containing ImmunoCult™ T Cell Activator, seed onto the coated plate, and incubate at 37°C.
5. After 3 - 4 days of culture, add CD8 SP T Cell Maturation Medium, without ImmunoCult™ T Cell Activator.
6. Incubate at 37°C and harvest cells after 7 days.