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Introduction

T cells are lymphocytes that provide defense against pathogens and tumors. As part of the adaptive immune system, T cells recognize a wide range of targets through their antigen-specific T cell receptors (TCRs) and exert effector functions, including cytokine secretion and cell killing. A crucial feature of T cells is the formation of memory cells, which helps establish a much faster response to reinfection than the primary response. While T cells can be isolated from peripheral blood, human pluripotent stem cells (hPSCs) offer a potentially unlimited source of immune cells. The ability to differentiate hPSCs to T cells provides a useful tool for developing adoptive immunotherapy in cancer patients and for research into the basic biology of these cells.

STEMdiff™ T Cell Kit (Catalog #100-0194) facilitates the differentiation of hPSCs to T cells, without the use of stromal cells and in serum-free culture conditions.

Differentiate hPSCs to T Cells

STEMdiff™ T Cell Kit comprises STEMdiff™ Hematopoietic - EB reagents paired with a StemSpan™ T Cell Generation Kit. The STEMdiff™ T Cell Kit is used in a four-stage protocol to differentiate hPSCs to T cells. In the first two stages, STEMdiff™ Hematopoietic - EB reagents are used to differentiate hPSCs to CD34⁺ hematopoietic progenitor cells (Figure 1). First, hPSCs are cultured for 3 days in EB Formation Medium and EB Medium A to induce mesoderm specification. This is done in ����������±����™, allowing for the formation of uniformly sized embryoid bodies (EBs). In the second stage, the cells are cultured for 9 days in EB Medium B, promoting their specification to hematopoietic progenitor cells. The generated EBs are then dissociated into single cells, and CD34⁺ cells are enriched using �������⳧���™ immunomagnetic cell isolation.

In the third stage, CD34⁺ cells are cultured for 14 days in StemSpan™ Lymphoid Progenitor Expansion Medium on plates coated with StemSpan™ Lymphoid Differentiation Coating Material to stimulate their proliferation and differentiation to lymphoid progenitor (LP) cells. Finally, LP cells are differentiated to CD4⁺ CD8⁺ double-positive (DP) T cells (Figure 2).

For DP T cell differentiation, LP cells are cultured for 14 days in StemSpan™ T Cell Progenitor Maturation Medium on plates coated with StemSpan™ Lymphoid Differentiation Coating Material. In this system, approximately 60 CD4⁺ CD8⁺ DP T cells can be generated per CD34⁺ cell (average of results from multiple embryonic stem [ES]/induced pluripotent stem [iPS] cell lines; see Figure 6).

STEMdiff™ T Cell Kit Components

The following products are components of STEMdiff™ T Cell Kit (Catalog #100-0194) and are also available for individual sale.

Protocol for Differentiation of hPSCs to T Cells

This protocol is designed to promote the differentiation of hPSCs to CD4⁺CD8⁺ DP T cells over 40 days of culture. The first two stages— mesoderm formation and hematopoietic specification—are shown in Figure 1. Stage three—lymphoid progenitor differentiation—and stage four—DP T cell differentiation—are shown in Figure 2. An optional protocol extension for further maturation of DP T cells to CD8⁺ single-positive (SP) T cells is shown in Figure 3.

This protocol has been optimized for use with ES and iPS cells; refer to the Technical Manual (Document #10000007541) for complete instructions. Optimal cell yields depend on maintenance of proper cell health, which is achieved by following the recommended schedule of feeding and medium changes.

Differentiation of hPSCs to CD34⁺ Hematopoeitic Progenitor Cells (Stages 1 and 2)

Day 0

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 2.5 mL of EB Formation Medium (for one well of a 6-well plate).
3. Harvest hPSCs and generate a single-cell suspension using ACCUTASE™.
4. Dilute hPSCs to 1.4 x 10⁶ cells/mL in 2.5 mL of EB Formation Medium, then seed into the AggreWell™ plate prepared in step 2. Incubate at 37°C for 2 days.

Day 2

5. Perform a half-medium change with EB Medium A. Incubate at 37°C for 24 hours.

Day 3

6. Prepare EB Medium B (STEMdiff™ Hematopoietic - EB Basal Medium + STEMdiff™ Hematopoietic - EB Supplement B). Perform a half-medium change with EB Medium B. Incubate at 37°C for 2 days.

Day 5

7. Harvest EBs, then filter and elute these with 2.5 mL EB Medium B, using a 37 μm reversible strainer.
8. Transfer eluted EBs to a non-tissue culture-treated plate. Incubate at 37°C for 2 days.

Day 7

9. Add 2.5 mL EB Medium B. Incubate at 37°C for 3 days.

Day 10

10. Perform a half-medium change with EB Medium B. Incubate at 37°C for 2 days.

Day 12

11. 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.

Differentiation of CD34⁺ Cells to CD4⁺CD8⁺ DP T Cells (Stages 3 and 4)

Day 0

12. Coat non-tissue culture-treated plates with StemSpan™ Lymphoid Differentiation Coating Material; refer to the Technical Manual (Document #10000007541) for complete instructions.
13. Prepare StemSpan™ Lymphoid Progenitor Expansion Medium (StemSpan™ SFEM II + StemSpan™ Lymphoid Progenitor Expansion Supplement).
14. Dilute CD34⁺ cells to 5 x 104 cells/mL in StemSpan™ Lymphoid Progenitor Expansion Medium and seed onto the coated plate.
15. Incubate at 37°C for 14 days, following instructions in the Technical Manual (Document #10000007541) for required half-medium changes and plate transfer on day 7.

Day 14

16. Harvest lymphoid progenitor cells (containing CD5⁺CD7⁺ cells; see Figure 5) for further differentiation to DP T cells.
17. Prepare StemSpan™ T Cell Progenitor Maturation Medium (StemSpan™ SFEM II + StemSpan™ T Cell Progenitor Maturation Supplement).
18. 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 12), incubate at 37°C, and follow instructions in the manual for required half-medium changes and additions.
    
    Note: Removing dead cells using fluorescence-activated cell sorting (FACS) at this stage may improve frequency and yield of DP T cells.

Day 28

19. Harvest cells containing DP T cells (see Figure 6) for use in downstream assays, or follow the optional protocol extension for further maturation to CD8⁺ SP T cells (see Figures 3 and 7).

Optional Protocol Extension>

An optional protocol to mature CD4⁺CD8⁺ DP T cells to CD8⁺ SP T cells is presented in Figure 3. This extended protocol uses StemSpan™ T Cell Progenitor Maturation Medium prepared with reagents included in STEMdiff™ T Cell Kit and must be combined with additional components, including either ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator or ImmunoCult™ Human CD3/CD28 T Cell Activator, and Human Recombinant IL-15.

Protocol for Optional CD8⁺ SP T Cell Maturation

1. Prepare a freshly coated plate (See step 12 of Protocol for Differentiation of hPSCs to T Cells on page 3).
2. Prepare 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™ Human CD3/CD28/CD2 T Cell Activator or ImmunoCult™ Human CD3/CD28 T Cell Activator at half of the recommended concentration to an aliquot of CD8⁺ SP T Cell Maturation Medium.
4. Dilute cells to 1 x 10⁶ cells/mL in CD8⁺ SP T Cell Maturation Medium + 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.

Analysis of Differentiation from hPSCs to T Cells

hPSCs are differentiated to CD34⁺ hematopoietic progenitor cells over 12 days (Figure 4). CD7⁺CD5⁺ lymphoid progenitor cells generated after 14 days of culture of hPSC-derived CD34⁺ cells (Figure 5), DP T cells generated during a second 14-day culture step (Figure 6), and CD8⁺ SP T cells generated by following the optional protocol extension (Figure 7) are identified by flow cytometry. Cells may also be stained with antibodies directed against cell surface markers CD3, CD4, CD5, CD7, CD8a, CD8b, CD34, CD144, and �հ��αβ for analysis of hematopoietic progenitor cells, lymphoid progenitor cells, and T cell subsets. In the representative flow cytometry plots shown, dead cells were excluded by light-scatter profile and DRAQ7™ staining. The average frequency and yield of the indicated hematopoietic progenitor, lymphoid progenitor, and T cell populations are shown in Figures 4C, 5C, 6E, and 7G. CD8⁺ SP T cells were further stimulated and their ability to degranulate and produce cytokines was assessed in Figure 8.

Product Information

Recommended Antibodies for Analysis*

Accessory Products*

The following products are components of STEMdiff™ T Cell Kit (Catalog #100-0194) and are also available for individual sale.