Table 2.
Derivation of T cells from human pluripotent stem cells.
| Author | Stem cell Type | Cell type | Time line in days | Culture conditions | Remarks |
|---|---|---|---|---|---|
| Good et al.[65] | iPSCs | Antigen-specific T cells | 35+ | Note: Feeder OP9/DLL1 cells were readied a week prior on 0.1% gelatin. D0–D12: MEF feeder-dependent iPSCs were co-cultured on OP9/DLL1 with OP9 medium (MEM + FBS + penicillin/streptomycin). Increased the ratio of OP9 medium compared with iPSC medium. On day 9, multilayered center structure evolved with dome-like shape. D13–D35: Harvested hematopoietic progenitor-like cells were resuspended in OP9 medium with SCF + FLT3 + hIL-7. Cell passage was done every 5–7 days. D35+: Enriched CD4+ cells were cultivated in OP9 medium with IL-2 + IL-7 + CD3 antibody + CD28 antibody. Stimulated cells were collected after 7 days and mixed with irradiated HLA-A*02:01 + LCL loaded with MART-1 peptide in the presence of MEM + IL-7 + IL-21. Obtained CD8-αβ CTL cells. |
Generation of antigen-specific T cells are more effective when EBs are used. CD43+ cells were analyzed by flow cytometry. CD8-αβ, MART1 tetramer checked. Antigen-specific T cells were derived from iPSCs using this protocol. |
| Guo et al.[66] | hPSCs | T lymphocytes | 33+ | D0–11: EBs generated with basic differentiation medium (BDM) + BMP4 and cultured in 15-cm dish within 2.5 days. After 2.5 days, VEGF was added and cells were cultured for 6 days. On day 7, 2% of condition medium prepared from AFT024-mlL-3, AFT024-mlL-6, AFT024-hFIt3L and AFT024-mSCF was added. Doxycycline was added from day 6. Medium was replaced on alternate days. D12–D21: Hematopoietic maturation (iHPC) was carried out by seeding with OP9-DL1 cells in EM medium containing α-MEM + FBS + monothioglycerol + GlutaMAX + ascorbic acid + 2% conditioned medium (excluding AFT024-MIL-6). Cells sorted and EM medium used on alternate days. D21–D33+: Matured hematopoietic cells co-cultured with OP9-DL1 feeder cells in T-cell induction medium (TIM + α-MEM + FBS + GlutaMAX) supplemented with 2% conditioned medium derived from AFT024-hFIt3L and AFT024-Hil-7 cell culture for next 12 days. |
Hematopoietic differentiation: Conditioned medium added until day 11. CD31+CD45-CD41(LOW) analyzed with flow cytometer. Hematopoietic maturation step: Cells co-cultured with OP9-DL1. TIM medium changed every third day. CD3 and CD45.2 expression analyzed with flow cytometer. |
| Montel-Hagen et al.[61] | hPSCs | T cells | 67+ | D0–D3: Generation and isolation of hEMPs done using X-VIVO 15 medium along with rh activin A + rhBMP4 + rhVEGF + ROCK inhibitor Y-27632 dihydrochloride. Cells numbering 3 × 106 cells per 3 mL were cultured. Medium changed to X-VIVO 15 + rhBMP4 + rhVEGF + rhFGF. On D3.5, CD32–CD56+ cells were isolated using FACS D3–D10: Induction of hematopoietic lineage was done by T-cell differentiation. Embryonic mesodermal organoids were generated by aggregating hEMPs with MS5-HDLL4 cells. These cells were introduced into hematopoietic induction medium along with EGM2 + ROCK inhibitor + SB431543. On D4, 5 × 105 MS5-HDLL4 cells were combined with 0.5–1 × 104 purified hEMPs per PSC-ATO and centrifuged and cultured in hematopoietic induction medium in the presence of EGM2 + SB431542. Medium was changed twice in a week. D10–D17: Medium changed to EGM2 containing SB431542 along with cytokines rhTPO + rhFLT3L5 + rhSCF. D17–D67: PSC-ATOs were initiated by changing the medium to RPMI medium supplemented with B27 + ascorbic acid + rhSCF + rhFLT3L + rhIL-7. Medium was changed completely every 3–4 days for 50 more days. |
3D organoid formed before processing to next step. MS5-HDLL1 can also be used instead of MS5-HDLL4. PSC-ATO protocol employs PSC-ATOs to efficiently pattern hPSCs to T cells |
| Nishimura et al.[67] | T-iPSCs | T cells | 60+ | Regeneration of hiPSCs done using T cells or CTL clones. T cells were activated by a-CD3/CD28 antibody-coated beads or PHA-L. Activated cells reprogrammed and cultured with RPMI 1640 medium containing 10% AB serum + glutamine + penicillin + streptomycin. On D12, The RH10 medium was replaced with DMEM/F12 FAM + 20% KOSR + glutamine + NEAA + mercaptoethanol + bFGF. D0–D15: iPSCs were transferred to irradiated C3H10T1/2 feeder cells in EB medium containing IMDM + 15% FBS + human insulin + transferrin+ sodium selenite + glutamine + monothioglycerol + ascorbic acid + VEGF + SCF + FLT-3L. D16–D45: Derived hematopoietic progenitors were collected and grown on feeder OP9-DL11 cells. The medium consisted of MEM + 15% FBS + glutamine + penicillin + streptomycin + FLT-3L + IL-7. D46–D60: The T lineage cells was harvested and mixed with irradiated HLA-A24 PBMCs and co-cultured using RH10 medium in the presence of IL-7 and IL-15. |
iPSC clones transfected with small interfering RNA L527 using Lipofectamine RNAiMAX for removal of SeV vectors from cytoplasm. T cells differentiated on OP9-DL1 cells during co-culture in OP9 medium. CD45+ cells isolated using FACS. |
| Nagano et al.[59] | T-iPSCs | CTLs | D0–D12: Differentiation was done on stromal OP9 feeder cells in α-MEM supplemented with 20% FBS. D13–D17: Enriched CD34+ progenitors were plated on OP-DLL1 stromal feeder cells with OP9 medium containing hIL-7, hFlt and hSCF hematopoietic factors. D18–D40: Floating cells were collected and transferred to new OP9-DLL1 feeder cells with OP0 medium containing hematopoietic factors. Passage on to a new OP9-DLL1 feeder layer was done every week. |
T-cell-generating potential of T-iPSCs evaluated by frequency of CD4+CD8+ DP cells; once DP cells are generated, CD8-αβ SP cells are derived by stimulating isolated DP cells. |
bFGF, basic fibroblast growth factor; DMEM, Dulbecco's Modified Eagle's Medium; DP, double-positive; EB, embryoid body; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FGF, fibroblast growth factor; hSCF, human stem cell factor; hEMPs, human embryonic mesodermal progenitors; iHPCs, induced hematopoietic progenitor cells; IMDM, Iscove's Modified Dulbecco's Medium; KOSR, knockout serum replacement; MEF, mouse embryonic fibroblast; MEM, Minimum Essential Medium; NEAA, non-essential amino acid; PHA-L, Phaseolus vulgaris leukoagglutinin; PSC-ATO, PSC–artificial thymic organoid; SCF, stem cell factor; SeV, Sendai virus; SP, single-positive; T-iPSCs, T lymphocyte iPSCs; VEGF, vascular endothelial growth factor.