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. 2023 Mar 1;56(4):e13425. doi: 10.1111/cpr.13425

TABLE 1.

Representative differentiation methods for generating PSC‐derived immune cells.

Generated cell type Cell source Methods Serum/feeder status Total days required Factors in culture medium Functional test Publication
T lineage cell hESC Feeder Serum‐containing Feeder: MEF, OP9, OP9‐DL1 43–61 days FLT3‐L, IL‐7, SCF In vitro TCR rearrangement and cytokine production assays 11
T lineage cell hiPSC Feeder Serum‐containing Feeder: C3H10T1/2, OP9‐DLL1 16–45 days VEGF, SCF, FLT3L, IL‐7, IL‐15 In vitro cell cytotoxicity assays 12
T lineage cell hiPSC Feeder Serum‐containing Feeder: MEF, OP9, OP9‐DLL1 ~40 days FLT3‐L, IL‐7, SCF, IL‐2, anti‐human CD3, anti‐human CD28 In vitro TCR rearrangement and cytokine production assays 13
T lineage cell hiPSC EB and DL4‐μbeads Serum‐free Feeder‐free ~50 days BMP4, bFGF, SB431542, CHIR99021, IL‐6, IL‐11, VEGF, IGF‐1, SCF, EPO In vivo thymus engraftment assay 14
T lineage cell hiPSC EB and immobilized DL4 protein Serum‐free Feeder‐free 35–42 days VEGF, BMP4, bFGF, SCF, TPO, FLT3L, SB431542, CHIR99021, IL‐7, OKT3 In vitro and in vivo cell cytotoxicity assays 15
T lineage cell hESC hiPSC Feeder and artificial thymic organoid (ATO) Serum‐free Feeder: MS5‐hDLL1/4 67 days Activin A, BMP4, VEGF, FGF, SCF, FLT3L, TPO, IL‐7, Ascorbic Acid In vitro TCR rearrangement and cell cytotoxicity assays 16
NKT cell hiPSC Feeder Serum‐containing Feeder: C3H10T1/2, OP9‐DLL1 35 days VEGF, FLT3L, SCF, IL‐7, IL‐2, IL‐15 In vitro cell cytotoxicity assay 17
NK cell hESC Feeder Serum‐containing Feeder: MEF, S17, AFT024 21–67 days IL‐15, IL‐3, IL‐7, SCF, FLT3L, In vitro cell cytotoxicity and cytokine production assays 18
NK cell hESC hiPSC EB Serum‐free Feeder‐free 39 days VEGF, BMP4, IL‐3, IL‐7, IL‐15, SCF, FLT3L In vitro specific killing assay 19
NK cell hESC hiPSC EB Serum‐free Feeder‐free 34 days VEGF, BMP4, IL‐3, IL‐7, IL‐15, SCF, FLT3L In vitro specific killing assay 20
NK cell hESC hiPSC Feeder and organoid Serum‐free Feeder: OP9, OP9‐DLL1/DLL4 27 days BMP4, ACTIVIN A, bFGF, CHIR99021, PIK‐90, A‐83‐01, C59, SB431542, Hydrocortisone, SGR‐SM, FLT3L, TPO, SCF, EGF, VEGF, IGF‐1, Ascorbic Acid In vitro and in vivo cell cytotoxicity assays 21
Macrophage hESC EB and Feeder Serum‐containing Feeder: S17 26–32 days GM‐CSF, M‐CSF In vitro phagocytosis, HIV infection, and cytokine production assays 22
Macrophage hESC EB Serum‐containing Feeder: MEF ~18 days for first harvest, multiple harvests M‐CSF, IL‐3 In vitro endocytosis, phagocytosis, cytokine production, and polarization assays 23
Macrophage hESC hiPSC EB Serum‐free Feeder‐free ~21 days for first harvest, multiple harvests BMP4, VEGF, SCF, M‐CSF, IL‐3 In vitro phagocytosis, HIV infection, and cytokine production assays 24
Macrophage hiPSC EB Serum‐free Feeder: MEF ~20 days for first harvest, multiple harvests M‐CSF, IL‐3 In vitro phagocytosis assay and in vivo bacterial infections 25 , 26
Macrophage hESC hiPSC Monolayer Serum‐free Feeder‐free 16–28 days BMP4, VEGF, SCF, bFGF, SCF, FL3, IL‐3, TPO, M‐CSF, GM‐CSF In vitro cytokine production, chemotaxis, and polarization assays 27
Macrophage hiPSC Monolayer Serum‐free Feeder‐free 15 days BMP4, Activin A, CHIR99021, SB431542, VEGF, bFGF, SCF, IL‐3, IL‐6, TPO, M‐CSF In vitro microfluidic flow adhesion, endocytosis, phagocytosis, polarization, and cytokine production and chemokines assays 28 , 29
Dendritic cell hESC Feeder Serum‐containing Feeder: OP9‐DL1 24–29 days GM‐CSF, IL‐4 In vitro antigen presentation assay 30
Dendritic cell hESC EB Serum‐free Feeder‐free 17–25 days BMP4, VEGF, SCF, GM‐CSF, IL‐4 In vitro phagocytosis and antigen presentation assays 31
Dendritic cell hiPSC Feeder Serum‐free Feeder‐free 28 days GM‐CSF, FLT3L, SCF, VEGF, BMP4 In vitro phagocytosis, antigen presentation, and cytokine production assays 32
B cell hiPSC Feeder Serum‐containing Feeder: OP9, MS5 21–42 days IL‐3, IL‐7, SCF, FLT3L In vitro VDJ rearrangement assay 33
B cell hESC hiPSC Feeder Serum‐containing Feeder: OP9, MS5 39 days bFGF, IL‐3, FLT3L, SCF, IL‐7 N/A 34
Microglia hESC hiPSC EB Serum‐free Feeder: MEF 74 days M‐CSF, IL‐34 In vitro cytokine production assay 35
Microglia hiPSC Monolayer Serum‐free Feeder‐free 26 days BMP4, VEGF, CHIR99021, FGF2, SCF, DKK1, IL‐3, IL‐6, M‐CSF In vitro phagocytosis and cytokine production assays 36