Differentiation of iPSC-derived iNK Cells

Human iPSC culture and differentiation to iCD34⁺ cells were performed as previously described (35 (link), 36 (link), 60 (link)). At the beginning of the iNK cell differentiation culture, iCD34 cells were plated on stroma cells in B0 media supplemented with cytokines that support NK cell differentiation from hematopoietic progenitors (61 (link)). After 21 days of culture, iNK cells were harvested and co-cultured with irradiated K562 cells transduced with membrane-bound IL-21 and 4–1BBL constructs (62 (link)) in supplemented B0 media for 2 – 3 weeks. K562 cells were propagated in RPMI 1640 media (Corning) containing 10% fetal bovine serum (FBS) (Hyclone). The following fluorescently-conjugated antibodies were used for phenotypic analysis of iNK cells: anti-KIR3DL1 (DX9), anti-KIR2DL1 (HP-MA4), anti-KIR3DL2/3 (DX27), anti-NKG2D (D11), anti-CD16 (3G8), anti-NKp44 (p44–8), anti-NKp46 (9E2), anti-NKp80 (5D12), anti-perforin (B-D48), anti-granzyme b (QA16A02), anti-2B4 (C1.7), anti-LFA-1 (HI111), anti-CD69 (FN50), anti-CD62L (DREG-56), anti-PD-1 (EH12.2H7), anti-LAG-3 (11C3C65), anti-TIGIT (A15153G) (all from Biolegend), anti-NKG2A (Z199; Beckman Coulter), anti-NKp30 (p30–15; BD Biosciences), and anti-NKG2C (134591; R&D Systems).

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Cichocki F., Bjordahl R., Gaidarova S., Mahmood S., Abujarour R., Wang H., Tuininga K., Felices M., Davis Z.B., Bendzick L., Clarke R., Stokely L., Rogers P., Ge M., Robinson M., Rezner B., Robbins D.L., Lee T.T., Kaufman D.S., Blazar B.R., Valamehr B, & Miller J.S. (2020). iPSC-derived NK cells maintain high cytotoxicity and enhance in vivo tumor control in concert with T cells and anti-PD-1 therapy. Science translational medicine, 12(568), eaaz5618.

Publication 2020

RPMI 1640 media anti-NKG2A anti-NKG2C

4 1bbl Antibodies Cd62l Cells Culture cell Cytokines Fetal bovine serum Granzyme b Hematopoietic cell progenitors Human Il 21 Ipsc K562 cells Kir2dl1 Kir3dl1 Lfa 1 Membrane Nkp44 Nkp46 Perforin Phenotypic Stroma cells Tigit

Corresponding Organization : Fate Therapeutics (United States)

Other organizations : Sanford Consortium for Regenerative Medicine, University of California, San Diego

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Variable analysis

independent variables

Culture conditions for iCD34+ cell differentiation to iNK cells
Co-culture of iNK cells with irradiated K562 cells transduced with membrane-bound IL-21 and 4–1BBL constructs

dependent variables

Phenotypic analysis of iNK cells (expression of KIR3DL1, KIR2DL1, KIR3DL2/3, NKG2D, CD16, NKp44, NKp46, NKp80, perforin, granzyme b, 2B4, LFA-1, CD69, CD62L, PD-1, LAG-3, TIGIT, NKG2A, NKp30, NKG2C)

control variables

Propagation of K562 cells in RPMI 1640 media containing 10% fetal bovine serum (FBS)

controls

Positive control: Irradiated K562 cells transduced with membrane-bound IL-21 and 4–1BBL constructs
Negative control: Not explicitly mentioned

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