US 12,410,400 B2
Methods for generating mimetic innate immune cells from pluripotent stem cells
Jieming Zeng, Singapore (SG); and Shu Wang, Singapore (SG)
Assigned to Agency for Science, Technology and Research, Singapore (SG)
Appl. No. 16/484,349
Filed by Agency for Science, Technology and Research, Singapore (SG)
PCT Filed Feb. 7, 2018, PCT No. PCT/SG2018/050051
§ 371(c)(1), (2) Date Aug. 7, 2019,
PCT Pub. No. WO2018/147801, PCT Pub. Date Aug. 16, 2018.
Claims priority of application No. 10201700937P (SG), filed on Feb. 7, 2017; and application No. 10201705582S (SG), filed on Jul. 6, 2017.
Prior Publication US 2019/0359940 A1, Nov. 28, 2019
Int. Cl. C12N 5/0783 (2010.01); A61K 40/15 (2025.01); A61K 40/42 (2025.01)
CPC C12N 5/0636 (2013.01) [A61K 40/15 (2025.01); A61K 40/42 (2025.01); C12N 5/0646 (2013.01); C12N 2501/125 (2013.01); C12N 2501/2307 (2013.01); C12N 2501/2315 (2013.01); C12N 2501/26 (2013.01); C12N 2502/1358 (2013.01); C12N 2502/45 (2013.01); C12N 2506/02 (2013.01); C12N 2510/00 (2013.01)] 6 Claims
 
1. A method of generating gamma-delta natural killer T cells (γδ NKT cells), the method comprising:
(a) expanding γδ T cells of human peripheral blood cells in PBMC culture medium containing an amino-bisphosphonate and interleukin 2 (IL2);
(b) transducing the expanded γδ T cells with reprogramming transcription factors to generate induced pluripotent stem cells (iPSCs);
(c) screening the TCRG and TCRD gene configuration of the iPSCs to identify γδ T cell-derived iPSCs;
(d) co-culturing γδ T cell-derived iPSCs with a stromal cell line deficient in expressing macrophage colony stimulating factor (M-CSF) to generate innate immune cell progenitors;
(e) co-culturing the innate immune cell progenitors with a stromal cell line deficient in expressing M-CSF and ectopically expressing Notch ligand, Delta like 1 (DLL1) in a media comprising stem cell factor (SCF), Fins-related tyrosine kinase 3 ligand (FLT3L), interleukin 7 (IL7) and interleukin 15 (IL15) to generate differentiated gamma-delta NKT cells;
(f) passaging the differentiated gamma-delta NKT cells weekly for 3 to 5 weeks to obtain gamma-delta NKT cells expressing gamma-delta TCRs (γδ TCRs); and
(g) detecting the expression profile of the gamma-delta NKT cells obtained in step (f); wherein the γδ NKT cells expressing gamma-delta TCRs (γδ TCRs), express NKp30 in 99% of the cells, NKp44 in 99% of the cells, NKp46 in 97% of the cells, NKG2D in 84% of the cells, DNAM-1 in 97% of the cells, TRAIL in 89% of the cells, and NKG2A/CD94 in 83% of the cells in combination with expression of at least any one of CD56, CD16, FASL and less than 5% expression of killer cell immunoglobulin-like receptors (KIR) are γδ NKT cells.