US 12,421,496 B2
Method for producing natural killer cell and use thereof
Yee Sook Cho, Daejeon (KR); Han-Seop Kim, Daejeon (KR); Binna Seol, Daejeon (KR); and In Pyo Choi, Daejeon (KR)
Assigned to KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, Daejeon (KR)
Appl. No. 16/649,417
Filed by KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, Daejeon (KR)
PCT Filed Sep. 21, 2018, PCT No. PCT/KR2018/011247
§ 371(c)(1), (2) Date Jul. 1, 2020,
PCT Pub. No. WO2019/059713, PCT Pub. Date Mar. 28, 2019.
Claims priority of application No. 10-2017-0121980 (KR), filed on Sep. 21, 2017; and application No. 10-2018-0113308 (KR), filed on Sep. 20, 2018.
Prior Publication US 2020/0407685 A1, Dec. 31, 2020
Int. Cl. C12N 5/0783 (2010.01); A61K 40/15 (2025.01); A61K 40/42 (2025.01); A61P 35/00 (2006.01)
CPC C12N 5/0646 (2013.01) [A61K 40/15 (2025.01); A61K 40/42 (2025.01); A61P 35/00 (2018.01); C12N 5/0638 (2013.01); A61K 2239/31 (2023.05); A61K 2239/38 (2023.05); A61K 2239/50 (2023.05); C12N 2501/2307 (2013.01); C12N 2501/2315 (2013.01); C12N 2501/602 (2013.01); C12N 2501/603 (2013.01); C12N 2501/604 (2013.01); C12N 2501/606 (2013.01); C12N 2501/727 (2013.01)] 11 Claims
 
1. A method for producing natural killer cells comprising directly reprogramming into natural killer cells, consisting of:
(a) introducing a reprogramming factor into isolated cells, wherein the reprogramming factor is Oct4, Sox2, Klf4, and Myc; and
(b) from the following day after the introduction, culturing the cells of step (a) in i) a first medium comprising cytokine, growth factor, and GSK3β inhibitor to increase the efficiency of direct reprogramming, and ii) a second medium comprising cytokine, growth factor, and aryl hydrocarbon receptor inhibitor to promote the production of natural killer cells, wherein the cytokine in the first medium is one or more selected from the group consisting of IL-2, IL-3, IL-6, IL-7, IL-11, IL-15, and IL-21; and the growth factor in the first medium comprises SCF (Stem Cell Factor), FLT3 (Fms-like Tyrosine Kinase 3) ligand, and TPO (Thrombopoietin) and is one or more selected from the group consisting of EPO (Erythropoietin), BMP4 (Bone Morphogenetic Protein 4), and EGF (Epidermal Growth Factor), wherein the cytokine in the second medium is one or more selected from the group consisting of IL-2, IL-7, and IL-15; and the growth factor in the second medium comprises SCF and FLT3 ligand,
wherein the isolated cells are peripheral blood mononuclear cells, skin fibroblasts or dental pulp cells,
wherein when the isolated cells are peripheral blood mononuclear cells, the first medium in step (b) includes IL-3, IL-6, SCF, FLT3, TPO, and CT 99021; and the second medium in step (b) includes IL-2, IL-7, IL-15, SCF, FLT3, and StemRegenin, wherein the peripheral blood mononuclear cells are further cultured in a medium including Serum-free medium supplement, Ascorbate, MTG (Monothioglycerol), L-glutamine, Human transferrin, SB431542, CT99021, IL-3, IL-6, IL-11, SCF, FLT3, TPO, and EPO before culturing in the second medium in step (b),
wherein when the isolated cells are skin fibroblasts or dental pulp cells, the first medium in step (b) includes FBS, Serum Replacement, NEAA (Non-Essential Amino Acids), β-mercaptoethanol, bFGF (basic Fibroblast Growth Factor), and CT99021, and the second medium in step (b) includes IL-2, IL-7, IL-15, SCF, FLT3, and StemRegenin, and wherein the skin fibroblasts or dental pulp cells are further cultured in a (a′) medium including Serum-free medium supplement, Ascorbate, MTG, L-glutamine, Human transferrin, SB431542, CT99021, IL-3, IL-6, IL-11, SCF, FLT3, TPO, and EPO, and then cultured in a (b′) medium including N2, neuronal cell culture supplement, BSA (Bovine Serum Albumin), Ascorbate, MTG (Monothioglycerol), L-glutamine, Human transferrin, BMP4, bFGF, SB431542, and CT99021, before culturing in the second medium in step (b).