A method for inducing differentiation into NK cells from human embryonic stem cells

A technology of human embryonic stem cells and NK cells, which can be applied to embryonic cells, biochemical equipment and methods, animal cells, etc., and can solve problems such as insufficient lethality

Active Publication Date: 2022-07-12
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] CN109415699A is a method for preparing CD4CD8 double-positive T cells applied by National University of Japan, which can be induced to differentiate into T cells by hESC / hiPSC cells, but does not involve the cultivation of NK cells
[0005] In summary, the existing technology uses embryonic stem cells to induce differentiation into NK cells, although they have high purity, but there is a technical problem of insufficient lethality

Method used

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  • A method for inducing differentiation into NK cells from human embryonic stem cells
  • A method for inducing differentiation into NK cells from human embryonic stem cells
  • A method for inducing differentiation into NK cells from human embryonic stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Recovery and passage of human embryonic stem cells

[0035] Human embryonic stem cell line H9, Vitronectin (human vitronectin), ROCK inhibitor Blebbistatin, and EDTA digest were purchased from Anhui Zhongsheng Suyuan Biotechnology Co., Ltd.

[0036] (1) Six-well plate coating

[0037] Thaw the coated protein Vitronectin at room temperature, aliquot into 120 μL / tube, add 120 μL of Vitronectin to 9 mL of DMEM / F12 medium, mix and dilute gently, and dispense 1.5 mL / well into a six-well plate, gently Shake and mix to obtain a six-well plate coated with Vitronectin coating solution. Use after standing at room temperature for 2 hours. When using, tilt the six-well plate and suck up the coating liquid with a pipette.

[0038] (2) Recovery of cells

[0039] Preheat the water bath to 37°C, take out a frozen human embryonic stem cell line H9 (1mL), place it in a 37°C water bath, shake it gently by hand, thaw it within 1 min, and observe with the naked eye that the ice...

Embodiment 2

[0050] Example 2 Induction of ESC to CD34 + hematopoietic stem cell differentiation

[0051] (1) Induction of mesoderm cells

[0052] A. When the confluence of the second generation embryonic stem cells obtained in Example 1 is more than 80%, the medium is aspirated and discarded, and E3 medium containing factors is added, wherein the cell density is 0.8×10 6 cells / mL, recorded as day 0, in an ultra-low attachment (ULA) flask at 37°C, 5% CO with continuous stirring at 15 rpm on a rocker platform 2 Incubator for 24h to form cell aggregates.

[0053] The factor-containing E3 medium is the addition of L-ascorbic acid 2-phosphate magnesium (Ascorbic acid 2-phosphate magnesium) and 5ng / mL sodium selenite (Sodiumselenite, 50 μg / mL) to the E3 medium. Purchased from Merck), 50ng / mL of FGF2 (human basic fibroblast growth factor), 50ng / mL of VEGF (vascular endothelial growth factor), 2 μM of CHIR 99021 (glycogen synthase kinase-3 inhibitor), 10 μM Blebbistatin (non-muscle myosin typ...

Embodiment 3

[0062] Example 3 Separation and purification of CD34 by immunomagnetic beads + hematopoietic stem cells

[0063] (1) Collect CD34 + Hematopoietic stem cells were digested in Accutase™ cell digestion solution at 37°C for 20 minutes to obtain a single cell suspension. Single cell suspensions were washed in MACs buffer (PBS containing 5 mg / mL BSA, 1 mM EDTA) and filtered through a 100 µm cell strainer to remove aggregates.

[0064] (2) Using CD34 paramagnetic microbeads produced by Miltenyi to label CD34 + Hematopoietic stem cells, operate according to the kit instructions, harvest CD34 + cells and counted. CD34 analysis by flow cytometry + cell purity, the results showed that the CD34 + The purity of hematopoietic stem cells is 96.9% (such as image 3 ).

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Abstract

The invention relates to a method for inducing differentiation into NK cells from human embryonic stem cells, belonging to the technical field of genetic engineering; the method includes the recovery and passage of human embryonic stem cells, inducing mesodermal cells, and inducing CD34 cells + Hematopoietic stem cells, isolation and purification of CD34 + Hematopoietic stem cells, induced to obtain NK cells, expanded and cultured; the human embryonic stem cells are human embryonic stem cell line H9; the recovery and passage of the human embryonic stem cells, the human embryonic stem cells are revived and passaged, passed to the second generation, and the cells are confluent When the ratio is more than 80%, it is used to induce mesodermal cells; the present invention uses human embryonic stem cells to induce differentiation to obtain NK cells, and when the effect-target ratio is 20:1, the killing rate of HELA tumor cell line The killing rate of the LOVO tumor cell line was 91.24%.

Description

technical field [0001] The invention relates to a method for inducing differentiation into NK cells from human embryonic stem cells, and belongs to the technical field of genetic engineering. Background technique [0002] Cell-based therapies for the treatment of relapsed or refractory cancers have aroused interest and attention. In addition to CAR-T cell studies, clinical trials using NK cells isolated from peripheral blood or umbilical cord blood are rapidly expanding. This approach requires NK cells to be collected for each patient, resulting in donor variability and heterogeneity of NK cells. sex. In contrast, human embryonic stem cell (hESC) or induced pluripotent stem cell (hiPSC)-derived NK cells provide a more homogeneous population of cells that can be produced on a clinical scale. These properties make hESC- or hiPSC-derived NK cells an ideal cell population for the development of standardized, "off-the-shelf" immunotherapy products. [0003] CN109415699A is the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783C12N5/0789C12N5/0735
CPCC12N5/0646C12N5/0647C12N2506/02C12N2500/38C12N2500/12C12N2501/115C12N2501/165C12N2501/727C12N2501/73C12N2501/155C12N2500/50C12N2501/998C12N2500/36C12N2500/32C12N2501/105C12N2501/125C12N2501/145C12N2501/26C12N2501/2303C12N2501/2307C12N2501/2302C12N2501/2315C12N2501/2321C12N2500/90
Inventor 刘明录金海锋强邦明王立新张传鹏冯建海韩庆梅许淼
Owner SHANDONG XINRUI BIOTECH CO LTD
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