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Method for induced differentiation of human embryonic stem cells into NK cells

A human embryonic stem cell, NK cell technology, applied in embryonic cells, biochemical equipment and methods, animal cells, etc., can solve problems such as insufficient lethality

Active Publication Date: 2022-05-17
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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  • Method for induced differentiation of human embryonic stem cells into NK cells
  • Method for induced differentiation of human embryonic stem cells into NK cells
  • Method for induced differentiation of human embryonic stem cells into NK 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 digestion solution were purchased from Anhui Zhongsheng Traceability Biotechnology Co., Ltd.

[0036] (1) Six-well plate coating

[0037] Thaw the coating protein Vitronectin at room temperature, aliquot into 120 μL / tube, take 120 μL Vitronectin and add 9 mL of DMEM / F12 medium, mix gently to dilute, and distribute in a six-well plate at 1.5mL / well, gently Shake and mix to obtain a six-well plate coated with Vitronectin coating solution. After standing at room temperature for 2 hours, use it. When using it, tilt the six-well plate and suck up the coating solution 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 with your hand, thaw within 1 min, and obs...

Embodiment 2

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

[0051] (1) Induction of mesoderm cells

[0052] A. When the second-generation embryonic stem cells obtained in Example 1 have a confluence of more than 80%, the medium is aspirated and discarded, and the 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, under continuous stirring at 15 rpm on a rocker platform, at 37 °C, 5% CO 2 Incubate in an incubator for 24 hours to form cell aggregates.

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

Embodiment 3

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

[0063] (1) Collection of CD34 + Hematopoietic stem cells were digested in Accutase™ Cell Digestion Solution at 37°C for 20 minutes to obtain a single cell suspension. The single cell suspension was 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) CD34 was labeled with CD34 paramagnetic microbeads produced by Miltenyi + For hematopoietic stem cells, operate according to the instructions of the kit, and harvest CD34 + cells and counted. Analysis of CD34 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 induced differentiation of human embryonic stem cells into NK cells, and belongs to the technical field of genetic engineering. The method comprises the following steps: resuscitation and passage of human embryonic stem cells, induction to obtain mesoderm cells, induction to obtain CD34 + hematopoietic stem cells, separation and purification of the CD34 + hematopoietic stem cells, induction to obtain NK cells, and multiplication culture. The human embryonic stem cells are human embryonic stem cell lines H9; the recovery and passage of the human embryonic stem cells are as follows: the human embryonic stem cells are recovered and then subjected to passage to the second generation, and when the cell confluence degree is more than 80%, mesoderm cells are obtained through induction; the NK cells are obtained through induced differentiation of human embryonic stem cells, and when the effect-target ratio is 20: 1, the killing rate of HELA tumor cell lines is 94.25%, and the killing rate of LOVO tumor cell lines is 91.24%.

Description

technical field [0001] The invention relates to a method for inducing differentiation of human embryonic stem cells into NK cells, belonging to the technical field of genetic engineering. Background technique [0002] Cell-based therapies for the treatment of relapsed or refractory cancers have attracted interest and attention. In addition to the study of CAR-T cells, clinical trials using NK cells isolated from peripheral blood or umbilical cord blood are rapidly expanding. This method requires the collection of NK cells for each patient, resulting in donor differences 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 homogenous cell population 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" immunotherapeutic products. [0003] CN109415699A is ...

Claims

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Application Information

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Patent Type & Authority Applications(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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