Method for differentiating human induced pluripotent stem cells into natural killer cells

A technology of natural killer cells and pluripotent stem cells, applied in the biological field, can solve problems such as difficulty in expanding scale and complicated operation, and achieve the effect of strong cell killing ability, strong cytokine secretion ability and tumor killing ability

Pending Publication Date: 2021-12-17
HELP STEM CELL INNOVATIONS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the technical problems are: 1) the Spin EB method is used to form embryoid bodies, which is complicated to operate and difficult to scale up; 2) animal-derived ingredients (Matrigel, Gelatin, etc.) are used in the process; 3) feeders are used Cells (OP9, K562)

Method used

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  • Method for differentiating human induced pluripotent stem cells into natural killer cells
  • Method for differentiating human induced pluripotent stem cells into natural killer cells
  • Method for differentiating human induced pluripotent stem cells into natural killer cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0143] The verification of embodiment 1 culture medium I

[0144] Control other parameters unchanged, only adjust the composition of medium I, select the basic medium AIM-V, and adjust cytokine I, as shown in Table 1. The results of flow cytometry are shown in Table 2, and the detection diagram is shown in figure 2 .

[0145] Among them, the components of medium II are AIM-V, SCF 30ng / ml, TPO 10ng / m, FLT3L 10ng / ml, IL1520ng / ml.

[0146] The composition of medium III is PRIME-XV, SCF 10ng / ml, IL7 10ng / ml, Human Serum Albumin 20%, human AB serum 15%.

[0147] Table 1 Verification of medium Ⅰ

[0148]

[0149]

[0150] Table 2 D39 flow cytometry results

[0151]

Embodiment 2

[0152] Verification of embodiment 2 medium II

[0153] Control other parameters unchanged, only adjust the composition of the medium II, select the basic medium AIM-V, adjust the cytokine II, as shown in Table 3, the results of the flow cytometry test are shown in Table 4, and the test figure is shown in image 3 .

[0154] Components of medium I: AIM-V, VEGF 40ng / ml, bFGF 20ng / ml, SCF 50ng / ml, IL-3 10ng / ml.

[0155] The composition of medium III is PRIME-XV, SCF 10ng / ml, IL7 10ng / ml, Human Serum Albumin 20%, human AB serum 15%.

[0156] Table 3 Validation of Medium II

[0157] Element SCF (ng / ml) FLT3L (ng / ml) IL12 (ng / ml) IL18(ng / ml) IL21(ng / ml) Culture medium II-1 \ \ 10 50 15 Culture medium II-2 20 25 \ \ \ Culture medium II-3 0 25 10 50 15 Culture medium II-4 20 25 0 50 15 Culture medium II-5 20 200 10 50 15 Culture medium II-6 1 35 25 1 200 Culture medium II-7 20 25 10 50 15 ...

Embodiment 3

[0161] Verification of Example 3 Medium III

[0162] Control other parameters unchanged, only adjust the composition of the medium III, select the basal medium AIM-V, and adjust the cytokine II, as shown in Table 5. The results of flow cytometry are shown in Table 6, and the detection diagram is shown in Figure 4 .

[0163] Components of medium I: AIM-V, VEGF 40ng / ml, bFGF 20ng / ml, SCF 50ng / ml, IL-3 10ng / ml.

[0164] Components of medium II: Optimizer, SCF 30ng / ml, FLT3L 30ng / ml, IL12 10ng / ml, IL1810ng / ml, IL21 10ng / ml.

[0165] Table 5 Verification of Medium III

[0166]

[0167] Table 6 flow detection results

[0168]

[0169] Embodiment 4 killing detection

[0170] Controlling other conditional parameters unchanged, only adjusting the components of medium I, medium II and medium III, and detecting NKG2D receptors by flow cytometry.

[0171] Verify that the medium composition is as follows:

[0172] Control group 1:

[0173] Medium I: PRIME-XV, SCF 20ng / ml;

...

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Abstract

The invention belongs to the technical field of biology, and relates to a method for differentiating human induced pluripotent stem cells into natural killer cells. The method comprises the following steps of 1, differentiating the human induced pluripotent stem cells to form endothelial progenitor cells with expression of KDR+CD73+; 2, performing endothelial-blood conversion on the endothelial progenitor cells with the expression of KDR+CD73+ to form NK progenitor cells with the expression of CD34-CD45+; and 3, further differentiating the NK progenitor cells into NK cells with expression of CD56+CD3-. The NK cells produced by the scheme provided by the invention highly express NKG2D receptors, and have stronger cytokine secretion ability and tumor killing ability.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a key technology for differentiation of human induced pluripotent stem cells (iPSCs) into immune cells, in particular to a method for differentiating human induced pluripotent stem cells into natural killer cells. Background technique [0002] Human natural killer cells (NK) are an important part of the body's innate immune system, and are the body's first line of defense against infection and killing tumor cells. NK cells can recognize tumor antigens through their cell surface cytotoxic receptors (NKp46, NKp44, NKp30), NKG2D and DNAM-1 (CD226), and through natural killing (through perforin, granzyme lysing tumor cells), secretion Cytokines, and antibody-dependent ADCC pathways kill and inhibit tumor cells. [0003] NK cells are very important for the body to defend and fight against tumors, but NK cells in tumor patients are usually functionally impaired. Therefore, NK cells with nor...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/074
CPCC12N5/0646C12N2506/45C12N2501/165C12N2501/115C12N2501/16C12N2501/155C12N2501/125C12N2501/2303C12N2501/2306C12N2501/145C12N2501/26C12N2501/2307C12N2501/2312C12N2501/2315C12N2501/2318C12N2501/2321C12N2501/2311
Inventor 葛文雪陈涛涛王嘉显
Owner HELP STEM CELL INNOVATIONS CO LTD
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