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Natural killer cell differentiated from human pluripotent stem cells as well as preparation method and application thereof

A natural killer cell and human pluripotent stem cell technology, applied in the field of stem cell biology, can solve problems such as complex operation and low efficiency of NK cell system

Pending Publication Date: 2021-04-06
深圳市济因生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to provide a fast, efficient and simple method for inducing differentiation of human pluripotent stem cells into natural killer cells in order to solve a series of problems such as the low efficiency and complicated operation of the existing human pluripotent stem cell differentiation to obtain NK cell system

Method used

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  • Natural killer cell differentiated from human pluripotent stem cells as well as preparation method and application thereof
  • Natural killer cell differentiated from human pluripotent stem cells as well as preparation method and application thereof
  • Natural killer cell differentiated from human pluripotent stem cells as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0084] A method for directionally inducing differentiation of human pluripotent stem cells into natural killer cells, comprising the following steps:

[0085] S1, forming embryoid bodies;

[0086] S2. Directed permanent hematopoietic differentiation of embryoid bodies to obtain hematopoietic precursor cells;

[0087] S3. Hematopoietic precursor cells differentiate to obtain natural killer cells;

[0088] S4: Expansion of natural killer cells.

[0089] Specific steps are as follows:

[0090] 1. Day-1 to day0: formation of embryoid bodies

[0091] The well-growing hPSCs were digested into single cells, resuspended in human pluripotent stem cell culture medium, and allowed to stand overnight in the presence of anti-apoptosis inhibitors to form EB spheres with uniform size and shape. The hPSCs used in the experiment are hiPSCs and commercialized hESCs, which have undergone strict pluripotency verification and are maintained in normal human pluripotent stem cell medium E8.

[...

Embodiment 2

[0125] A method for directionally inducing differentiation of human pluripotent stem cells into natural killer cells, the specific steps are as follows:

[0126] 1. Day-1 to day0: formation of embryoid bodies

[0127] The well-growing hPSCs were digested into single cells, resuspended in human pluripotent stem cell culture medium, and allowed to stand overnight in the presence of anti-apoptosis inhibitors to form EB spheres with uniform size and shape. The hPSCs used in the experiment are hiPSCs and commercialized hESCs, which have undergone strict pluripotency verification and are maintained in the normal human pluripotent stem cell medium mTeSR.

[0128] The hPSCs cultured by the above method were subjected to the embryoid body formation experiment when the growth density was about 80%. The specific method is: use Accutase to completely digest hPSCs into a single cell suspension, and add the anti-apoptosis inhibitor HA100, the concentration of the inhibitor is 1 μM, and the...

Embodiment 3

[0153] A method for directionally inducing differentiation of human pluripotent stem cells into natural killer cells, the specific steps are as follows:

[0154] 1. Day-1 to day0: formation of embryoid bodies

[0155] The well-growing hPSCs were digested into single cells, resuspended in human pluripotent stem cell culture medium, and allowed to stand overnight in the presence of anti-apoptosis inhibitors to form EB spheres with uniform size and shape. The hPSCs used in the experiment are hiPSCs and commercialized hESCs, which have undergone strict pluripotency verification and are maintained in the normal human pluripotent stem cell medium mTeSR.

[0156] The hPSCs cultured by the above method were subjected to the embryoid body formation experiment when the growth density was about 80%. The specific method is: use EDTA to completely digest hPSCs into a single cell suspension, and add the anti-apoptosis inhibitor Blebbistatin, the concentration of the inhibitor is 50 μM, and...

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Abstract

The invention relates to the field of stem cell biology, and discloses a preparation method of natural killer cells differentiated from human pluripotent stem cells. The preparation method comprises the steps of S1, forming an embryoid body; S2, performing directional permanent hematopoiesis differentiation on the embryoid body to obtain hematopoietic precursor cells; and S3, differentiating the hematopoietic precursor cells to obtain natural killer cells. The invention also discloses application of the natural killer cell prepared by the method as a pharmaceutical composition for preventing and / or treating tumors by cells. According to the method, efficient and stable differentiation of the NK cells is achieved, EB adherent differentiation is achieved in the induced differentiation period, TGFB inhibitor treatment is prolonged, 10% or above of CD34+CD45+permanent hematopoietic HPCs can be obtained through rapid induction. The method has the advantages of being efficient, stable, low in cost and suitable for large-scale cell preparation production; and the method is easy and convenient to operate, the NK cells obtained through differentiation have typical NK cell surface marking molecules, the anti-tumor function of the NK cells is superior to that of the NK cells derived from umbilical cord blood, and great scientific research and clinical application potentials are achieved.

Description

technical field [0001] The invention relates to the field of stem cell biology, in particular to a natural killer cell differentiated from human pluripotent stem cells and a preparation method and application thereof. Background technique [0002] Natural killer (Nature Killer, NK) cells have a killing effect on tumor cells, especially chimeric antigen receptor NK (CAR-NK) cells, which have been clinically reported to have a good effect on eliminating cancer cells. However, it is difficult to isolate and expand NK cells directly from umbilical cord blood or peripheral blood, and T cells need to be eliminated, otherwise severe graft-versus-host disease (GVHD) will occur. In particular, many patients have too few NK cells, which may lead to the failure of NK cell isolation and expansion. At the same time, the quality of NK cells from different donors varies greatly, making it difficult to form standardized products. [0003] Human pluripotent stem cells (human pluripotent st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/0735C12N5/10A61K35/17A61P35/00
CPCC12N5/0646A61K35/17A61P35/00C12N2501/155C12N2501/11C12N2501/165C12N2501/115C12N2501/125C12N2501/26C12N2501/2303C12N2501/2306C12N2501/105C12N2501/145C12N2501/135C12N2501/15C12N2501/10C12N2501/22C12N2501/14C12N2501/2302C12N2501/2301C12N2501/2307C12N2501/2312C12N2501/2315C12N2501/2318C12N2501/2321C12N2501/2327C12N2500/38C12N2506/02C12N2506/45
Inventor 不公告发明人
Owner 深圳市济因生物科技有限公司
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