A method and application of differentiating human pluripotent stem cells into natural killer cells

A natural killer cell and cell culture technology, applied in the field of stem cell biology, can solve the problems of long differentiation cycle, low differentiation efficiency and purity, and dependence on feeder layer cells in differentiation methods, and achieve low preparation cost, low cost, and high cell viability Effect

Active Publication Date: 2021-08-10
安徽中盛溯源生物科技有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0014] Although the process of in vitro differentiation from pluripotent stem cells into natural killer cells has been relatively clear at the principle level, and there are currently many methods for inducing differentiation, but the existing differentiation methods may have long differentiation cycles, low differentiation efficiency and purity, and poor differentiation. Unknown components, dependence on feeder cells, complex operation and high cost, so it is difficult to expand production and clinical use

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  • A method and application of differentiating human pluripotent stem cells into natural killer cells
  • A method and application of differentiating human pluripotent stem cells into natural killer cells
  • A method and application of differentiating human pluripotent stem cells into natural killer cells

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Embodiment 1

[0137] This embodiment discloses a method for preparing natural killer cells, comprising the following steps:

[0138] S1: Formation of embryoid bodies;

[0139] S2: Differentiation of embryoid bodies to hematopoietic progenitor cells;

[0140] S3: differentiation of hematopoietic progenitor cells into NK cells;

[0141] S4: Maturation and expansion of NK cells.

[0142] Specifically, the flow chart is as figure 1 As shown, the steps are as follows:

[0143] (1) Day-1~Day 0: Formation of embryoid body (EB)

[0144] 1) Culture of human pluripotent stem cells (iPSCs)

[0145] The human iPSCs used in the experiment have undergone strict pluripotency verification (expressing various pluripotency markers, and can form teratomas including inner, middle and outer germ layers in immunodeficient mice). iPSCs are normally cultured in iPSC maintenance medium, the medium used is E8 or TeSR or other similar medium.

[0146] The human iPSC is prepared by the method disclosed in the ...

Embodiment 2

[0194] This example studies the effects of different EB culture methods on the EB differentiation rate. In the experimental group, the culture method of 3D suspension culture to form embryoid bodies was adopted throughout the induction and differentiation process, that is, the iPSCs were directly formed into smaller volume EBs on a 3D shaker, and then replaced with different special differentiation media at different differentiation stages ( Concrete steps refer to embodiment 1). During the culture process, the volume of EB gradually increased, and the cells continued to proliferate. Compared with the 2D differentiation scheme (see patent CN107429230A), the 3D differentiation condition greatly saves the culture space and culture volume, and the number of cells obtained under the same culture system is significantly increased, which is conducive to the scale-up of hPSCs to multipotential blood precursor cells Production. Compared with the differentiation scheme of Spin EB (US...

Embodiment 3

[0196] The present invention finds that in 3D induction culture, the use of small molecule reagents, cytokines and cell matrix can replace serum and trophoblast cells, and accelerate the differentiation process and improve the differentiation efficiency, which is beneficial to the production of subsequent clinical-grade cell preparations.

[0197] In our differentiation method, in addition to using 3D to improve differentiation efficiency, the combined use of small molecule reagents, cytokines, and cell substrates not only avoids the use of serum and trophoblast cells, but also further increases the proportion of blood precursor cells. CHIR99021 was selected as the GSK3β inhibitor, BMP4 was used as the BMP signaling pathway activator, SB431542 was used as the Nodal inhibitor, and DLL4-Fc recombinant protein was used as the Notch signaling pathway activator in the cell matrix. On days 6-12, the ratio of blood progenitor cells (CD34+) obtained can be as high as 20-80%, which is h...

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Abstract

The invention relates to the field of stem cell biology, in particular to a method and application for differentiating human pluripotent stem cells into natural killer cells. The invention discloses a natural killer cell derived from pluripotent stem cells, which expresses CD56, Nkp30, Nkp44 and Nkp46, and also expresses markers CD16 and CD94 of mature natural killer cells. The invention also discloses a method for preparing natural killer cells, comprising the following steps: S1: forming embryoid bodies; S2: differentiation of embryoid bodies to hematopoietic progenitor cells; S3: differentiation of hematopoietic progenitor cells to NK cells; S4: NK cells maturation and expansion. The differentiation method provided by the present invention is based on a culture medium with defined components and an optimized combination of cytokines, which can rapidly, efficiently, simply and cost-effectively differentiate induced pluripotent stem cells into natural killer cells.

Description

technical field [0001] The present invention relates to the field of stem cell biology, in particular to the lineage-specific differentiation of multipotent or multipotent stem cells, in particular to a method and application for differentiating human pluripotent stem cells into natural killer cells. Background technique [0002] According to the latest national cancer report in 2018, the estimated number of new cases of malignant tumors nationwide in 2014 was 3.804 million, and an average of more than 10,000 people were diagnosed with cancer every day. The incidence rate of tumor was 278.07 / 100,000, the cumulative incidence rate of 0-74 years old was 21.58%, and the cumulative mortality rate was 12.00%. In 2013, "Science" magazine listed tumor immunotherapy as the first of the top ten scientific breakthroughs. Immunotherapy has become a new generation of tumor treatment after surgery, chemotherapy, radiotherapy, and tumor targeted therapy, bringing new benefits to the treat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783C12N5/074A01N1/02A61K35/17A61P35/00
CPCA01N1/0221A61K35/17A61P35/00C12N5/0646C12N2500/80C12N2501/105C12N2501/11C12N2501/115C12N2501/125C12N2501/135C12N2501/14C12N2501/145C12N2501/155C12N2501/165C12N2501/22C12N2501/2301C12N2501/2302C12N2501/2303C12N2501/2306C12N2501/2307C12N2501/2312C12N2501/2318C12N2501/2321C12N2501/2327C12N2501/26C12N2501/33C12N2501/405C12N2501/58C12N2506/45C12N2513/00C12N2533/50C12N2533/52
Inventor 不公告发明人
Owner 安徽中盛溯源生物科技有限公司
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