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Method for differentiating natural killer cells from pluripotent stem cells based on single cell sequencing rational design and application of CSF1R inhibitor

A technology of natural killer cells and pluripotent stem cells, applied in the field of high-resolution cell atlas, can solve the problems of long differentiation time, blind optimization of differentiation methods, and unstable system, and achieve the effect of promoting differentiation

Pending Publication Date: 2022-03-01
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, iPSCs face a series of challenges in differentiating NK cells. First, the differentiation system is not stable enough. Second, the purity of differentiated cells has not been accurately measured. Third, the differentiation time is too long.
Most importantly, there is currently very limited understanding of the "black box" in the process of iPSC differentiation of NK cells. The expression of key genes, key transcription factors, and changes in gene regulatory networks that determine the process of differentiation have not been systematically described. , so the traditional optimization of the differentiation method is relatively blind, and there may be unknown components in the differentiated NK cells, which may cause potential clinical treatment risks

Method used

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  • Method for differentiating natural killer cells from pluripotent stem cells based on single cell sequencing rational design and application of CSF1R inhibitor
  • Method for differentiating natural killer cells from pluripotent stem cells based on single cell sequencing rational design and application of CSF1R inhibitor
  • Method for differentiating natural killer cells from pluripotent stem cells based on single cell sequencing rational design and application of CSF1R inhibitor

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preparation example Construction

[0053] The method for preparing natural killer cells provided by the present invention, simple and convenient, can be stably, and quickly obtain high purity natural kill cells.

[0054] In some preferred embodiments, the CSF1R inhibitor includes, but is not limited to, BLZ 945 and / or PLX3397, for example, LINIFANIB (ABT-869), OSI-930, GW2580, PLX5622, and the like. In the present invention, it is possible to suppress the expression of CSF1R in the cells and does not affect the substance of cell survival.

[0055] It should be noted that "BLZ945 and / or PLX3397" means that the CSF1R inhibitor may include only BLZ945, or may include only PLX3397, and may be BLZ945 and PLX3397.

[0056] In some preferred embodiments, the step of obtaining multi-capable cell induction culture to obtain a pyrogenic bobbin comprises performing a plurality of stem cells in the first medium;

[0057] The first medium includes: base medium, Y27632 8 to 12 μm, BMP416 to 24 ng / ml, VEGF 16 to 24 ng / ml,...

Embodiment 1

[0075] Preheat (15-25 ° C) is sufficient amounts of MTESR1.

[0076] IPS cells were digest into single cells with Tryple.

[0077] 1. Use 1ml without CA 2+ And MG 2+ PBS wash original hole.

[0078] 2. Insert PBS and add 200 μL of tryple to digest for 5 minutes.

[0079] 3. Centrifuge 200 × g after digestion for 5 minutes.

[0080] 4. Revenue with the following medium:

[0081] Basic medium: STEMDIFF TMApel TM 2 contained Y27632 (10 μM), BMP4 (20 ng / mL), VEGF (20 ng / mL) and SCF (40 ng / mL).

[0082] 5. Adjust the cell density to 80,000 tells / ml, add 100 μl / pore cell suspension in low adsorbed 96-well plates.

[0083] 6.300 × g Centrifuge for 5 minutes in 37 ° C, 5% CO 2 The cell culture box was cultured for 6 days, and EB (ambugula), on day 7, 20-40 EBs were placed in a 6-well plate having a matrix glue (1 mg / ml or 0.1% degass), with the following Culture culture medium:

[0084] Basic medium: STEMSPAN TM -XF, containing SCF (20 ng / ml), IL-7 (20 ng / mL), IL-15 (10 ng...

Embodiment 2

[0088] The cells were collected in Example 1 to last 30th day, and the CSF1R inhibitor BLZ9450.5 μm and PLX3397 1 μm were treated, respectively, and the control group (not treated, normal cultured) was provided, and the CD45 was collected and detected after 8 days. + CD56 + The ratio of NK cells, Figure 4 Indicated (shown) Figure 4 The expression of A is NK differentiation to the 24th day of the CSF1R in the UMAP map, Figure 4 B is CSF1R changes in the amount of mRNA expression during macrophages in vitro, and in vitro. Figure 4 C is the results of control group flow cytometry analysis, Figure 4 D is the result of the analysis of the BLZ945 treatment of group flow cytometry. Figure 4 The E is the results of PLX3397 treatment of group flow cytometry analysis.

[0089] Such as Figure 4 The A, CSF1R is highly expressed in the in vitro differentiation system in myeloid cells (mononuclear cells, dendritic cells, etc.); almost no expression in strand cells (NK cell precursors, NK cells,...

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Abstract

The invention provides a method for differentiating natural killer cells from pluripotent stem cells based on single cell sequencing rational design and application of a CSF1R inhibitor, and relates to the technical field of biology. The inventor opens a'black box 'in the process of differentiating the iPSC into the NK according to single cell transcriptome data, provides a rational design method for differentiating the iPSC into the NK, and rationally designs the application of a small-molecule inhibitor in the process of differentiating the NK from the in-vitro pluripotent stem cells. Research finds that the CSF1R inhibitor treatment can improve the NK proportion by inhibiting the differentiation of myeloid cells and shorten the time required for differentiation, so that the CSF1R inhibitor can be used for promoting the differentiation of in-vitro pluripotent stem cells to prepare natural killer cells. The invention provides a preparation method of natural killer cells, which is simple and convenient, and can stably and quickly obtain high-purity natural killer cells.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular based on a single-cell sequencing technique to draw NK differentiated high resolution cell maps, involving CSF1R-related inhibitors in promoting sputum poly-energy stem cell differentiation. Application of natural killer cells. Background technique [0002] NK cells are immunocytes with killing tumor cell function. The engineered modified expression Car's NK cells have the ability to target tumor cells expressing certain antigens. Since CAR-T cells, CAR-NK cells have lower cytokines storms, so there is a good application prospect. NK cells (natural kill cells) (natural kill cells) (natural kill cells) (natural kill cells) (induced multi-capable cells) were capable of obtaining highly homogeneous products, and sufficient amounts of cells can be obtained to form a similar spot product. Therefore IPSC differentiated NK cells are ideal for future spot immune cell products. However, IPSC ...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/22C12N2506/45
Inventor 张进朱雨晴张丽
Owner ZHEJIANG UNIV
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