Method for inducing and amplifying decidua-like natural killer cells from hematopoietic stem cells in vitro

A technology of natural killer cells and hematopoietic stem cells, applied in the field of biomedicine, can solve the problems of insufficient source of decidual NK cells and difficult industrialized production, and achieve the effects of broad application prospects and value, efficient differentiation and stable quality.

Active Publication Date: 2021-06-29
UNIV OF SCI & TECH OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] This application provides a method for inducing and expanding decidual-like NK cells from hematopoietic stem cells derived from umbilical cord blood or autologous bone marrow in order to solve the problems of insufficient sources of decidual NK cells and difficulty in industrial production in the prior art

Method used

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  • Method for inducing and amplifying decidua-like natural killer cells from hematopoietic stem cells in vitro
  • Method for inducing and amplifying decidua-like natural killer cells from hematopoietic stem cells in vitro
  • Method for inducing and amplifying decidua-like natural killer cells from hematopoietic stem cells in vitro

Examples

Experimental program
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Embodiment 1

[0026] Embodiment 1: Separation of umbilical cord blood (or bone marrow) mononuclear cells

[0027] Cord blood and bone marrow samples were obtained from healthy blood donors in the First Affiliated Hospital of University of Science and Technology of China. Fresh human umbilical cord blood (or bone marrow) was collected in an anticoagulant tube, and mononuclear cells were separated after dilution according to the ratio of umbilical cord blood (or bone marrow): sterile 1×PBS=1:1. Take the sample with a 10ml pipette, extend it to 0.5cm above the liquid surface of human peripheral blood lymphocyte separation medium (Tianjin Haoyang Company, article number: LTS1077, hereinafter referred to as Ficoll), the sample slides naturally onto the surface of the Ficoll liquid, and then gently Gently load the entire sample onto the Ficoll. Centrifuge at 600g for 25 minutes at 20°C. After the centrifugation is completed, the centrifuge tube has obvious layers, which are respectively from bo...

Embodiment 2

[0028] Example 2: CD34 + HSC isolation and purification

[0029] Use CD34 Magnetic Beads Separation Kit (Miltenyi Company, Cat. No.: 130-046-702) for CD34 + For the sorting of HSC, the specific steps are as follows. According to the counting results, the cell pellet obtained in Example 1 was resuspended using MACS buffer (see the instruction manual of the sorting kit for the configuration method), and every 1×10 7 Add 30 μl MACS buffer to resuspend the cells. per 1×10 7 cells, add 10 μl FcR Blocking Reagent, and mix thoroughly. Next, every 1×10 7Add 10 μl CD34 Micro Beads to each cell and mix well. Place in a 4°C refrigerator and incubate for 30 minutes. After incubation, add 10ml MACS buffer, mix well, and centrifuge at 300g, 4°C for 10 minutes. Discard the supernatant and resuspend the cells in 2ml MACS buffer. Put the MS separation column (Miltenyi company, article number: 130-042-201) into the magnet, add 500 μl of MACS buffer to wash the column, and then add the ...

Embodiment 3

[0030] Example 3: Detection of CD34 by flow cytometry + HSC sorting purity

[0031] The method for flow cytometry antibody-labeled samples is as follows: collect the cell suspension in a 1.5ml EP tube, add 1×PBS to a volume of 1ml, mix well, centrifuge at 500g, 4°C for 5 minutes, discard the supernatant; add to each tube 100 μl of 1×PBS to resuspend cells, and add 2 μl of fluorescein-coupled flow cytometry antibody CD34-PE (BD Company, catalog number: 555822), mix well, and incubate at 4°C for 30 minutes; add to 1.5ml EP tube 1ml 1×PBS, after mixing, centrifuge at 500g, 4°C for 5 minutes, discard the supernatant, add 200μl 1×PBS to each tube to resuspend the cells; Detection by multi-laser flow cytometry. The experimental results were analyzed using FlowJo_V10 software. The test results showed that in the sorted cells, CD34 + The proportion of cells is more than 95%, the analysis and statistical results are shown in figure 1 .

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Abstract

The invention discloses a method for inducing and amplifying decidua-like natural killer (NK) cells from hematopoietic stem cells in vitro. The method comprises the following steps: culturing CD34+ hematopoietic stem cells in four stages in vitro, namely amplifying the CD34+ hematopoietic stem cells in stage I, inducing differentiation of the CD34+ hematopoietic stem cells into NK cell precursors in stage II, inducing differentiation of the NK cell precursors into NK cells in stage III, inducing amplification and maturation of the NK cells and collecting CD3-CD56+NK cells in stage IV, and thus obtaining decidua-like natural killer cells, wherein the culture is started from a fresh basic culture medium containing a cytokine combination I, fresh basic culture mediums containing the cytokine combination I, combination II, combination III and combination IV are used in culture stages I, II, III and IV respectively for half-quantity liquid change culture, the cytokine combination I is composed of Flt3L and SCF; the combination II is composed of Flt3L, SCF and IL-15; the combination III is composed of Flt3L, SCF and IL-15; and the combination IV is IL-15.

Description

technical field [0001] This application belongs to the technical field of biomedicine, in particular, it relates to the CD34 derived from cord blood (Cord blood, CB) or autologous bone marrow (Bone marrow, BM) + A method for inducing and expanding hematopoietic stem cells (Hematopoietic stem cells, HSC) in vitro to obtain decidual-like natural killer (Natural killer, NK) cells. Background technique [0002] As a semi-allograft of the mother, the fetus can escape the rejection of the mother's immune system during pregnancy, which is the prerequisite for the establishment of pregnancy. With the invasion of trophoblast cells, the decidualization of the endometrium is gradually completed, forming the maternal-fetal interface where the mother and fetus interact. During early pregnancy, immune cells account for about 30% of the decidual tissue at the maternal-fetal interface, among which decidual natural killer (dNK) cells exist in large numbers, accounting for up to 70%. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/0789
CPCC12N5/0646C12N5/0647C12N2506/11C12N2501/26C12N2501/125C12N2501/2315
Inventor 傅斌清杜祥慧魏海明孙汭田志刚
Owner UNIV OF SCI & TECH OF CHINA
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