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Mait-like cells and method for manufacturing same

A production method and cell therapy technology, applied in animal cells, vertebrate cells, genetically modified cells, etc., can solve problems such as difficult animal function analysis, unknown cell lines with this characteristic, and lack of cell proliferation ability

Active Publication Date: 2015-09-02
DAIICHI SANKYO CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] As mentioned above, in mice, which are frequently used as experimental animals, MAIT cells are a very rare cell population, and it is difficult to perform functional analysis using this animal
On the other hand, MAIT cells are abundant in humans compared to mice, but there is a limit to the production of MAIT cells in large quantities from human biological samples such as peripheral blood.
In addition, in this method, the number and properties of the obtained MAIT cells are highly likely to fluctuate greatly, and there are difficulties in the stability and reproducibility of experiments using these cells.
Furthermore, MAIT cells usually have almost no cell proliferation ability, and the factors and stimuli that induce their proliferation have not been determined, so they cannot proliferate under in vitro conditions (Dusseaux et al., 2011)
Therefore, as a solution to widely conduct research using MAIT cells, for example, the use of model cells similar in function to MAIT cells is considered, but currently, few cell lines with such characteristics are known

Method used

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  • Mait-like cells and method for manufacturing same
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  • Mait-like cells and method for manufacturing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1: Establishment of iPS cells from human-derived MAIT cells

[0105] Mononuclear cells were prepared from human cord blood using Ficoll. The mononuclear cells were mixed with a biotin-labeled substance of the monoclonal antibody 3C10 (gifted by Dr. Olivier Lantz of L'Institut Curie, France, or Biolegend) that specifically recognizes TCR (Vα7.2) of MAIT cells, Cells reactive with the 3C10 antibody were positively selected using a MACS column (manufactured by Miltenyi Biotech) using avidin magnetic beads to concentrate MAIT cells. This operation was performed using umbilical cord blood from 3 different donors. As a result, the purity of MAIT cells defined as 3C10 positive cells by FACS analysis was 96%, 88%, and 78%, respectively.

[0106] The 200,000 3C10-positive cells thus purified were infected with SeVdp(KOSM)302L (gifted by Dr. Makoto Nakanishi from the Institute of Advanced Industrial Research) serving as a vector for production of human iPS cells for 2 ...

Embodiment 2

[0111] Example 2: Characteristic analysis of MAIT-iPS cells

[0112] The MAIT-iPS cell lines 1-3D, 2-5D, and 4-6D individually established and isolated in Example 1 showed a single layer, flat outline, and clear colonies on MEF feeding, which showed that they were different from ordinary human ES / iPS cells. Cells with very similar morphology ( Figure 2A ).

[0113] In addition, the expression of markers specific to human ES / iPS cells was investigated using MAIT-iPS cells thus subcultured. For fixed MAIT-iPS cells, anti-alkaline phosphatase (ALP) antibody, anti-SSEA4 antibody, anti-Oct-3 / 4 antibody, anti-Nanog antibody (above, R&D Systems Inc.), anti-TRA-1 antibody as primary antibody -60 antibody (BD Bioscience) or anti-TRA-1-81 antibody (Santa Cruz) was reacted, and rhodamine-labeled secondary antibody (Jackson ImmunoResearch) was used for staining. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI: 4',6-diamidino-2-phenylindole) solution (1 µg / mL). The stai...

Embodiment 3

[0133] Example 3: Production of MAIT cells from MAIT-iPS cells

[0134] Regarding stem cells such as ES cells, it is known that by co-culturing OP9 cells as feeder cells, differentiation can be induced into CD34-positive blood cell / lymphocyte progenitor cells, and further, by co-culture with OP9 cells that forcibly express Notch ligand DLL1 (OP9 / DLL1) co-culture, can differentiate into cells of T cell series (Schmitt TM et al., Nat. Immun.5, 410-417 (2004); Wakao H et al., FASEB J.22, 2223-2231 ( 2008); Wakao H et al., WO2008 / 038579; Watarai H et al., J.Clin.Invest.120, 2610-2618 (2010); WO2010 / 027094; Timmermans F et al., J.Immunol.182, 6879-6888 (2011)).

[0135] In this example, induction of differentiation from MAIT-iPS cells into MAIT cells was attempted based on this method.

[0136] As OP9 cells and OP9 / DLL1 cells, cells purchased from Riken Cell Bank were used. First, on the OP9 cells 3 to 7 days after fusion, a substance that disperses the colony of MAIT-iPS cell...

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Abstract

The object of the present invention is to provide a method for preparing induced pluripotent stem cells from MAIT cells, and providing induced pluripotent stem cells derived from MAIT cells. Another object of the present invention is to provide a method for preparing MAIT-like cells from induced pluripotent stem cells, and providing MAIT-like cells. Differentiation of the induced pluripotent stem cells having a MAIT cell-specific TCR gene can be induced to establish MAIT-like cells.

Description

technical field [0001] The present invention relates to a method for producing artificial pluripotent stem cells from MAIT cells, and artificial pluripotent stem cells derived from MAIT cells. In addition, the present invention relates to a method for producing MAIT-like cells from artificial pluripotent stem cells, and MAIT-like cells obtained thereby. Background technique [0002] MAIT cells (Mucosal associated invariant T cells, mucosa-associated constant T cells) can produce various cytokines to control various immune responses, and are known as innate immune T cells as cells that undertake the "bridge role" between natural immunity and acquired immunity A type of lymphocyte. MAIT cells are abundant in humans, for example, 20-50% of T cells in the liver, lamina propria lymphocytes (LPL) in the intestinal mucosa or peripheral blood mononuclear cells (peripheral blood mononuclear cells: 1-10% of PBMCs), on the other hand, are rare cells in mice (Dusseaux et al., 2011; Le...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0783C12Q1/02A61K35/17A61P31/04A61P31/10
CPCA61K35/17C12N2510/00C07K14/7051C12N5/0636C12N5/0696C12N2501/125C12N2501/2307C12N2501/26C12N2501/602C12N2501/603C12N2501/604C12N2501/606C12N2502/1394C12N2506/11C12N2506/45A61P31/04A61P31/10A61K2239/38A61K39/4611A61K2239/31A61K39/464817
Inventor 若尾宏藤田博美小清水右一吉清和则
Owner DAIICHI SANKYO CO LTD