Method for producing cd4/cd8 double-positive t cells

A double-positive, pluripotent stem cell technology, applied in the field of preparation of CD4CD8 double-positive T cells, can solve the problems of difficult differentiation regulation, low self-regeneration ability, low gene transfer efficiency, etc., and achieve the effect of efficient preparation

Pending Publication Date: 2019-03-01
KYOTO UNIV
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AI Technical Summary

Problems solved by technology

In these attempts, as gene transfer cells, CD34-positive cells as bone marrow progenitor cells, natural T lymphocytes, etc. were used, but there were many problems: their self-regeneration ability in Ex-vivo was low, gene transfer efficiency was low, It is difficult to regulate differentiation by gene introduction, etc.
[0007] However, the efficiency of producing T lymphocytes from pluripotent stem cells by these methods is not sufficient, and improvements are desired

Method used

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  • Method for producing cd4/cd8 double-positive t cells
  • Method for producing cd4/cd8 double-positive t cells
  • Method for producing cd4/cd8 double-positive t cells

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preparation example Construction

[0080] In the production of CD4CD8 double-positive T cells, hematopoietic progenitor cells can be cultured using feeder cells, but preferably cultured without using feeder cells.

[0081] Hematopoietic progenitor cells can be cultured adherently or in suspension, but adherent culture is preferred in this step. In the case of adherent culture, the culture container can be coated and used. For example, as a coating agent, Matrigel can be cited (Niwa A, et al.PLoS One.6(7):e22261, 2011) , collagen, gelatin, laminin, heparan sulfate proteoglycan, RetroNectin, Fc-DLL4 or nestin, and combinations thereof.

[0082] In addition, when hematopoietic progenitor cells are obtained by suspension culture of embryoid bodies, it is preferable to perform adherent culture after dissociation into single cells.

[0083] In the present invention, the culture temperature conditions for culturing hematopoietic progenitor cells for the preparation of CD4CD8 double-positive T cells are not pa...

Embodiment 1

[0132] On ultra-low adhesion treated 6-well plates (CORNING: #3471), 3x10 5 ~6x10 5 Each well was inoculated with TkT3V1-7, FfI-01, Ffl-14 or KhES-3 (day 0), and added 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 5ng / ml sodium selenite, 2mM L-glutamine, 45mMα-monothioglycerol, 50μg / ml ascorbic acid) added 10ng / ml BMP4, 5ng / ml bFGF, 15ng / ml VEGF, 2μM SB431542 and in hypoxia Under the condition (5%O 2 ) were cultured for 5 days (Day 5).

[0133] Next, 50 ng / ml of SCF, 30 ng / ml of TPO, and 10 ng / ml of Flt3L were added, and further culture was carried out for 5 to 9 days (to day 14).

[0134] The obtained hematopoietic progenitor cells were plated on a 48-well plate coated with Fc-DLL4 (5 μg / ml) (Sino Biological Inc.) and Retronectin (5 μg / ml) (Takara Bio Co., Ltd.), and added with 50 ng / ml of SCF, OP9 medium (15% FBS, 2mM L-glutamine, 100Uml penicillin , 100ng / ml streptomycin, 55μM 2-mercaptoethanol, 50μg / ml ascorbic acid, 10μg / ml human insulin, 5.5μg / ml hum...

Embodiment 2

[0139] CD34-positive hematopoietic progenitor cells from umbilical cord blood (HemaCare #CBCD34C-1) were coated with Fc-DLL4 (5 μg / ml) and Retronectin (5 μg / ml) on a 48-well plate, cultured in OP9 medium supplemented with 50ng / ml SCF, 50ng / ml IL-7, 50ng / ml Flt3L, 100ng / ml TPO, 15μMSB203580, and 30ng / ml SDF-1α for 21 days (Day 35). On the 35th day, CD45(+), CD3(+), CD4(+), and CD8(+) components were separated by FACS, and CD4CD8 double positive (Double positive) cells (called DP cells) were obtained. The results are shown in Image 6 . In addition, in Image 6 The upper part of the figure shows the results of culturing hematopoietic progenitor cells without adding SB203580 and SDF-1α. From these results, even when CD34-positive hematopoietic progenitor cells derived from umbilical cord blood were used, CD4CD8 double-positive T cells could be efficiently obtained by culturing in a medium containing a p38 inhibitor and SDF-1.

[0140] In Examples 1 and 2, at the time ...

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Abstract

A method for producing CD4 / CD8 double-positive T cells including the following steps: (1) a step that cultures pluripotent stem cells in culture fluid and induces hematopoietic progenitor cells and (2) a step that cultures the hematopoietic progenitor cells obtained in step (1) in culture fluid containing a p38 inhibitor and / or SDF-1 and induces CD4 / CD8 double-positive T cells.

Description

technical field [0001] The invention relates to a method for preparing CD4CD8 double-positive T cells from hematopoietic progenitor cells, a method for preparing CD4CD8 double-positive T cells from pluripotent stem cells, and a method for preparing CD8-positive T cells from the CD4CD8 double-positive T cells. Background technique [0002] T cells are considered to play a central role in the immune system against foreign pathogens such as bacteria and viruses or abnormal cells such as cancer cells, but the function of T cells decreases due to various reasons, leading to susceptibility to infection or cancer. If it is possible to supplement and regenerate immune cells and the like for such a disease, it will be an extremely effective means for improving the condition of the disease, improving the therapeutic effect, and the like. In such immune cell supplement therapy, functional supplementation and regeneration of T lymphocytes responsible for cellular immunity are strongly r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/0735C12N5/074C12N5/10
CPCA61K2039/5158A61K2039/5156C07K14/7051C07K2319/03C12N5/0636C12N2510/00C12N2506/45C12N2500/25C12N2500/38C12N2501/125C12N2501/21C12N2501/26C12N2533/50C12N2533/52C12N2501/115C12N2501/155C12N2501/165C07D401/04C12N5/0696C12N5/10C12N15/85C12N2506/03
Inventor 金子新安井裕入口翔一上田树
Owner KYOTO UNIV
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