WO2008056734A1 - Procédé de production de cellules dendritiques à partir de cellules souches d'embryons humains - Google Patents

Procédé de production de cellules dendritiques à partir de cellules souches d'embryons humains Download PDF

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WO2008056734A1
WO2008056734A1 PCT/JP2007/071700 JP2007071700W WO2008056734A1 WO 2008056734 A1 WO2008056734 A1 WO 2008056734A1 JP 2007071700 W JP2007071700 W JP 2007071700W WO 2008056734 A1 WO2008056734 A1 WO 2008056734A1
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cells
cell
embryonic stem
human embryonic
culture
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PCT/JP2007/071700
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Japanese (ja)
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Satoru Senju
Yasuharu Nishimura
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National University Corporation Kumamoto University
Mitsubishi Tanabe Pharma Corporation
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Publication of WO2008056734A1 publication Critical patent/WO2008056734A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464454—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • C12N5/064—Immunosuppressive dendritic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • the present invention relates to a method for differentiating dendritic cells from human embryonic stem cells, a method for producing dendritic cells from human embryonic stem cells, and dendritic cells obtained by the production method.
  • Dendritic cells phagocytose antigen proteins, break them down into peptides, and present the resulting peptides to T cells as a complex with major histocompatibility antigen (MHC) (hereinafter also referred to as "antigen presentation"! / Then, T cells are stimulated to activate the T cells in an antigen-specific manner. Further, the dendritic cells are the cells having the highest antigen presenting ability in the living body. On the other hand, the dendritic cells suppress the function of T cells reactive to self antigens and are also involved in maintaining immunological self tolerance. Thus, dendritic cells play a central role in regulating immune responses in vivo.
  • MHC major histocompatibility antigen
  • Dendritic cells are differentiated and produced from hematopoietic stem cells in the bone marrow in vivo.
  • hematopoietic stem cells in bone marrow differentiate into erythrocytes, platelets, neutrophils, eosinophils, basophils, macrophages, lymphocytes, etc. in addition to dendritic cells due to the presence of growth factors.
  • human dendritic cells have been obtained by isolating dendritic cells from human peripheral blood, or culturing monocytes, which are dendritic cell precursor cells in peripheral blood, and inducing differentiation, etc. It is known to be obtained.
  • monocytes which are dendritic cell precursor cells in peripheral blood, and inducing differentiation, etc.
  • monocytes which are dendritic cell precursor cells in peripheral blood, and inducing differentiation, etc. It is known to be obtained.
  • monocytes which are dendritic cell precursor cells in peripheral blood, and inducing differentiation, etc. It is known to be obtained.
  • monocytes which are progenitor cells.
  • it is necessary to perform a blood cell separation operation involving extracorporeal blood circulation There are drawbacks.
  • Non-patent Document 1 a method for obtaining dendritic cells by inducing differentiation of mouse embryonic stem cells has been reported.
  • Patent Document 2 a method for obtaining dendritic cells of human embryonic stem cells.
  • Patent Document 1 Japanese Unexamined Patent Application Publication No. 2004-313038
  • Patent Document 2 WO2006 / 022330 Nonfret
  • Non-Patent Document 1 S. Senju et al., Blood, Vol. 101, p. 3501 to p. 3508, May 1, 003
  • One aspect of the present invention is to efficiently obtain dendritic cells derived from humans, to stably supply human dendritic cells, and to a means for antigen-specific control of human immune responses (for example, Providing a means of strongly stimulating the response of cytotoxic T cells to specific antigens, or a means of proliferating immunosuppressive T cells to specific antigens, etc., and antigen-specific control of immune responses Supply treatments for diseases (eg, malignant tumors, infectious diseases, autoimmune diseases, allergic diseases, etc.) that are expected to have therapeutic effects, rejection in organ transplants and graft versus host disease (GVHD: Graft versus Host)
  • the present invention relates to providing a method for differentiating dendritic cells from human embryonic stem cells, which enables at least one of providing means for preventing or treating diseases).
  • another aspect of the present invention is to obtain a large amount of dendritic cells derived from humans, efficiently obtain dendritic cells derived from humans, and stably supply human dendritic cells. And obtaining at least one dendritic cell that specifically controls the immune response of an individual (for example, a dendritic cell that strongly activates a cytotoxic T cell response to a specific antigen). It relates to providing a method for producing dendritic cells from human embryonic stem cells.
  • Yet another aspect of the present invention is to activate a human immune response in an antigen-specific manner (for example, to strongly activate a cytotoxic ⁇ cell response to a specific antigen).
  • the present invention relates to providing dendritic cells capable of at least one of obtaining a therapeutic effect for a disease for which a therapeutic effect is expected by antigen-specific activation of a response.
  • a therapeutic effect is expected by antigen-specific control of the immune response.
  • the present invention relates to providing the use of the dendritic cells for the manufacture of a medicament that enables at least one of treatment for a disease to be caused.
  • the present invention it is possible to suppress at least one of antigen-specific immunosuppression, for example, treatment of diseases such as autoimmune diseases and allergic diseases.
  • the present invention relates to providing an immune response control agent.
  • Other problems of the present invention are also apparent from the description of the present specification.
  • the gist of the present invention is as follows:
  • step (B) Cell group A obtained in step (A) and cells having the property of inducing differentiation and proliferation of blood cells in the presence of granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor To obtain a cell group B, and
  • step (C) a step of culturing the cell group B obtained in the step (B) in the presence of granulocyte macrophage colony stimulating factor and interleukin 4,
  • step (B) Prior to step (B), a human embryonic stem cell derived from a co-culture of human embryonic stem cells and cells having the property of inducing differentiation and proliferation of blood cells and mesoderm
  • the cells collected in step (A) are left in a culture vessel to remove adherent cells, the above [1] Differentiation method,
  • step (A) and step (B) are mitomycin C-treated V, and OP9 cells whose proliferation ability has been lost by irradiation.
  • the method includes (D) adding the tumor necrosis factor ⁇ and lipopolysaccharide to the culture obtained in the step (C), and further culturing.
  • the differentiation method according to any one of the above,
  • step (B) Cell group A obtained in step (A) and cells having the property of inducing differentiation and proliferation of blood cells in the presence of granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor Co-cultured to obtain cell group B,
  • step (C) culturing the cell group B obtained in step (B) in the presence of granulocyte-macrophage colony-stimulating factor and interleukin 4, and
  • step (D ′) separating dendritic cells derived from human embryonic stem cells from the culture obtained in step (C),
  • step (E) adding the tumor necrosis factor ⁇ and lipopolysaccharide to the culture obtained in step (C) and further culturing;
  • step (ii) The production method according to [7], wherein in step (ii), a CD40 ligand is further added,
  • dendrites derived from humans there is an excellent effect that cells can be supplied efficiently and stably.
  • a means for antigen-specific control of an individual's immune response for example, a means for strongly activating a cytotoxic T cell response to a specific antigen
  • an immune response It has an excellent effect that it can supply treatment means for diseases (for example, malignant tumors, self-immune diseases, allergic diseases, etc.) that are expected to have a therapeutic effect by antigen-specific control.
  • dendritic cells derived from humans can be efficiently and stably produced in large quantities, and the burden and danger to cell donors. It has the excellent effect of being able to be supplied without incurring any damage.
  • dendritic cells that specifically control an individual's immune response for example, dendritic cells that strongly activate the response of cytotoxic T cells to a specific antigen
  • dendritic cells that specifically activate the response of cytotoxic T cells to a specific antigen are obtained. be able to.
  • the dendritic cells of the present invention can control an individual's immune response in an antigen-specific manner (eg, strongly activate a cytotoxic T-cell response to a specific antigen). It has an excellent effect of being able to obtain a therapeutic effect for a disease for which a therapeutic effect is expected by controlling the reaction antigen-specifically or by antigen-specific control of the immune response.
  • an antigen-specific manner eg, strongly activate a cytotoxic T-cell response to a specific antigen.
  • the immune response can be controlled in an antigen-specific manner, for example, treating diseases such as malignant tumors, autoimmune diseases, and allergic diseases. If you can use force S! /, You will have an excellent effect.
  • Dendritic cells having immunosuppressive activity are produced from human embryonic stem cells and administered to humans, thereby immunizing against alo (allogeneic) antigens derived from the inherent genetic background of human embryonic stem cells. Expected to induce tolerance. This makes it possible to transplant differentiated therapeutic cells derived from the same embryonic stem cells without causing rejection, and the problem of rejection, which is the biggest problem in regenerative medicine using embryonic stem cells. Can be solved.
  • step (B) Cell group A obtained in step (A) above and the ability to induce differentiation and proliferation of blood cells Co-cultured cells having a quality in the presence of granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor to obtain cell group B, and
  • step (C) a step of culturing the cell group B obtained in the step (B) in the presence of granulocyte macrophage colony stimulating factor and interleukin 4,
  • the differentiation method of the present invention has one major feature in that human embryonic stem cells are used. Therefore, according to the differentiation method of the present invention, compared to the case where dendritic cells are isolated from a living body or the case of differentiation from monocytes, the cell donor (donor) has a large amount without causing physical burden and risk. The excellent effect of being able to be supplied is demonstrated.
  • the differentiation method of the present invention since the embryonic stem cells are used, if necessary, is a gene that is expected to exhibit a therapeutic effect (hereinafter referred to as “therapeutic gene”) safe? It exhibits an excellent effect that it can be easily introduced and expressed. Furthermore, according to the present invention, since human embryonic stem cells are used, it is suitable for application to so-called cell therapy.
  • the human embryonic stem cells used in the present invention are not particularly limited, for example, human moon cake ten stem cells established and distributed at the Research Center for Stem Cell Medicine, Institute of Regenerative Medicine, Kyoto University ( http://www.shigen.nig.ac.jp/escell/human/top.jsp) and various human embryonic stem cells.
  • the human embryonic stem cells can be appropriately selected according to the intended use of the obtained dendritic cells.
  • the above-mentioned "cells having the property of inducing differentiation and proliferation of blood cells", ie feeder cells, include, for example, OP9 cells (RIKEN BioResource Center deposit number: RCB1124), ST2 cells (RIKEN Bioresource Center deposit number: RCB0224), PA6 cells (RIKEN BioResource Center deposit number: RCB1127).
  • OP9 cells are preferable from the viewpoint of improving the efficiency of inducing differentiation into blood cells.
  • the "differentiation and proliferation of blood cells” is performed during the co-culture of the "human embryonic stem cells” and the “cells having a property of inducing differentiation and proliferation of blood cells".
  • the “differentiation and proliferation of blood cells” is performed.
  • Cells having the property of inducing '' were cultured in a culture container containing an appropriate medium under the culture conditions according to the feeder cell, and grown to the extent that the bottom surface of the culture container was almost covered. Later, after cell growth was lost by mitomycin c treatment or irradiation,
  • the medium that can be used for the production of the feeder cell is appropriately selected depending on the type of cell used as the feeder cell as long as it is a medium suitable for culturing adherent mammalian cells. Examples thereof include a MEM, DMEM [Dulbecco's modified Eagle medium (culture solution)] and the like.
  • the culture vessel used in the step (ii) is a tissue culture from the viewpoint of promoting the differentiation of human embryonic stem cells into mesodermal cells, the viewpoint of stably adhering feeder cells for a long period of time, and the like.
  • Any culture vessel provided with a coating suitable for the above may be used.
  • the coating can be performed with gelatin, fibronectin or the like, for example.
  • the culture condition of the feeder cell can be appropriately set according to the type of cell used as the feeder cell. For example, in the case of ⁇ 9 cells, etc., there are conditions for culturing at 37 ° C and 5% by volume CO in a culture container coated with 0.1% gelatin solution, etc., in a medium supplemented with 20% by volume fetus serum. Can be mentioned.
  • the cell density of the human embryonic stem cells at the time of seeding is the mesodermal property obtained per medium (culture medium) used from the viewpoint of sufficiently exerting the differentiation ability. From the viewpoint of maximizing the number of cells, etc., it is preferable that the area is 1 ⁇ 10 5 to 5 ⁇ 10 5 cells / 60 cm 2 culture vessel bottom area (or ⁇ ⁇ ⁇ ⁇ ⁇ culture solution).
  • the culture medium used for co-culture of human embryonic stem cells and feeder cells in the step (ii) may be any culture medium suitable for mammalian cell culture. Can be appropriately selected depending on the type, and examples include ⁇ ⁇ , DMEM (Dulbecco's modified Eagle's medium), IMDM (Iscob's modified Dulbecco's medium), and the like. Such a medium can also be used in the following steps (B) to (D).
  • Culture conditions during co-culture of human embryonic stem cells and feeder cells in the step (A) are appropriately determined depending on the type of human embryonic stem cells used, the composition of the culture solution, and the like. For example, conditions such as around 37 ° C (especially 37 ° C), 5 vol% CO, etc.
  • the human embryonic stem cells and feeder cells in the step (A) are co-cultured with sufficient differentiation of the human embryonic stem cells into mesodermally differentiated cells. In order to maximize the number of differentiated cells, about 15 to 18 days is desirable. During this period, the culture medium is replaced with a new one every 2 to 3 days.
  • the cell group A obtained by! / In the step (A) shows properties of mesodermal cells, and can be obtained as a cell group containing a cluster of cells having a round shape.
  • the differentiation method of the present invention comprises transforming human embryonic stem cells into human embryonic stem cells from a co-culture of cells having the property of inducing differentiation and proliferation of blood cells.
  • the cells collected in step (A) are left in the culture vessel to remove the adherent cells. It is preferable to do this.
  • step (A) cells that have differentiated into the mesodermal system are selectively collected, and from the viewpoint of improving the efficiency of differentiation into dendritic cells, the cells are fed to the feeder cell and the plastic surface. Strong adherence! / It is preferable to remove cell group A and separate cell group A.
  • Phosphate buffered saline containing ethylenediaminetetraacetic acid is added to the co-culture and at 37 ° C for an appropriate time to separate cells that have differentiated into mesodermal cells, e.g., 20-40 minutes
  • EDTA ethylenediaminetetraacetic acid
  • the collected cells are transferred to a centrifuge tube and precipitated by centrifugation at 1,500 rpm for 10 minutes, the supernatant is removed by aspiration, and a new culture solution is added to suspend. It is preferable to inoculate a newly prepared culture dish and maintain at 37 ° C., for example, for 2 hours or more.
  • mesodermally differentiated cells are weakly adherent or buoyant, so such a cell group is collected as a cell group A by pipetting and suspended in a freshly prepared culture medium.
  • the cells that adhere strongly to the bottom of the culture dish while maintaining at 37 ° C for 2 hours or more contain many cells other than mesodermally differentiated cells. Discard them.
  • the cell mass with a size of 100 Hm or more contained in the suspension of cells collected by pipetting is a nylon mesh ( ⁇ D. FALCON, Cell Strainer, 100 ⁇ m nylon). It is desirable to remove it using etc.! / [0032] It is desirable to remove many floating dead cells by exchanging the culture medium the day after the start of the culture in step (B).
  • cell group A containing mesodermally differentiated cells obtained in step (A), and cells having the property of inducing the differentiation and proliferation of blood cells is one major feature.
  • the ratio of blood cells, particularly myeloid cells (myeloid cells) can be further improved in the force, step (B). Derived dendritic cells can be obtained more efficiently and can be supplied stably.
  • the conditions of the culture gas phase in the co-culture of the cell group A and the feeder cell in the step (B) are the same as those in the step (A) according to the composition of the culture medium used. Can be set appropriately.
  • step (B) the cell density of cell group A is adjusted from the standpoint of fully exerting the differentiation ability, from the standpoint of maximizing the number of produced cells per culture medium to be used, and the like.
  • The it is desirable to transfer the cell group A collected from step (A) to a culture container having a volume of about 2 to 4 times the culture container in step (A) and culture in step (B).
  • the content of granulocyte-macrophage colony-stimulating factor in the medium in the step (B) is 50 to 200 ng / ml, preferably 75 to 75, from the viewpoint of producing dendritic cells with strong T cell stimulating activity. A range of 150ng / ml is desirable! /.
  • the content of the macrophage colony-stimulating factor in the medium in the step (B) is 25 to 50 ng / ml, preferably 30 to 50 ng / ml, from the viewpoint of producing dendritic cells with strong T cell stimulating activity. A range is desirable.
  • the time for co-culture of the cell group A and the feeder cell in the step (B) is as follows: From the viewpoint of sufficiently improving the proportion of blood cells, particularly myeloid (myeloid) cells, in cell group A, in order to maximize the number of produced cells per culture medium used, and finally to stimulate T cells. From the viewpoint of producing strong dendritic cells, it is preferably 7 to 10 days.
  • step (B) medium replacement or medium addition may be performed as appropriate during the culture period.
  • the cell group B obtained by! / In the step (B) shows the properties of blood cells and may vary depending on the type of animal from which the embryonic stem cells used are derived. For example, it can be obtained as a cell group containing floating cells having a round shape as shown in panel E of FIG.
  • the culture vessel used in the step (B) is preferably coated with gelatin, fibronectin or the like from the viewpoint of stably attaching feeder cells for a long period of time.
  • the cell group B obtained in the step (B) is preferably collected by, for example, pipetting, etc. S is preferable. Usually, 2 10 5 to 8 10 5 cells are obtained in the culture medium 101111. It is done. This cell group B is used in the following step (C).
  • the cell group B obtained in step (B) is cultured in the presence of granulocyte macrophage colony-stimulating factor and interleukin-4 [step (C)].
  • step (C) the differentiation method of the present invention, by carrying out this step (C), the proportion of dendritic cell-like cells is further improved, and dendritic dendritic activity with high stimulating activity and antigen presenting activity on T cells is obtained. Since cells can be produced, dendritic cells derived from humans can be obtained more efficiently and can be stably supplied.
  • step (C) The conditions of the culture gas phase at the time of co-culture in the step (C) depend on the type of human embryonic stem cells used, the composition of the medium, and the like in the steps (A) and (B). It can be set as appropriate. In step (C), it is desirable not to use OP9 cells.
  • the medium used in the step (C) contains granulocyte-macrophage colony-stimulating factor and interleukin-4.
  • the content of granulocyte-macrophage colony-stimulating factor in the medium in step (C) is preferably 50 to 200 ng / ml, from the viewpoint of maximizing the number of cells produced per culture medium and the amount of stimulating factor. Is preferably in the range of 75 to 150 ng / ml.
  • the content of interleukin 4 in the medium in the step (C) is 5 to 20 ng / ml, preferably 10 from the viewpoint of maximizing the number of production cells per culture medium to be used and the amount of stimulating factor. ⁇ ; A range of 15 ng / ml is desirable.
  • the co-culture time in the step (C) is such that the cell group B is sufficiently differentiated into irregularly shaped dendritic cell-like cells having protrusions, and finally produced in the cells. From the viewpoint of sufficiently improving the ratio of such dendritic cell-like cells, and from the viewpoint of maximizing the number of cells produced per culture medium and the amount of stimulating factor, about 3 to 6 days is desirable.
  • a part of the cell group C obtained in the step (C) shows the nature of dendritic cells, for example, expression of CD40, CD86, etc. As such, it can be obtained as a cell group containing floating cells that are morphologically heterogeneous and show a form having protrusions.
  • the culture vessel used in step (C) is preferably a cell with low cell adhesion, for example, a non-coated polystyrene vessel, but a culture vessel with non-cell-adhesive coating may also be used. It can be used.
  • the cell density of the cell group B in the suspension is determined from the viewpoint of fully exerting the differentiation ability, the culture medium used, and the yield of dendritic cells per stimulating factor. From the viewpoint of maximizing the amount of cells, it is desirable that the solution is 3 X 10 5 to 6 X 10 5 cells / 8 m
  • the culturing time in the step (C) is 2 to 5 days from the viewpoint of producing dendritic cells having stronger T cell response control activity, and the degree of differentiation is evaluated depending on the cell morphology. I want to decide.
  • Dendritic cells obtained by the differentiation method of the present invention have the activity of strongly stimulating T cells by phagocytosing and degrading antigenic proteins and presenting the resulting peptides to T cells.
  • the T cells are killer T cells (Tc) that recognize antigen peptides presented on MHC class I molecules and T helper cells that recognize antigen peptides presented on MHC class II molecules. It is a cell (Th).
  • Th cells When Th cells are activated by presenting antigens from dendritic cells, they produce various site ins, which can activate B cells and macrophages.
  • a tumor necrosis factor is added to the culture obtained by performing the steps (A) to (C).
  • Step (D) By adding ⁇ and lipopolysaccharide and further culturing [Step (D)], it is possible to produce dendritic cells (mature dendritic cells) with improved cell stimulation. In addition, in the step (D), it is possible to produce a preferred dendritic cell (mature dendritic cell) for which it is preferable to further add CD40 ligand.
  • step (D) in addition to granulocyte macrophage colony stimulating factor and interleukin-14 in step (C), tumor necrosis factor ⁇ and lipopolysaccharide are used V. It exhibits the excellent effect of being able to produce dendritic cells (mature dendritic cells) with improved cell-stimulating properties. Further, in the step (D), when a preferable dendritic cell (mature dendritic cell) can be produced by further adding CD40 ligand, an excellent effect is exhibited.
  • the culture vessel used in step (D) and the conditions of the culture gas phase can be appropriately set in the same manner as in steps (i) and (ii) according to the composition of the culture solution.
  • step (D) is the same as in steps (B) and (C) except that tumor necrosis factor a (TNF-a) and lipopolysaccharide or CD40 ligand are further added. ) Is the same as the medium used for co-culture.
  • TNF-a tumor necrosis factor a
  • CD40 ligand lipopolysaccharide or CD40 ligand
  • the culture time in the step (D) may be 2 to 4 days from the viewpoint of producing dendritic cells having stronger T cell stimulating activity.
  • step (D) if it is a factor that promotes dendritic cell maturation, instead of or in addition to tumor necrosis factor a (TNF- ⁇ ) and lipopolysaccharide Alternatively, bacterial extracts, fungal extracts, mycoplasma extracts, double-stranded RNA, CD40 ligand, etc. may be used. In particular, CD40 ligand is preferably used.
  • TNF- ⁇ tumor necrosis factor a
  • CD40 ligand is preferably used.
  • step (D) the amount of TNF-a added is preferably stronger than the T cell stimulating activity! / From the viewpoint of producing dendritic cells, 5 to 20 ng, preferably 7.5 to 20 to 20 ng.
  • step (D) the amount of lipopolysaccharide added is stronger than T cell stimulating activity! From the viewpoint of producing cells, 3 to 5 g, preferably 3 to 4 g is desired per 1 ml of the culture solution.
  • step (D) the amount of CD40 ligand added is stronger than the T cell stimulating activity! / ⁇
  • 10-30 ng with respect to 1 ml of the culture solution Preferably, it is 15 to 25 ng.
  • cells can be recovered from the culture (step (X)).
  • the method of collecting is not particularly limited, and examples thereof include pipetting operations.
  • means for antigen-specific control of an individual's immune response for example, a strong cytotoxic T cell reaction to a specific antigen such as a microbial antigen or a cancer cell antigen.
  • a strong cytotoxic T cell reaction to a specific antigen such as a microbial antigen or a cancer cell antigen.
  • Preventive reactions associated with organ transplantation and cell transplantation, preventive measures for diseases that are expected to have therapeutic effects eg, autoimmune diseases, allergic diseases, etc.
  • a means of treatment and the like can be provided.
  • control of the immune response is intended to mean a concept including both suppression and activation of the immune response.
  • the present invention provides steps (A) to (C) in the differentiation method, and
  • step (D ′) separating dendritic cells derived from human embryonic stem cells from the culture obtained in step (C),
  • a method for producing dendritic cells from human embryonic stem cells A method for producing dendritic cells from human embryonic stem cells.
  • step (E) adding the tumor necrosis factor ⁇ and lipopolysaccharide to the culture obtained in step (C) and further culturing;
  • the method for producing dendritic cells from human embryonic stem cells is preferred. Moreover, it is preferable to further add a CD40 ligand in the step (ii).
  • the production method of the present invention since steps (ii) to (C) of the differentiation method are performed, dendritic cells derived from humans are obtained from the same viewpoint as the differentiation method of the present invention. It has the excellent effect of being able to supply a large amount efficiently and stably.
  • the production method of the present invention is such that cells having the property of inducing differentiation and proliferation of blood cells in step (A) and step (B) are treated with mitomycin C or OP9 cells that have lost their ability to proliferate upon irradiation are preferred! /.
  • the contents of tumor necrosis factor ⁇ , lipopolysaccharide and CD40 ligand in the medium are preferably the same as in step (D) of the differentiation method. .
  • a marker specific to dendritic cells is used. Examples thereof include cell sorting by flow cytometry using an antibody against the antibody, cell sorting using magnetic microbeads coated with the antibody, and the like.
  • markers specific to the dendritic cells include HLA-DR, CD40, CD86, CDla, CD83, CD80, HLA-Classl (HLA-A, HLA-B, HLA-C) and the like. I can get lost.
  • a dendritic cell can exhibit a desired property by introducing a gene such as a therapeutic gene into a human embryonic stem cell as a raw material in advance. Therefore, in another embodiment, the production method of the present invention preferably introduces a nucleic acid containing a gene to be introduced into human embryonic stem cells prior to performing step (A).
  • Examples of the gene to be introduced include the therapeutic gene, specifically, for example, a gene for immunosuppression, a gene for immunostimulation, a gene for an antigen, and the like.
  • genes for antigen loading genes for factors that induce T cell migration, genes for factors that enhance T cell responses, genes for factors that suppress T cell responses, and the like can be mentioned.
  • an antigen is a protein or peptide that is a target of treatment or diagnosis, and includes, for example, various bacteria, various viruses, proteins constituting them, and proteins that are specifically expressed in cancer cells. (Tumor antigen protein), peptides that are part of the tumor antigen protein, molecules that become the target of recognition by the immune system in autoimmune diseases and allergic diseases, and the like.
  • the introduction of the nucleic acid can be performed by a conventional method, for example, the electopore position method, the lipofusion method or the like. From the standpoint of fully exerting the differentiation performance of human embryonic stem cells, the electoral position method is desirable.
  • the nucleic acid may be a so-called naked nucleic acid or may be linked to a plasmid vector! /.
  • the vector may contain elements effective for promoting transcription, such as various promoters and enhancers, if necessary.
  • non-viral vectors such as It is desirable to introduce a plasmid vector using an electoral position.
  • differentiation is induced from conventional peripheral blood monocytes or hematopoietic stem cells, etc., in order to differentiate into dendritic cells by introducing a plasmid vector into the dendritic cells at the stage of human embryonic stem cells.
  • a plasmid vector into the dendritic cells at the stage of human embryonic stem cells.
  • dendritic cells can be stably used for treatment.
  • viral vectors there are restrictions on the number (type) and size (size) of genes that can be introduced.
  • a normal plasmid vector can be used, which is excellent in that a plurality of genes can be introduced by co-transfection of genes or stepwise introduction using a plurality of drug resistance genes.
  • the present invention is excellent in that it can also perform target destruction / modification / introduction of a gene. These properties are particularly useful when more complex genetic modifications are required, such as for the purpose of suppressing or qualitatively controlling the immune response, not just for the purpose of enhancing the immune effect. .
  • the present invention relates to a dendritic cell obtained by the production method.
  • the immune response of an individual can be controlled in an antigen-specific manner (for example, by strongly activating the response of cytotoxic T cells to a specific antigen).
  • Antigen-specific control makes it possible to obtain a therapeutic effect on a disease for which a therapeutic effect is expected.
  • dendritic cells obtained by the above production method in the manufacture of a medicament for the treatment of a disease capable of obtaining a therapeutic effect by specifically controlling the immune response can also be provided.
  • Examples of the "disease whose therapeutic effect can be obtained by antigen-specific control of immune response” include, for example, autoimmune diseases, tumors, allergic diseases, infectious diseases, rejection associated with organ transplantation, and the like. Examples include graft-versus-host disease (GVHD).
  • GVHD graft-versus-host disease
  • an auxiliary agent capable of stably maintaining the dendritic cells of the present invention such as a medium, may be used as appropriate.
  • an immune response control agent comprising dendritic cells obtained by the above production method as an active ingredient can also be provided.
  • the immune response control agent of the present invention since the dendritic cell of the present invention is contained, Response can be suppressed or activated.
  • the immune response control agent of the present invention may contain the auxiliary agent capable of stably holding dendritic cells as an active ingredient.
  • the pharmacological evaluation of the immune response control agent of the present invention can be evaluated using T cell stimulation activity measured by T cell proliferation assay as an index.
  • FIG. 1 shows an outline of the schedule for inducing differentiation of human embryonic stem cells into dendritic cells.
  • the cells collected in Step B were partially frozen and stored.
  • the cells in each step were analyzed using an inverted microscope (trade name: 1X70, manufactured by Olympus Corporation).
  • OP9 cells supplied by RIKEN BioResource Center
  • DMEM fetal calf serum
  • the human embryonic stem cells are suspended in a MEM (a essential medium) supplemented with 20% by volume urine fetal serum (FCS) at a density of 3 ⁇ 10 5 cells // ⁇ medium. It was.
  • FCS urine fetal serum
  • the obtained cell suspension is seeded on the mitomycin C-treated ⁇ 9 cells of the above culture dish, 37 ° C, 5% by volume CO 2.
  • the induction of differentiation of the embryonic stem cells was started.
  • the culture medium was removed and replaced with a new culture medium.
  • Panel A in FIG. 2 is undifferentiated human embryonic stem cells, and panels B to D show the morphology of cells derived from human embryonic stem cells that are differentiating in step (A).
  • Panel B shows cells on day 3 after initiation of differentiation
  • Panel C shows cells on day 11, and
  • Panel D shows day 15 cells.
  • the differentiation from undifferentiated human embryonic stem cells to mesodermal cells progressed as the number of culture days passed, and the density of cells differentiated into mesodermal cells increased.
  • the surface of the feeder layer is almost derived from human embryonic stem cells by about 10 days after the start of differentiation induction Of epithelial cell-like cells.
  • Panel B at the third day after the start of differentiation induction, many cells formed epithelial cell-like large flat cell mass surrounded by round cells.
  • Panel C on the 11th to 12th days after the initiation of differentiation induction, the round cell mass that appeared to have differentiated into mesoderm appeared around the epithelial cell-like cell mass. When the medium was changed every two to three days, the mesoderm-like round cell mass gradually increased.
  • 10 to 20% of the surface of the feeder layer was covered with a mesoderm-like round cell mass 15 days after the initiation of differentiation induction.
  • step (A) of the above (1) cells derived from human embryonic stem cells collected from one culture dish were transplanted onto two culture dishes on a mitomycin C-treated OP9 feeder.
  • Panel E in Fig. 2 shows the morphology of cells differentiated from human embryonic stem cells (5 days after transplantation, 22 days after initiation of differentiation).
  • the cells derived from human embryonic stem cells collected in step (B) of (2) were suspended at a density of 5 ⁇ 10 5 cells / 8 ml culture solution. 8 ml of the obtained cell suspension was transferred to a single culture dish (diameter 6 cm) without stromal (feeder) cells, and further cultured at 37 ° C and 5 vol% CO
  • GM-CSF granulocyte macrophage colony stimulating factor
  • step (C) of (3) in the culture, final concentration 20 ng / ml tumor necrosis factor a (TNF- a; P e protec Inc.) to a final concentration of S ⁇ g / ml Lipopolysaccharide (LP S, derived from E. coli; manufactured by SIGMA) and further cultured at 37 ° C, 5% by volume CO
  • step (A) in Example 1 above in place of mitomycin C-treated OP9 cells, mitomycin C-treated ST2 cells or mitomycin C-treated PA6 cells were used.
  • Step (A) was performed as in the case of the OP9 cells.
  • step (A) Use of mitomycin C-untreated OP9 cells in step (A)
  • Step (A) in Example 1 the case where OP9 cells were pretreated with mitomycin C was compared with the case where V was not pretreated.
  • Step (B) the blood cell differentiation that appeared in step (B) was observed. Spherical cell force shown was very few.
  • step (B) in Example 1 above in the same manner as in the case of OP9 cells, mitomycin C-treated ST2 cells or mitomycin C-treated PA6 cells were used instead of mitomycin C-treated OP9 cells. I did it.
  • step (C) compared to the case of using mitomycin C-treated OP9 cells of Example 1, the number of floating cells obtained after step (C) was higher when mitomycin C-treated ST2 cells or PA6 cells were used. Less than half.
  • step (C) After (number of cells: about 200,000) and step (X) (number of cells: about 200,000), cells were treated in Fc block reagent (Miltenyi Biotec) for 5 minutes. Thereafter, the obtained product was divided into the following fluorescein isothiocyanate (FITC) -conjugated monoclonal antibody (mAb) (manufactured by Pharmagen): anti-human CD80 (clone L307.4, mouse IgGl), anti-human CD83 ( Clone H B15e, mouse IgGl), anti-human CD86 (clone FUN—1, mouse IgGl), anti-human CD40 (clone 5C3, mouse IgGl) and anti-human histocompatibility leukocyte antigen (HLA) —DR (clone L243, mouse Stained with IgG2a). As isotype-matched controls, mouse IgG2a (clone G155-178) and mouse IgGl (clone MOPC-21) were used
  • FIG. 4 shows an outline of the schedule for inducing differentiation from human embryonic stem cells to dendritic cells.
  • step (C) in Example 1 (A) was performed in the same manner as in step (C)
  • the final concentration of 10 ng was added to the culture on the 3rd to 4th day after transplantation in step (C) of Example 1 (3).
  • TNF—a Tumor necrosis factor a
  • LPS lipopolysaccharide
  • the floating cells in the culture obtained in C) were stimulated. As a result, compared to Example 1, the ratio of dendritic cell-like cells contained in the floating cells increased, and some of the attached cells became floating cells.
  • step (C) cell number: about 200,000
  • step (X) cell number: about 200,000
  • Fc block reagent Miltenyi Biotec
  • the resulting product was analyzed using a cell analysis device equipped with CellQuest software (Example 2) except that a FIT C-conjugated anti-human HLA-Class I monoclonal antibody (clone G46-2. 6 BD Biosciences) was used.
  • a FIT C-conjugated anti-human HLA-Class I monoclonal antibody clone G46-2. 6 BD Biosciences
  • FACScan manufactured by Becton Dickinson
  • the human embryonic stem cell-derived dendritic cell-like cells recovered in step (X) express CD40, CD80, CD83, CD86, HLA-DR, or HLA-classI, respectively, and in step (C) It was expressed higher than cells (right column).
  • This can be differentiated into more mature dendritic cells when additional CD40 ligand is added, and by using flow cytometry, human embryonic properties can be achieved in steps (D ') and (F) of the present invention. It means that stem cell-derived dendritic cells can be separated.
  • TNF tumor necrosis factor
  • IL interleukin
  • CD40 ligand final concentration 20 ng / ml; manufactured by Peprotec
  • LPS final concentration 3 g / ml
  • OK432 Group A type 3 Streptococcus pyogenes Su strain penicillin lyophilized powder Product name:
  • Density of dendritic cell-like cells were cultured in 1. 2xl 0 5 pieces of conditions per 150 1 of culture solution. ELISA was performed using a kit manufactured by Pierce. As a result, as shown in FIG. 6, dendritic cell-like cells derived from human embryonic stem cells produce TNF- ⁇ upon stimulation of lipopolysaccharide and IL-12 upon stimulation of ⁇ 432. Confirmed to do. This means that the dendritic cell-like cells derived from human embryonic stem cells are stimulated with a foreign substance, and the force S is generated to produce cyto force-in.
  • Dendritic cell power derived from human embryonic stem cells S T cells are stimulated to proliferate, whether they have the activity to proliferate, T cells when co-cultured with Aro (allogeneic) T cells The growth was examined by quantification. Human reactive blood T cells were used as reactive T cells. Also, As the stimulator cells, use the cells obtained by X-ray irradiation (40 Gy) of each of the cells after step (C) and after step (C) on the 8th day of step (B) obtained in Example 1. It was.
  • Mononuclear cell groups were isolated from human heparinized blood using a trade name: Ficoll-Paque PLUS (manufactured by Amersham Biosciences).
  • CD14-cells were isolated from a mononuclear cell group using magnetic beads coated with an anti-human CD14 antibody (trade name, supermagnetic Micro Beads, manufactured by Miltenyi Biotec).
  • an anti-human CD14 antibody trade name, supermagnetic Micro Beads, manufactured by Miltenyi Biotec.
  • these molecules are expressed using the magnetic beads as described above.
  • the cell group was removed and the remaining cells were used as sputum cells.
  • Example 1 step (X) For the dendritic cells obtained in Example 1 step (X), the antigen presenting ability to the human T cell line SA32.5 which specifically recognizes the GAD65 antigen-derived peptide and shows a proliferative reaction was examined.
  • the dendritic cells obtained in Step (X) of Example 1 take up protein antigens into the cells, restrict them to produce peptides, and display them on HLA-DR molecules on the cell surface. Whether or not it has the activity to be examined.
  • the DNA fragment encoding the GAD65 p96-174 protein fragment was ligated to the prokaryotic expression vector pGEX-4T-3 (Amersham Biosciences), and then the resulting vector was used to isolate E. coli DH5a. Transformed, thereby the E. coli DH In 5 ⁇ , gnoretathione S transferase fusion GAD65 protein (GST-GAD) was expressed. Recombinant proteins were extracted from bacterial inclusion bodies by inducing recombinant protein production in Escherichia coli DH5 ⁇ by the method reported by Fragioni and Neel [Anal. Biochem., 210, 179 187 (1993)].
  • the recombinant protein was purified using dartathionagarose (manufactured by SIGMA). The purity and amount of the fusion protein were confirmed by sodium lauryl sulfate polyacrylamide gel electrophoresis. The obtained recombinant protein was concentrated using a trade name: Centricon-lC Millipore, separated from a low molecular weight peptide fragment, and the buffer was replaced with a medium by dialysis.
  • step (X) of Example 1 The dendritic cells obtained in step (X) of Example 1 were irradiated with X-rays (40 Gy), and the cells that lost their proliferation ability were antigen-presenting cells. Used as
  • SA32.5 (3 X 10 4 / well) and dendritic cells (3 X 10 4 / well) in the presence of GST or the GST-GAD protein obtained in (1) above (0 ⁇ 01-3 ⁇ 1), 96-well plate flat culture plate, RPMI-1640 medium supplemented with 10% human plasma, cultured at 37 ° C, 5% CO by performing T cell proliferation assay It was.
  • a plasmid vector was introduced into human embryonic stem cells by electroporation.
  • undifferentiated human embryonic stem cells maintained on mouse embryo-derived fibroblasts were harvested, the Darube'co's Modified Eagle's culture medium (DMEM) O. 2 ml, floated 2 X 10 6 cells, plasmid DNA 60 ⁇ g was added, and electroporation was performed with a 4 mm gap cuvette under the conditions of 150 V and 150 ⁇ F.
  • DMEM Darube'co's Modified Eagle's culture medium
  • floated 2 X 10 6 cells floated 2 X 10 6 cells
  • plasmid DNA 60 ⁇ g was added, and electroporation was performed with a 4 mm gap cuvette under the conditions of 150 V and 150 ⁇ F.
  • a gene pulser manufactured by Bio-Rad was used as the electroporation apparatus.
  • the cells were cultured in a culture medium containing a selective drug G418 (20011 g / ml), and then a gene transfer cell clone was isolated.
  • a gene transfer cell clone was isolated.
  • pi i-GAD a vector that has the effect of presenting human glutamate decarboxylase 65 (GAD65) pi ll- 131 (LQ DVMNILLQYVVKSFDRSTK; SEQ ID NO: 1) on the HLA class II molecule, was used. . From this vector, a protein having a structure in which a part of the human invariant chain is replaced with a GAD peptide is expressed.
  • the transgenic human embryonic stem cell clone obtained in Example 9 above was differentiated into dendritic cells in the same manner as in Example 1, and the GAD65 antigen-derived peptide used in Examples 7 and 8 was specifically used.
  • dendritic cells derived from human embryonic stem cells can be efficiently and stably supplied in large quantities.
  • FIG. 1 shows a schematic diagram of a differentiation method (schedule) from human embryonic stem cells to dendritic cells.
  • FIG. 2 shows micrographs of cells over time when human embryonic stem cells were differentiated into dendritic cells.
  • panel A is before differentiation induction
  • panel B is day 3 after differentiation initiation
  • panel C is day 11
  • panel D is day 15
  • panel E is day 22 (steps) B Day 5)
  • Panel F is Day 26 (Step C Day 1)
  • Panel G is Day 28 (Step C Day 3)
  • Panels H and I are Day 32 (Step D Day 4) Eye) cells.
  • Fig. 3 shows suspension cells in the culture medium after differentiation from human embryonic stem cells to dendritic cells (Step C-derived cells (left column) and Step X-derived cells (right column in Fig. 1)). Column)) shows the results of analyzing the expression of cell surface molecules CD80, CD83, CD86, CD40, and HLA-DR (vertical axis: number of cells, horizontal axis: fluorescence intensity).
  • the histogram power S of the thick solid line S the fluorescence intensity of the dye stained with the antibody specific to the molecule to be analyzed
  • the thin line histogram is the control with the isotype antibody, non-specific The fluorescence intensity in the case of simple antibody staining is shown.
  • FIG. 4 shows a schematic diagram of a differentiation method (schedule) from human embryonic stem cells to dendritic cells.
  • Figure 5 shows suspension cells (Step C-derived cells in Figure 4 (left column) and Step X-derived cells (right) in Figure 4) when human embryonic stem cells were differentiated into dendritic cells. Column)) shows the results of analyzing the expression of cell surface molecules CD80, CD83, CD86, CD40, HLA-DR, and HLA-Classl (vertical axis: number of cells, horizontal axis: fluorescence intensity).
  • a thick solid line histogram shows the fluorescence intensity of the antibody stained with the molecule specific to the analysis target, and the thin line histogram is a control with an isotype antibody and non-specific. The fluorescence intensity in the case of antibody staining is shown.
  • the left column (excluding HLA-Classl) in Fig. 3 and Fig. 5 shows the same data.
  • FIG. 6 shows dendritic cell-like cells derived from human embryonic stem cells obtained in Example 3, that is, cells after step (C), the same as 040 ligand (2013 ⁇ 4 / 1111), lipo Stimulate with polysaccharide (1 ⁇ 3: 3; ⁇ / 1111) or 0X432 (10 / ⁇ / 1111), collect the culture after 72 hours
  • FIG. 6 is a graph showing the results of measuring the concentrations of TNF-a and IL 12 in a culture solution by the LISA method.
  • Fig. 7 is a graph showing the results of analysis of aro (allogeneic) T cell stimulating activity by cells induced to differentiate from human embryonic stem cells.
  • circles indicate floating cells in the culture medium after step (X)
  • diamonds indicate floating cells in the culture medium after step (C)
  • squares indicate the 8th day of step (B). Shows floating cells in the culture medium, and shows the T cell proliferation response when each floating cell is irradiated with X-rays and used as stimulating cells.
  • Fig. 8 shows the results of examining the antigen-presenting ability of dendritic cells differentiated from human embryonic stem cells.
  • Panel A shows a T cell that recognizes the GA D65 antigen-derived peptide by loading differentiated dendritic cells with a GAD65 antigen-derived peptide (concentration 6 M), irradiating with X-rays, thereby deteriorating the proliferation ability.
  • the growth response of SA32.5 when cultured with strain SA32.5 (squares) is shown.
  • panel A the value obtained by measuring the incorporation of thymidine into chromosome DNA by SA32.5 using a scintillation counter for / 3-wire measurement is shown.
  • FIG. 1 As a negative control, dendritic cells without GAD65 antigen-derived peptide are cultured with SA32.5 (diamonds).
  • Panel B shows a culture containing GST-fused GAD65 protein antigen (GST-GAD) produced by genetic recombination by irradiating dendritic cells differentiated from human embryonic stem cells with X-rays. The growth response of SA32.5 when cultured with T cell line SA32.5 in the solution is shown (square). In panel B, as in panel A, the value obtained by quantifying the proliferation reaction by 3 ⁇ 4 thymidine incorporation is shown. As a negative control, the results when cultured in a culture medium containing GST protein instead of GST-GAD are shown (diamonds).
  • FIG. 9 shows stimulation of GAD65-specific T cells by dendritic cells derived from human embryonic stem cells into which a gene expression vector that presents a peptide antigen derived from GAD65 has been introduced.
  • Differentiation-induced dendritic cells are irradiated with X-rays, so that the proliferation ability is lost, and the proliferation response of SA32.5 is shown when cultured with the T cell line SA32.5 that recognizes GAD65 antigen-derived peptide (square). ).
  • SA32 According to 5, the uptake of 3 H-thymidine chromosomal DNA, (a value measured using a scintillation counter for three-wire measurement. Further, as a negative control, human embryo that does not introduce a gene expression vector Dendritic cells derived from sex stem cells The results when cultured with 32.5 are shown (diamonds).
  • SEQ ID NO: 1 is a partial sequence of human GAD65 protein

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Abstract

Procédé de différenciation de cellules dendritiques issues de cellules souches d'embryons humains, comprenant les étapes consistant à : (A) obtenir un groupe de cellules A en co-cultivant des cellules souches d'embryons humains et des cellules ayant la propriété d'induire une différentiation et une prolifération de cellules sanguines ; (B) obtenir un groupe de cellules B en co-cultivant le groupe de cellules A obtenu à l'étape (A) et les cellules ayant une propriété d'induction de la différentiation et de la prolifération de cellules sanguines en présence d'un facteur de stimulation des colonies de granulocytes-macrophages et d'un facteur de stimulation des colonies de macrophages ; et (C) cultiver le groupe de cellules B obtenues à l'étape (B) en présence d'un facteur de stimulation des colonies de granulocytes-macrophages et d'interleukine 4.
PCT/JP2007/071700 2006-11-08 2007-11-08 Procédé de production de cellules dendritiques à partir de cellules souches d'embryons humains WO2008056734A1 (fr)

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JP5861191B2 (ja) * 2010-09-30 2016-02-16 国立大学法人 熊本大学 ミエロイド系血液細胞の製造方法
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WO2020045368A1 (fr) 2018-08-27 2020-03-05 マイキャン・テクノロジーズ株式会社 Procédé d'évaluation de médicaments anti-infectieux, vaccins, etc. utilisation de cellules monocytaires immortalisées et de cellules induites

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