WO2006022330A1 - 霊長類動物胚性幹細胞から樹状細胞の製造方法 - Google Patents
霊長類動物胚性幹細胞から樹状細胞の製造方法 Download PDFInfo
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- 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 primate embryonic stem cells into rod cells, a method for producing rod cells from primate embryonic stem cells, the rod cells obtained by the method, and the rod shape Cells, use of said rod cells for the manufacture of a medicament for the treatment of a disease capable of obtaining a therapeutic effect by antigen-specific control of the immune response, and a cellular medicament for the treatment of the disease About.
- Spider cells phagocytose antigen proteins, break them down into peptides, and present the resulting peptides to T cells as a complex with the major histocompatibility antigen (MHC) (hereinafter also referred to as “antigen presentation”). )
- MHC major histocompatibility antigen
- the rod-shaped cells are cells having the highest antigen-presenting ability in the living body.
- the above-mentioned rod cells suppress the function of T cells reactive to self antigens and are also involved in maintaining immunological self tolerance. Thus, rod cells play a central role in regulating the immune response in vivo.
- rod-shaped cells are also produced by differentiation of hematopoietic stem cell force in the bone marrow.
- Hematopoietic stem cells in the bone marrow differentiate into erythrocytes, platelets, neutrophils, eosinophils, basophils, macrophages, lymphocytes, etc., in addition to rod-like cells, due to the presence of growth factors.
- rod cells can be obtained by isolating existing rod cells from peripheral blood, inducing differentiation of hematopoietic stem cells in bone marrow (for example, Patent Document 1). ing.
- Patent Document 1 discloses that mouse hematopoietic stem cells are induced to differentiate to obtain rod-shaped cells.
- hematopoietic stem cells are difficult to grow for a long period of time, and it is necessary to use a viral vector for gene transfer into rod-shaped cells prepared by this method. is there.
- Non-patent Document 1 attempts have been made to obtain rod-shaped cells by inducing mouse embryonic stem cells (Non-patent Document 1).
- Non-patent Document 1 when the method described in Non-Patent Document 1 is applied to other organisms, for example, primates, there is a disadvantage that mature rod-shaped cells do not occur.
- Patent Document 1 Japanese Patent Application Laid-Open No. 2004-16697
- Non-Patent Document 1 S. Senju et al., Blood, 101, pp. 3501 to 3508, 2 May 2003
- One aspect of the present invention is to efficiently obtain a rod-like cell derived from a primate animal, to stably supply a rod-like cell of a primate animal, and to make an immune response of an individual antigen specific.
- Supplying a means to control for example, a means to strongly activate a cytotoxic T cell response to a specific antigen
- a disease for which a therapeutic effect is expected by antigen-specific control of an immune response for example, self At least one of the following: providing treatment for immune diseases, allergic diseases, etc., and providing for the prevention or treatment of rejection in organ transplantation and Graft versus Host Diosease (GVHD)
- the present invention relates to providing a method for dividing primate animals into embryonic stem cell force rod-shaped cells.
- another aspect of the present invention is to obtain a large amount of rod-like cells derived from primates, to obtain rod-like cells derived from primates efficiently, A stable supply of cells, and to obtain rod-shaped cells that specifically control an individual's immune response (for example, rod-shaped cells that strongly activate the response of cytotoxic T cells to a specific antigen).
- the present invention relates to providing a method for producing rod-shaped cells from primate embryonic stem cells, which enables at least one of the following.
- Yet another aspect of the present invention is to control an individual's immune response in an antigen-specific manner (for example, to strongly activate a cytotoxic ⁇ cell response to a specific antigen), an immune response
- the present invention relates to providing a rod-shaped cell that enables at least one of obtaining a therapeutic effect on a disease for which a therapeutic effect is expected by antigen-specific control of the antigen.
- the preparation for the manufacture of a medicament that enables at least one of treatments for diseases for which a therapeutic effect is expected by antigen-specific control of an immune response is provided. Relates to providing the use of dendritic cells.
- the present invention it is possible to at least one of antigen-specific control of immune response, 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) a step of obtaining a cell group B by co-culturing the cell group A obtained in the step (A) and a newly prepared cell having a property of inducing differentiation and proliferation of blood cells;
- step (D) culturing the cell group C obtained in the step (C) in the presence of granulocyte macrophage colony stimulating factor and interleukin 4,
- step (A) from the co-culture of primate embryonic stem cells and cells having the property of inducing differentiation and proliferation of blood cells to primate embryonic stem cells.
- Cells having the property of inducing differentiation and proliferation of blood cells in step (A) are ST2 cells, and newly prepared having the property of inducing differentiation and proliferation of blood cells in step (B) (1) or (2) above, wherein the newly prepared cells that are OP9 cells and have the property of inducing differentiation and proliferation of blood cells in step (C) are OP9 cells.
- Differentiation method
- step (E) adding the tumor necrosis factor ⁇ and lipopolysaccharide to the culture obtained in step (D) and further culturing, [1] ⁇ [3] Any Or the differentiation method according to claim 1,
- step (B) a step of obtaining a cell group B by co-culturing the cell group A obtained in the step (A) and a newly prepared cell having a property of inducing differentiation and proliferation of blood cells;
- step (D) a step of culturing the cell group C obtained in the step (C) in the presence of a granulocyte macrophage colony stimulating factor and interleukin 4, and ( ⁇ ⁇ ′) obtained in the step (D). Isolating rod-shaped cells derived from primate embryonic stem cells from the culture;
- step (Ii) adding the tumor necrosis factor ex and lipopolysaccharide to the culture obtained in step (D) and further culturing;
- step (F) separating a rod-shaped cell derived from a primate embryonic stem cell from the culture obtained in step (ii);
- the therapeutic effect can be obtained by antigen-specifically controlling the immune response, comprising the rod-shaped cells obtained by the production method according to any one of [5] to [10] as an active ingredient.
- Cell medicines for the treatment of diseases obtainable,
- the differentiation method of embryonic stem cells from rodent stem cells of the primate of the present invention into rodent cells the excellent effect of efficiently and stably supplying rodent cells derived from primate animals is achieved. Play.
- a means for specifically controlling an individual's immune response for example, a primate rod-like cell, a cytotoxic ⁇ cell response to a specific antigen is strongly enhanced. It is possible to provide therapeutic means for diseases (for example, autoimmune diseases, allergic diseases, etc.) that are expected to have therapeutic effects by antigen-specific control of immune responses! Play.
- a rod-like cell derived from a primate animal can be efficiently and stably supplied in large quantities. There is an excellent effect.
- a rod-shaped cell that specifically controls an individual's immune response eg, a rod-shaped cell that strongly activates a cytotoxic ⁇ cell response to a specific antigen
- I can do this.
- an individual's immune response can be controlled in an antigen-specific manner (for example, it can strongly activate a cytotoxic ⁇ cell response to a specific antigen). It has an excellent effect that it can obtain a therapeutic effect on a disease for which a therapeutic effect is expected by controlling antigen-specific immune response of the body and antigen-specific control of immune response.
- the immune response control agent of the present invention can be antigen-specifically controlled. For example, it is possible to treat diseases such as autoimmune diseases and allergic diseases! / ⁇ ⁇ Excellent effect.
- FIG. 1 shows a schematic diagram of an example of a method for differentiation of embryonic stem cells into rod-shaped cells in primates.
- FIG. 2 is a diagram showing micrographs of cells over time when differentiated into primate embryonic stem cell-powered rod cells.
- panel A is before the start of differentiation induction
- panel B is the third day after the start of differentiation induction
- panels C and D are the sixth day
- panels E and F are the ninth day
- panel No. Gi 16
- the cells of day 28, day No. Hi, 22th, Noh Noh, 26th, Noh No J, K and U are shown.
- the scale bar indicates 100 ⁇ m.
- FIG. 3 shows the results of analyzing the expression of cell surface molecules DR, CD40 and CD86 in suspension cells in culture medium when primate embryonic stem cells were differentiated into rod-shaped cells.
- FIG. 6 is the end of step (B) in FIG. 1
- Day22 is the end of step (C)
- Day26 is the end of step (D)
- Day28 is the end of step E.
- the cell analysis results at the end are shown.
- a thick solid line histogram shows the fluorescence intensity of the antibody stained for the molecule to be analyzed
- the thin dotted line histogram is a negative control and non-specific. The fluorescence intensity in the case of antibody staining is shown.
- FIG. 4 shows the results of analyzing the expression of CD80 and IL-12p40 mRNA by RT-PCR.
- mRNA for HPRT indicates the relative amount of RNA sample used for analysis.
- FIG. 5 is a graph showing the results of analysis of T cell stimulating activity by cells induced to differentiate from embryonic stem cells of primates.
- diamonds represent floating cells in the culture medium after step (D)
- white circles represent floating cells in the culture medium after step (E)
- squares represent undifferentiated embryonic stem cells.
- FIG. 6 is a diagram showing the results of examining the expression of a foreign gene in cells induced to differentiate from a primate embryonic stem cell into which a foreign gene has been introduced.
- Panel A shows the structure of a plasmid DNA vector encoding the 13 strand of the HLA-DR53 molecule.
- Panel B The results of detection of mRNA derived from the transgene by RT-PCR are shown.
- cES is an undifferentiated embryonic stem cell
- cES—53-23 is an undifferentiated embryonic stem cell into which a gene has been introduced
- cES—DC is an embryonic stem cell force distribution.
- the obtained rod cells, “cES-DC-53-23” indicate rod cells that have been induced to differentiate from the embryonic stem cells into which the gene has been introduced.
- HP RT mRNA indicates the relative amount of RNA sample used in the analysis.
- FIG. 7 shows the results of examining the antigen-presenting ability of rod-shaped cells differentiated from embryonic stem cells into which DR53 ⁇ gene was introduced.
- Panel ⁇ is loaded with GAD65 antigen-derived peptides (concentrations 1.25, 2.5, 5. ⁇ / ⁇ ⁇ ) on rod-shaped cells differentiated from DR53 ⁇ -transfected embryonic stem cells and irradiated with X-rays
- GAD65 antigen-derived peptides concentration 1.25, 2.5, 5. ⁇ / ⁇ ⁇
- primate embryonic stem cells and cells having the property of inducing differentiation and proliferation of blood cells are co-cultured to obtain a cell group ⁇
- B Cell group A obtained in step (A) above and the property of inducing differentiation of blood cells.
- step (D) culturing the cell group C obtained in the step (C) in the presence of granulocyte macrophage colony stimulating factor and interleukin 4,
- the present invention relates to a method for separating embryonic stem cell power rod-shaped cells of primates.
- the separation method of the present invention has one major feature in that primate embryonic stem cells (ES cells) are used. Therefore, according to the differentiation method of the present invention, an excellent effect of being able to be supplied in a large amount is exhibited as compared with the case of isolating rod-shaped cells from a living body or the case of differentiation from hematopoietic stem cells. Further, according to the differentiation method of the present invention, since the ES cell is used, a gene expected to show a therapeutic effect (hereinafter also referred to as “therapeutic gene”) can be easily introduced if necessary. It exhibits an excellent effect that it can be expressed. Furthermore, according to the present invention, since primate ES cells are used, it is suitable for application to so-called cell therapy.
- ES cells primate embryonic stem cells
- the ES cell force of the mouse is different from that for dividing cells into rod cells.
- ES cell of a primate animal is used.
- One major feature is that co-cultured cells with cells that have the property of inducing the differentiation and proliferation of blood cells.
- ES cells can be differentiated into mesodermal cells.
- the term “primate animal” includes humans, monkeys, and the like.
- the monkeys include power-cynomolgus monkeys, rhododendron monkeys, diphone monkeys, marmoset, chimpanzees and the like.
- the primate ES cells used in the present invention are not particularly limited, and examples thereof include force-quizal ES cell line CMK6 cells, human ES cells and the like.
- the ES cells can be appropriately selected depending on the intended use of the obtained rod-shaped cells.
- Examples of the "cell having the property of inducing differentiation and proliferation of blood cells” include, for example, OP9 cell (RIKEN BIORESOURCE Center (RIKEN BIORESOURCE CENTER: 305-0074 Ibaraki, Tsukuba Takanodai, 3-chome, 1-1-1) Cell number: R CB1124), ST2 cells (RIKEN BIORESOURCE CENTER cell number: RCB0224), PA6 cells (RIKEN BioResource Center ( RIKE N BIORESOURCE CENTER) cell number: RCB1127) and the like.
- ST2 cells are preferable from the viewpoint of improving the efficiency of inducing differentiation into blood cells.
- the “feeder cell” when co-culturing the “primate animal ES cell” and the “feeder cell”, the “feeder cell” is cultured in an appropriate medium.
- the cells can be cultured under the culture conditions corresponding to the feeder cell and grown to an extent that substantially covers the bottom surface of the culture container, thereby forming a feeder cell layer.
- the medium that can be used for the production of the feeder cell is appropriately selected according to the type of the 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 modified Eagle medium (culture medium)] and the like.
- the culture vessel used in the step (ii) is a tissue culture culture from the viewpoint of promoting the differentiation of ES cells into mesodermal cells, the viewpoint of stably adhering feeder cells for a long period of time, and the like. If it is a culture vessel with a coating suitable for it.
- the coating can be performed with, for example, gelatin, fibronectin or the like.
- the culture condition of the feeder cell can be appropriately set according to the type of the cell used as the feeder cell. For example, in the case of ST2 cells, ⁇ 9 cells, etc., in a culture container coated with 0.1 wt% gelatin solution etc., culture at 37 ° C, 5 vol% CO in a medium supplemented with 10 vol% urchin fetal serum The conditions to do are mentioned.
- the cell density of the ES cell maximizes the number of mesodermal cells obtained per medium (culture solution) from the viewpoint of sufficiently exerting the differentiation ability.
- the culture vessel is preferably a culture vessel of 5 ⁇ 10 4 to 1 ⁇ 10 5 cells / diameter 100 mm
- the medium used for the co-culture of ES cells and feeder cells in the step (A) may be any ES cell species that can be used as long as it is a culture solution suitable for culturing mammalian cells. It can be appropriately selected depending on the type, and examples thereof include a MEM, DMEM, IMDM (Iscob modified Dulbecco medium) and the like. Such a medium can also be used in the following steps (B) to (E).
- the conditions of the culture gas phase during the co-culture of the ES cells and the feeder cells in the step (A) can be appropriately set according to the type of ES cells used, the composition of the culture solution, and the like. For example, conditions such as around 37 ° C (particularly 37 ° C), 5 vol% CO, and the like can be mentioned.
- the co-culture time of the ES cell and the feeder cell in the step (A) is such that the ES cell is sufficiently differentiated into a cell differentiated into the mesoderm system, and the cell differentiated into the mesoderm system is used. In order to maximize the number, 9 to 11 days are preferred.
- step (A) the medium is changed every other day after the fourth day after the start of the culture.
- the cell group A obtained in the step (A) shows properties of mesodermal cells and may vary depending on the type of animal from which the ES cells used are used. It can be obtained as a cell group containing a mass of cells exhibiting
- embryos of primates Cell group A is isolated as a cell group containing cells derived from primate embryonic stem cells and differentiated into mesodermal cells from a co-culture of sexual stem cells and the feeder cell I prefer that.
- Isolation of the cell group A having the co-culture force may be performed by, for example, adding phosphate buffered saline containing 0.25 wt% trypsin ZlmM ethylenediaminetetraacetic acid (EDTA) to the co-culture, and It can be carried out by treating with C for a time suitable for separating cells differentiated into mesodermal cells, for example, for 8 to 15 minutes.
- EDTA ethylenediaminetetraacetic acid
- the cell group A obtained in the step (A) is a newly prepared cell (feeder cell) that induces differentiation and proliferation of blood cells.
- feeder cell a newly prepared cell that induces differentiation and proliferation of blood cells.
- the ratio of mesoderm cells, in particular, blood cells can be further improved, so that rod-like cells derived from primates are used. Can get more efficiently in And exhibits an excellent effect of being able to supply stably.
- the feeder cell mentioned in the step (A) can be used.
- OP 9 Cells are desirable.
- the “feeder cell” is used as the “feeder cell” used in the step (A). Similar to “cells”, the cells can be grown to the extent that they almost cover the bottom surface of the culture vessel, thereby forming a feeder-one cell layer.
- the culture conditions used for the culture vessel and the “feeder single cell” used in the step (B) are the same as those in the step (A).
- step (B) the cells were collected from step (A) from the viewpoint of sufficiently exerting the ability to separate and the number of produced cells per culture medium used.
- the cell group A has a larger area than in the case of step (A), specifically about 4 to 8 times the culture vessel in step (A) (for example, the area of the feeder cell layer is the same as in step (A). 4 to 8 times the case).
- the conditions of the culture gas phase during the co-culture of the cell group A and the feeder cell in the step (B) depend on the type of ES cell used, the composition of the culture solution, and the like. ) Can be set as appropriate.
- the medium used in the step (B) is the same as the medium used for the co-culture in the step (A).
- the co-culture time of the ES cell and the feeder cell in the step (B) is such that the ES cell is sufficiently differentiated into a mesodermal cell, particularly a blood cell, and a mesodermal cell,
- a mesodermal cell particularly a blood cell
- a mesodermal cell particularly a blood cell
- a mesodermal cell particularly a blood cell
- a mesodermal cell particularly a blood cell
- step (B) medium replacement or medium addition may be performed as appropriate during the culture period.
- the cell group B obtained is a mesodermal cell, particularly a blood cell.
- a mesodermal cell particularly a blood cell.
- it has a relatively uniform, small and round shape as shown in panel G of Fig. 2, although it may vary depending on the type of animal from which the ES cells are used. It can be obtained as a group of cells containing the floating cells shown.
- floating cells are collected by pipetting or the like, so that cells that have been differentiated in a direction other than insufficiently differentiated cells and cells other than blood cells (for example, including epithelial cells)
- the cell group sorted in the direction of the blood cell lineage cell can be efficiently collected.
- the differentiation method of the present invention the cell group B obtained in the step (B), and a newly prepared cell (feeder) having the property of inducing differentiation and proliferation of blood cells. 1 cell) is co-cultured in the presence of granulocyte-macrophage colony-stimulating factor [Step (C)]. Therefore, according to the differentiation method of the present invention, by performing such step (C), it is possible to induce the differentiation into the myeloid (myelocyte) system and the differentiation into the rod-like cells. Therefore, it is possible to obtain rod-like cells derived from primates more efficiently and to provide a stable supply.
- granulocyte-macrophage colony-stimulating factor [Step (C)]. Therefore, according to the differentiation method of the present invention, by performing such step (C), it is possible to induce the differentiation into the myeloid (myelocyte) system and the differentiation into the rod-like cells. Therefore, it is possible to obtain rod-like cells derived from primates more efficiently and to provide a stable supply.
- the culture vessel used in the step (C) is preferably one coated with gelatin, fibronectin or the like from the viewpoint of stably attaching feeder cells for a long period of time.
- the feeder cell mentioned in the step (A) can be used, for example, OP9 cells are preferred.
- the conditions of the culture gas phase during the co-culture in step (C) are the same as in steps (A) and (B) depending on the type of ES cells used, the composition of the medium, and the like. It can be set as appropriate.
- the medium used in step (C) is the same as the medium used for the co-culture in steps (A) and (B) except that it contains granulocyte-macrophage colony-stimulating factor.
- the content of granulocyte-macrophage colony-stimulating factor in the medium in the step (C) is selected from the viewpoint of maximizing the number of cells produced per culture medium and the amount of stimulating factor used. A range of ⁇ 200ngZml is desired!
- the cell density of the cell group B is from the viewpoint of sufficiently exerting the differentiation ability, from the viewpoint of maximizing the number of cells produced per amount of the culture medium to be used and the stimulating factor. (3 x 10 5 to 6 x 10 5 Z8ml medium Z diameter 100mm culture dish)!
- the co-culture time in the step (C) is such that the cell group B is sufficiently differentiated into irregularly shaped rod-like cells having protrusions, and finally produced in the cells. From the viewpoint of sufficiently improving the ratio of such rod-like cells and from the viewpoint of maximizing the number of cells produced per culture medium and the amount of stimulating factor, 4 to 6 days is desirable.
- a macrophage colony-stimulating factor may be used from the viewpoint of promoting differentiation into myeloid cells.
- the content of the macrophage colony stimulating factor in the medium in the step (C) is preferably in the range of 50 to 200 ngZml from the viewpoint of strong T cell stimulating activity and production of rod-shaped cells.
- step (C) from the viewpoint of sufficiently improving the proportion of the rod-like cells that are finally produced in the cells, preferably on the third or fourth day, It is desirable to add the medium containing granulocyte macrophage colony stimulating factor and no macrophage colony stimulating factor to the culture in half the original medium (4 ml Z diameter 100 mm culture dish).
- step (C) unlike the case of mouse ES cells, by adding a macrophage colony-stimulating factor temporarily at the time of induction of differentiation into rod cells, It is possible to further improve the efficiency of induction of the rod-shaped cells.
- step (C) during the culture period, the medium is replaced with a medium containing granulocyte macrophage colony stimulating factor and not containing macrophage colony stimulating factor in the medium, or granulocyte macrophage colony stimulating factor is replaced. It is also possible to add a medium that contains and does not contain a macrophage colony stimulating factor.
- part of the cell group C obtained is an ES cell that exhibits the properties of rod-shaped cells, such as the expression of DR, CD40, CD86, CD80, etc. Forces that may vary depending on the animal species from which they originate, for example as shown in panel H of Figure 2 In addition, it can be obtained as a group of cells containing floating cells that are morphologically heterogeneous and have protrusions.
- the cell group C obtained in the step (C) is added in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4).
- IL-4 interleukin-4
- the culture vessel used in the step (D) should have a low cell adherence, for example, a coated / polystyrene vessel is desirable, but has a coating power for cell culture. Cultured containers can also be used.
- the conditions of the culture gas phase during the culture in step (D) are the same as those in steps (A) and (B), depending on the type of ES cells used, the composition of the culture medium, and the like. Can be set as appropriate.
- step (D) The medium used in step (D) is used for the co-culture of steps (A) and (B) except that it contains granulocyte-macrophage colony-stimulating factor and interleukin-14. Same as the medium.
- the cell density of the cell group C is used from the viewpoint of sufficiently exerting differentiation ability, and from the viewpoint of maximizing the yield of rod-shaped cells per culture medium to be used and stimulating factor. From the above, it is desirable that 3 10 5 -6 10 5 cells 72.5 ml culture solution Z culture diameter 60 mm culture vessel.
- the culture time in step (D) is 2 to 5 from the viewpoint of further improving the proportion of rod-like cells in cell group C and producing rod-shaped cells with stronger T cell stimulating activity. It is desirable to evaluate the degree of differentiation according to the cell morphology and determine it appropriately within a day.
- the content of granulocyte-macrophage colony-stimulating factor in the medium in the step (D) is preferably in the range of 50-200 ng Zml from the viewpoint of maximizing the yield of rod cells per stimulating factor used.
- the content of interleukin-4 in the medium in the step (D) is preferably 5 to 20 ngZml from the viewpoint of producing rod-shaped cells with stronger T cell stimulating activity.
- step (D) MHC class II of the finally produced rod-shaped cells is used.
- FLT-3L fms-like tyrosine kinase 3 ligand
- IL 4 is not essential for the differentiation induction of rod cells, and when IL 4 is added to rod cells at an immature stage, the final state of rod cells Force that prevents maturation in the step In step (D), IL 4 is essential in the process until differentiation is induced in the rod cells.
- step (D) it is possible to obtain rod-shaped cells exhibiting the properties of rod-shaped cells, for example, the expression of DR, CD40, CD86, etc., morphologically heterogeneous, and having protrusions.
- the rod-shaped 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.
- Tc killer T cells
- Th cells When Th cells are activated by presenting antigens from rod cells, they produce various cytokines, which can activate B cells and macrophages.
- the rod-shaped cells obtained by performing the steps (A) to (D) are added with a tumor necrosis factor ex and lipopolysaccharide, and further subjected to a culturing step [step (E).
- a tumor necrosis factor ex and lipopolysaccharide are added with a tumor necrosis factor ex and lipopolysaccharide, and further subjected to a culturing step [step (E).
- the tumor necrosis factor a and lipopolysaccharide are used in addition to the interleukin-1 and the optional fms-like tyrosine kinase 3 ligand in the step (D). Therefore, it is possible to produce rod-shaped cells (mature rod-shaped cells) with improved T cell stimulating properties!
- the culture vessel used in step (E) and the conditions of the culture gas phase are the same as those in steps (A) and (B) depending on the type of ES cells used, the composition of the culture medium, and the like. Can be set as appropriate.
- the medium used in the step (E) contains GM-CSF, interleukin-1 4, tumor necrosis factor a (TNF- ⁇ ), lipopolysaccharide, and optionally FLT-3L. Except for this, it is the same as the medium used for the co-culture of the steps ( ⁇ ) and ( ⁇ ).
- the culture time in the step (v) is 1-2 days from the viewpoint of producing rod-shaped cells with stronger cell-stimulating activity.
- a CD40 ligand or the like may be used instead of TNF-a as long as it is a factor that promotes maturation of rod cells.
- the amount of TNF-a added is preferably stronger than T cell stimulating activity, from the viewpoint of producing a dendritic cell, 5 to 20 ng is desirable.
- step (E) if the factor promotes the maturation of rod cells, instead of lipopolysaccharide, a bacterial extract, a fungal extract, a mycoplasma extract, two Strand RNA or the like may be used.
- step (E) the amount of lipopolysaccharide added is preferably 3 to 5 / zg with respect to 1 ml of the culture solution from the viewpoint of stronger T cell stimulating activity and production of rod-shaped cells. .
- means for antigen-specific control of an individual's immune response for example, the reaction of cytotoxic T cells against a specific antigen using primate rod-shaped cells.
- Preventive reactions associated with organ transplantation treatment methods for diseases that are expected to have therapeutic effects (for example, autoimmune diseases, allergic diseases, etc.) And a means of treatment and the like can be provided.
- control of an immune response is intended to mean a concept that includes both suppression and activation of an immune response.
- the present invention provides steps (A) to (D) in the sorting method.
- step ( ⁇ ') a step of isolating rod-shaped cells derived from primate embryonic stem cells from the culture obtained in step (D), And a method for producing a rod-shaped cell of embryonic stem cell power of a primate animal.
- the production method of the present invention is replaced with the step (').
- step (Ii) a step of further culturing by adding interleukin-4, tumor necrosis factor oc, lipopolysaccharide and optionally fms-like tyrosine kinase 3 ligand to the culture obtained in step (D), and
- step (F) separating a rod-shaped cell derived from a primate embryonic stem cell from the culture obtained in step (E),
- step (E) is the same as the step (E) in the sorting method of the present invention.
- Advantageous embodiments are advantageous in that rod cells can be supplied with higher efficiency and more stability.
- means for separating the rod-like cells derived from primate embryonic stem cells from the culture include, for example, rod-like cells.
- examples include a affinity column using an antibody against a specific marker, cell sorting by flow cytometry using the antibody, and cell sorting using magnetic microbeads coated with the antibody.
- markers specific to the rod-shaped cells include HLA-DR, CD40 and CD.
- the antibody can be easily prepared by a conventional method using a polypeptide or a fragment thereof, which is a marker specific to rod cells.
- a polypeptide or a fragment thereof which is a marker specific to rod cells.
- Such an antibody may be a commercially available antibody.
- the production method of the present invention by introducing a gene such as a therapeutic gene into an ES cell as a raw material in advance, desired properties can be exerted on the rod-shaped cell. Therefore, in another embodiment, the production method of the present invention introduces a nucleic acid containing a gene to be introduced into a primate embryonic stem cell prior to performing step (A).
- the gene to be introduced include the therapeutic gene, specifically, for example, a gene for immunosuppression, a gene for immunostimulation, etc. More specifically, Examples include genes encoding antigens, 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.
- the resulting rod-shaped cells have an extremely high antigen-specific T cell stimulating activity. Is advantageous in that it expresses the activity of specifically suppressing the T cell reaction.
- 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 these, and cancer cells. Examples include proteins that are expressed (tumor antigen proteins), peptides that are part of tumor antigen proteins, and molecules that are targets of recognition by the immune system in autoimmune and allergic diseases.
- 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 ES cell sorting performance, the electopore position method is desirable.
- the nucleic acid may be V, a so-called naked nucleic acid, or may be linked to a vector (viral vector or non-viral vector).
- a vector viral vector or non-viral vector.
- examples of the vector include a phage and a plasmid.
- the vector may contain elements effective for transcription promotion, such as various promoters, enhancers, and terminators, as necessary.
- a non-viral vector for example, a plasmid vector by an electoral position from the viewpoint of ease of operation, therapeutic use, and the like.
- the production method of the present invention induces differentiation from conventional peripheral blood monocytes or hematopoietic stem cells in order to introduce a plasmid vector by electoporation at the stage of ES cells and differentiate it into rod-shaped cells.
- Gene transfer to the rod-shaped cells prepared by using a viral vector Compared to the method, the following points are excellent!
- the present invention relates to a rod-shaped 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.
- an auxiliary agent capable of stably holding the rod-shaped cells of the present invention such as a medium, may be used as appropriate.
- an immune response in a subject is antigen-specifically controlled, wherein a therapeutically effective amount of a rod-shaped cell obtained by the production method is administered to the subject.
- a method for treating a disease capable of obtaining a therapeutic effect is provided.
- Examples of the "disease whose therapeutic effect can be obtained by antigen-specific control of immune response” include, for example, autoimmune disease, tumor, allergic disease, infectious disease, rejection associated with organ transplantation, and the like. Examples include graft-versus-host disease (GVHD).
- GVHD graft-versus-host disease
- the "subject” is preferably a human in need of treatment for a disease that can obtain a therapeutic effect by antigen-specific control of the immune response as described above. It may be an organism that requires treatment of a disease capable of obtaining a therapeutic effect by controlling the immune response as described above in an antigen-specific manner, for example, pet animals such as Inu and cats.
- the "therapeutically effective amount” means that when the rod-like cells obtained by the above production method are administered to the subject, the immunity is compared with that of the subject not administering the rod-like cells.
- the amount of the rod-shaped cells that can control the response in an antigen-specific manner and thus can have a therapeutic effect on the above-mentioned diseases.
- the specific therapeutically effective amount is not generally determined as long as it is appropriately set depending on the administration form of the rod cells, the administration method, the purpose of use and the age, weight, symptom, etc. of the subject.
- the number of rod cells is 200,000 to 1,000,000 even / kg body gravity per treatment for a human (eg, an adult) per person!
- examples of a method for administering a therapeutically effective amount of the rod-like cells obtained by the production method to a subject include subcutaneous injection, intralymphatic injection, intravenous injection, or malignant tumor. This includes, but is not limited to, direct local injection.
- an immune response control agent comprising rod-shaped cells obtained by the production method as an active ingredient.
- the immune response control agent of the present invention may contain the above-mentioned adjuvant capable of stably holding the rod-shaped cells as the 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 differentiation-inducing culture from ES cells to rod-shaped cells.
- the cells collected in the following steps were partially frozen and stored.
- the cells in each step were analyzed using an inverted microscope (trade name: 1X70, manufactured by Olympus Corporation).
- CMK6 cells As embryonic stem cells (ES cells) of primates, CMK6 cells, an ES cell line established from force-cynomolgus monkeys, were used.
- ST2 cells (physical research) were prepared with DMEM supplemented with 10% by volume urchin fetal serum on a culture dish (diameter 10cm, manufactured by Falcon) previously coated with 0.1% gelatin solution. Supplied) was cultured at 37 ° C and 5% CO by volume. The ST2 cells touch the bottom of the culture dish
- the ES cells were suspended in ⁇ MEM (a essential culture medium) supplemented with 20% by volume urine fetal serum (FCS) at a density of 8 ⁇ 10 4 cells Z8 ml culture solution.
- FCS urine fetal serum
- Panels A to F in Fig. 2 show the results of observing the morphology of cells generated by induction of ES cell differentiation over time.
- Panel A in Fig. 2 shows undifferentiated ES cells, and panels B to F show the morphology of ES cell-derived cells that are being separated in step (A).
- Panel B shows cells on day 3 after initiation of differentiation induction, panels C and D on day 6, and panels E and F on day 9. Shown in Figure 2 As the number of culture days passed, the differentiation of undifferentiated ES cells into mesoderm cells progressed, and the density of cells separated into mesoderm cells increased.
- the surface of the feeder layer is almost like an ES cell-derived epithelial cell by 6 days after the start of differentiation induction. Covered with cells.
- step (A) of (1) above ES cell-derived cells recovered from one culture dish are respectively transplanted onto OP9 cell layers prepared in 4 to 8 other culture dishes.
- the cells were further cultured at 37 ° C. and 5% by volume CO 2 in ⁇ containing 20 vol% urine fetal serum.
- the OP cell layer was obtained by growing until the bottom surface of the culture dish was almost covered, similar to the ST2 cells.
- the ES cell-derived cells recovered from the four culture dishes in the step (B) of (2) were used as final concentration lOOngZml granulocyte macrophage colony-stimulating factor (GM-CSF; manufactured by Pep rotec) and final concentration lOOngZml macrophage.
- GM-CSF granulocyte macrophage colony-stimulating factor
- M-CSF colony stimulating factor
- the cells were suspended in a MEM containing 20% by volume urine fetal serum.
- the resulting cells transplants City on OP9 cell layer that had been prepared on one another culture dish (diameter 10 cm), and cultured at 37 ° C, 5 volume 0/0 CO.
- the culture dish contains a final concentration of lOOngZml GM-CSF and no M-CSF, and a new culture solution [composition: ex MEM containing 20% by volume of fetus serum] 4 ml was added.
- the ES cell-derived cells collected in step (C) of (3) were suspended at a density of 5 ⁇ 10 5 cells Z2.5 and 5 ml culture solution. 2.5 ml of the obtained cell suspension was transplanted to one culture dish (diameter 6 cm) without stromal cells, and further cultured at 37 ° C. and 5 vol% CO.
- the final concentration of lOOngZml GM—CSF (manufactured by Peprotec) and the final concentration of 10ng / ml Interleukin-4 (IL-4; manufactured by Peprotec), final concentration lOngZml FLT-3L (fms-like tyrosine kinase 3 ligand; manufactured by Peprotec), and ⁇ MEM containing 20% by volume fetus serum .
- step (D) of (4) above the final concentration of 10 ng / ml tumor necrosis factor a (TNF—manufactured by Peprotec) and final concentration of 3 / z gZml lipopolysaccharide ( LPS, derived from E. coli (manufactured by SIGMA) and culturing at 37 ° C and 5 vol% CO
- step (A) in Example 1 OP9 cells or PA6 cells were used instead of ST2 cells, and step (A) was performed in the same manner as in the case of ST2 cells.
- step (A) of Example 1 (1) bone morphogenetic protein 4 (BMP
- TPO Thrombopoietin
- SCF Stem cell stimulating factor
- Fit—3L vascular endothelial growth
- step (A) even when the site force-in is used, a force that does not show a remarkable effect of promoting the differentiation from ES cells to mesodermal cells. I got it.
- the cell surface molecules that are physically present and expressed in the rod-shaped cells are ES in Example 1 above. Whether it was also expressed in cell-derived cells was examined.
- ES cell-derived rod-like cells obtained in Example 1 were treated in Fc blocking reagent (Miltenyi Biotec) for 5 minutes. Thereafter, the obtained product was divided into the following fluorescein isothiocyanate (FITC) -binding monoclonal antibody (mAb) (Pharmingen): anti-human histocompatibility leukocyte antigen (HLA) -DR (clone L243, mouse IgG2a), Anti-human CD86 (clone FUN-1, mouse IgGl) and anti-human CD40 (clone 5C3, mouse IgGl) were used for staining. Said anti-human mAb cross-reacts with force-cynomolgus cells. As an isotype-matched control, mouse IgG2a (clone G155-178) and mouse IgGl (clone MOPC-21) were used for staining. And incubated at 4 ° C for 30 minutes
- a cell analyzer equipped with CellQuest software (trade name:
- FACScan manufactured by Becton Dickinson.
- the cells after step (B) (“dayl6” in FIG. 3) and Cells after step (C) (“day 22” in FIG. 3) hardly expressed these molecules.
- the ES cell-derived rod-like cells recovered in step (D) express HLA-DR, CD40, and CD86 at low levels. It was.
- cells stimulated with TNF-a and LPS in step (E) expressed these molecules at a high level.
- the product was homogenized with a product name: Qiashredder (Qiagen), and total RNA was extracted using a product name: RNeasy kit (Qiagen).
- RNA equivalent to 1 ⁇ g random hexamer primer (Gibco—BRL) and Superscript TM n reverse transcriptase (Gibco—BRL), 42 ° C CDNA was synthesized under the conditions of incubation for 50 minutes.
- Nucleic acid encoding Rhizobium CD80 SEQ ID NOs: 3 and 4
- the thermal profile in PCR is 95 ° C for 5 minutes, followed by 35 cycles of 95 ° C for 1 minute, 56 ° C for 1 minute and 72 ° C for 1 minute, and 72 ° C for 5 minutes. It is a condition to incubate.
- step (E) the cells stimulated with TNF-a and LPS are interleukin-12 (site force-in having an action of activating T cells). IL-12) was also expressed. Therefore, it was suggested that the cells obtained in Example 1 were rod-shaped cells.
- ES cell-derived rod-like cell force Quantifies T cell proliferation when co-cultured with Aro (allogeneic) T cells, whether they have the activity of stimulating T cells and causing a proliferative response This was considered.
- Reactive T cells were used as separate T-cell monkey peripheral blood T cells from those used to establish ES cells.
- X-irradiated ES cells in the undifferentiated state (negative control), ES cell-derived rod cells after step (D), and ES cell-derived rod cells after step (E) were used as stimulating cells ( 40Gy) was used.
- CD14 cells were isolated from mononuclear cells using magnetic beads coated with anti-human CD14 antibody (trade name, supermagneticMicro Beads, Miltenyi Biotec), and this was isolated from tissue culture. The plate was cultured at 37 ° C. for 1 hour in a medium (composition: RPMI-1640 medium containing 2% by volume heat-inactivated rabbit embryo serum). [0156] Thereafter, cells that do not adhere to the culture dish are collected, and the nylon wool column (made by filling a 10 ml plastic syringe with nylon wool manufactured by Wako Pure Chemical Industries, Ltd.) at 37 ° C, 5 volume% CO Left for 1 hour. The cells that did not adhere to the nylon wool column
- HLA—DR ⁇ 4 * 0103 cDNA a type of human histocompatibility leukocyte antigen (HLA) class II ⁇ -chain gene, is activated by the CAG promoter and contains an internal ribosome entry site (I RES) —neomycin resistance gene cassette Cloning into the animal expression vector pCAG-IR ES-Neo gave pCAG-DRB4-IN.
- Panel A in FIG. 6 shows a schematic diagram of the pCAG-DRB4 IN.
- Undifferentiated force-quizal ES cells (1.0 ⁇ 10 7 cells) were suspended in 0.4 ml of Dulbecco's modified culture medium (DMEM; manufactured by Gibco-BRL). The obtained suspension was mixed with 50 ⁇ g of the above pC AG-DRB4-IN. The obtained mixture was put into a 4 mm gap cuvette (trade name: BM6400, manufactured by BTX), and the cuvette was set in a product name: Gene Pulser (manufactured by Bio-Rad). After that, gene transfer was performed by electroporation under conditions of 250V and 500F.
- DMEM Dulbecco's modified culture medium
- the ES cell into which the gene has been introduced is transferred to a PEF (mouse embryonic fibroblast) layer on a 10 cm culture dish.
- PEF mouse embryonic fibroblast
- Above medium Composition: 20 vol% KSR (Gibco BRL Co.) in containing DMEM, and incubated at 37 ° C, 5 volume 0/0 CO. From day 2 after gene transfer, G418 (150 ⁇
- Drug-resistant cells ie, transgenic cells, were selected by adding g / ml) to the medium.
- transfectants On day 11 after gene introduction, clones of transgenic cells (transfectants) were collected as colonies of G418-resistant cells and transferred to a 24-well culture plate in which PEF had been cultured in advance. Furthermore, the cells were grown by continuing the culture in the presence of G418 (150 g / ml), and a part of the cells was stored frozen. In addition, the remaining part was cultured at 37 ° C, 5 vol% CO in a medium containing 3 mgZml G418 (composition: DMEM containing 20 vol% KSR (Gibco BRL)), and in the presence of such a high concentration of G418. But the transfectant claw that can grow
- PCR was performed using 5'-ctgactgaccgcgttactcccaca-3 '(SEQ ID NO: 7) and ttg gttatagatgtatctgatcaggt-3' (SEQ ID NO: 8). The expression of was examined. As a control, HPRT gene expression was examined. The thermal profile for PCR is 95 ° C for 5 minutes, followed by 35 cycles of 95 ° C for 1 minute, 56 ° C for 1 minute, and 72 ° C for 1 minute, followed by 72 ° C for 5 minutes. It is a condition.
- HLA—SA32.5 (5 ⁇ 10 4 / well), a human CD4 + T cell clone that recognizes GAD65 pil—131 bound to DR53 molecule (DRA * 0101 + DRB4 * 0103), and antigen ( The above-mentioned synthetic peptide) -loaded rod cells (2 ⁇ 10 4 Zwell) were placed on a 96-well flat-bottom culture plate in RPMI-1640 medium supplemented with 10% human plasma at 37 ° C. The T cell proliferation assay was performed by culturing at 5 vol% CO.
- the DR53 ⁇ gene-introduced rod cells obtained in Example 7 have the activity of taking a protein antigen into the cell, decomposing it into a peptide, producing a peptide, and displaying it on the DR molecule on the cell surface. Then, we examined whether or not.
- the DNA fragment encoding the GAD65 ⁇ 96-174 protein fragment was ligated to the prokaryotic expression vector pGEX-4 ⁇ -3 (Amersham Biosciences), and the resulting vector was used for E. coli DH5 a was transformed, thereby allowing the E. coli DH5a to express a glutathione 1 S transferase fusion GAD65 protein (GST-GAD).
- GST-GAD glutathione 1 S transferase fusion GAD65 protein
- the recombinant protein was purified using dartathione-agarose (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-10 (manufactured by Millipore), separated from a low molecular weight peptide fragment, and the buffer was replaced with a medium by dialysis.
- DR53 ⁇ obtained in Example 7 was used.
- the transfected rod cells were cultured at 37 ° C. in the presence of GST or GST-GAD protein obtained in (1) above (18 / z M) with final concentration of lOngZml TNF- ⁇ and final concentration of 3 gZml LPS. Incubate with 5% CO for 20 hours, wash 3 times with medium, and X-ray irradiation (40
- the SA32.5 (5 X 10 Suel) and the rod-shaped cells (2 X 10 4 Z uer) loaded with the antigen (the recombinant protein) were mixed on a 96-well flat-bottom culture plate at 10% by volume.
- T cells were cultured in RPMI-1640 medium supplemented with human plasma at 37 ° C and 5% CO by volume.
- rod-like cells derived from primates can be efficiently and stably supplied in large quantities, and a normal plasmid vector can be electroporated without using a viral vector. Genetic modification by introduction by the law becomes possible. According to the present invention, genetic modification of rod-shaped cells can be easily and efficiently performed. By administering such genetically modified rod cells to a living body, it becomes possible to control an individual's immune response in an antigen-specific manner. Therefore, various therapeutic effects are expected by antigen-specific control of the immune response. It becomes possible to treat such diseases.
- SEQ ID NO: 1 is the sequence of the HPRT primer.
- SEQ ID NO: 2 is the sequence of the HPRT primer.
- SEQ ID NO: 3 is the sequence of the CD80 primer.
- SEQ ID NO: 4 is the sequence of the CD80 primer.
- SEQ ID NO: 5 is the sequence of the IL-12p40 primer.
- SEQ ID NO: 6 is the sequence of the IL-12p40 primer.
- SEQ ID NO: 7 is the sequence of the HPRT primer.
- SEQ ID NO: 8 is the sequence of the HPRT primer.
- SEQ ID NO: 9 is a partial sequence of GAD65.
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006130651A3 (en) * | 2005-06-01 | 2007-03-29 | Wisconsin Alumni Res Found | Method of forming dendritic cells from embryonic stem cells |
WO2008056734A1 (fr) * | 2006-11-08 | 2008-05-15 | National University Corporation Kumamoto University | Procédé de production de cellules dendritiques à partir de cellules souches d'embryons humains |
WO2010122961A1 (ja) | 2009-04-24 | 2010-10-28 | 国立大学法人熊本大学 | 細胞医薬の製造方法 |
US8034613B2 (en) | 2005-06-01 | 2011-10-11 | Wisconsin Alumni Research Foundation | Multipotent lymphohematopoietic progenitor cells |
WO2012115276A1 (en) | 2011-02-23 | 2012-08-30 | Kyoto University | Method for producing dendritic cells from pluripotent stem cells |
WO2014148646A1 (ja) | 2013-03-21 | 2014-09-25 | 国立大学法人京都大学 | 神経分化誘導用の多能性幹細胞 |
WO2016159875A1 (en) * | 2015-03-31 | 2016-10-06 | Agency For Science, Technology And Research | Method for antigen loading of dendritic cells and vaccine |
CN114107203A (zh) * | 2021-12-01 | 2022-03-01 | 上海健康医学院 | 一种dc体外诱导扩增体系及诱导造血干细胞分化为树突状细胞的方法 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000028000A2 (en) * | 1998-11-05 | 2000-05-18 | Isis Innovation Limited | Method for producing dendritic cells |
WO2003083089A2 (en) * | 2002-03-28 | 2003-10-09 | Revivicor Inc. | Tolerogenic antigen-presenting cells |
JP2004313038A (ja) * | 2003-04-14 | 2004-11-11 | Kumamoto Technology & Industry Foundation | 哺乳動物のes細胞由来樹状細胞の産生方法 |
-
2005
- 2005-08-25 JP JP2006531978A patent/JP4695087B2/ja not_active Expired - Fee Related
- 2005-08-25 WO PCT/JP2005/015438 patent/WO2006022330A1/ja active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000028000A2 (en) * | 1998-11-05 | 2000-05-18 | Isis Innovation Limited | Method for producing dendritic cells |
WO2003083089A2 (en) * | 2002-03-28 | 2003-10-09 | Revivicor Inc. | Tolerogenic antigen-presenting cells |
JP2004313038A (ja) * | 2003-04-14 | 2004-11-11 | Kumamoto Technology & Industry Foundation | 哺乳動物のes細胞由来樹状細胞の産生方法 |
Non-Patent Citations (6)
Cited By (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006130651A3 (en) * | 2005-06-01 | 2007-03-29 | Wisconsin Alumni Res Found | Method of forming dendritic cells from embryonic stem cells |
GB2440494A (en) * | 2005-06-01 | 2008-01-30 | Wisconsin Alumni Res Found | Method of forming dendritic from embryonic stem cells |
US9624470B2 (en) | 2005-06-01 | 2017-04-18 | Wisconsin Alumni Research Foundation | Multipotent lymphohematopoietic progenitor cells |
US8785189B2 (en) | 2005-06-01 | 2014-07-22 | Wisconsin Alumni Research Foundation | Method of forming dendritic cells from embryonic stem cells |
GB2440494B (en) * | 2005-06-01 | 2010-07-28 | Wisconsin Alumni Res Found | Method of forming dendritic from embryonic stem cells |
US7811821B2 (en) | 2005-06-01 | 2010-10-12 | Wisconsin Alumni Research Foundation | Method of forming dendritic cells from embryonic stem cells |
US8435785B2 (en) | 2005-06-01 | 2013-05-07 | Wisconsin Alumni Research Foundation | Method of forming dendritic cells from embryonic stem cells |
AU2006252576B2 (en) * | 2005-06-01 | 2011-01-20 | Wisconsin Alumni Research Foundation | Method of forming dendritic cells from embryonic stem cells |
US8034613B2 (en) | 2005-06-01 | 2011-10-11 | Wisconsin Alumni Research Foundation | Multipotent lymphohematopoietic progenitor cells |
US8133732B2 (en) | 2005-06-01 | 2012-03-13 | Wisconsin Alumni Research Foundation | Method of forming myeloid precursor cells from human embryonic stem cells |
JPWO2008056734A1 (ja) * | 2006-11-08 | 2010-02-25 | 国立大学法人 熊本大学 | ヒト胚性幹細胞からの樹状細胞の製造方法 |
WO2008056734A1 (fr) * | 2006-11-08 | 2008-05-15 | National University Corporation Kumamoto University | Procédé de production de cellules dendritiques à partir de cellules souches d'embryons humains |
WO2010122961A1 (ja) | 2009-04-24 | 2010-10-28 | 国立大学法人熊本大学 | 細胞医薬の製造方法 |
US8551472B2 (en) | 2009-04-24 | 2013-10-08 | National University Corporation Kumamoto University | Method of making macrophage expressing an antibody directed against β-amyloid |
WO2012115276A1 (en) | 2011-02-23 | 2012-08-30 | Kyoto University | Method for producing dendritic cells from pluripotent stem cells |
EP2678425A4 (en) * | 2011-02-23 | 2015-01-21 | Univ Kyoto | METHOD FOR THE PRODUCTION OF DENDRITIC CELLS FROM PLURIPOTENTIAL STEM CELLS |
US9499789B2 (en) | 2011-02-23 | 2016-11-22 | Kyoto University | Method for producing dendritic cells from pluripotent stem cells |
EP2678425A1 (en) * | 2011-02-23 | 2014-01-01 | Kyoto University | Method for producing dendritic cells from pluripotent stem cells |
WO2014148646A1 (ja) | 2013-03-21 | 2014-09-25 | 国立大学法人京都大学 | 神経分化誘導用の多能性幹細胞 |
WO2016159875A1 (en) * | 2015-03-31 | 2016-10-06 | Agency For Science, Technology And Research | Method for antigen loading of dendritic cells and vaccine |
CN108026513A (zh) * | 2015-03-31 | 2018-05-11 | 新加坡科技研究局 | 树突状细胞的抗原加载的方法和疫苗 |
CN114107203A (zh) * | 2021-12-01 | 2022-03-01 | 上海健康医学院 | 一种dc体外诱导扩增体系及诱导造血干细胞分化为树突状细胞的方法 |
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