JPWO2006022330A1 - Method for producing dendritic cells from primate embryonic stem cells - Google Patents

Abstract

An object of the present invention is to provide a means for antigen-specific control of an individual's immune response. Accordingly, the present invention provides a method for differentiating primate embryonic stem cells into dendritic cells, a method for producing dendritic cells from primate embryonic stem cells, a dendritic cell obtained by the production method, and the dendritic cell. The present invention relates to the use of the dendritic 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 to the cell medicine for the treatment of the disease.

Description

  The present invention relates to a method for differentiating primate embryonic stem cells into dendritic cells, a method for producing dendritic cells from primate embryonic stem cells, a dendritic cell obtained by the production method, the dendritic cell, immunity The present invention relates to the use of the dendritic cells for the manufacture of a medicament for the treatment of a disease whose therapeutic effect can be obtained by antigen-specific control of the response, and to the cell medicine for the treatment of the disease.

  Dendritic cells phagocytose antigen protein, break it down into peptides, and present the resulting peptide as a complex with major histocompatibility antigen (MHC) to T cells (hereinafter also referred to as “antigen presentation”). Stimulates cells and activates the T cells in an antigen-specific manner. In addition, the dendritic cells are cells having the highest antigen presenting ability in vivo. On the other hand, the dendritic cells suppress the function of T cells reactive to self antigens and are involved in maintaining immunological self tolerance. Thus, dendritic cells play a central role in controlling immune responses in vivo.

  In vivo, dendritic cells are differentiated and produced from hematopoietic stem cells in the bone marrow. Hematopoietic stem cells in the bone marrow differentiate into erythrocytes, platelets, neutrophils, eosinophils, basophils, macrophages, lymphocytes and the like in addition to dendritic cells due to the presence of growth factors.

  Conventionally, it is known that dendritic cells can be obtained by isolating existing dendritic cells from peripheral blood, inducing differentiation of hematopoietic stem cells in bone marrow (for example, Patent Document 1). However, since the abundance of dendritic cells in peripheral blood is small, there is a drawback that it is difficult to obtain a large amount of dendritic cells. Further, the method described in Patent Document 1 discloses that dendritic cells are obtained by inducing differentiation of mouse hematopoietic stem cells. However, hematopoietic stem cells are difficult to grow for a long period of time, and there is a drawback that it is necessary to use a viral vector in order to introduce genes into dendritic cells prepared by this method.

In addition, attempts have been made to obtain dendritic cells by inducing differentiation of mouse embryonic stem cells (Non-patent Document 1). However, when the method described in Non-Patent Document 1 is applied to other organisms, for example, primates, there is a disadvantage that mature dendritic cells do not occur.
Japanese Patent Laid-Open No. 2004-16697 S. Senju et al., Blood, Vol. 101, pages 3501-3508, issued May 1, 2003

  One aspect of the present invention is a method for efficiently obtaining dendritic cells derived from primates, stably supplying primate dendritic cells, and antigen-specific control of individual immune responses. (For example, a means for strongly stimulating cytotoxic T cell responses to a specific antigen, etc.), diseases that are expected to have therapeutic effects by antigen-specific control of immune responses (eg, autoimmune diseases, Providing a means of treating allergic diseases, etc., and providing a means of preventing or treating rejection in organ transplantation and graft versus host disease (GVHD). And a method for differentiating primate embryonic stem cells into dendritic cells.

  Furthermore, another aspect of the present invention is to obtain a large amount of dendritic cells derived from a primate animal, to efficiently obtain a dendritic cell derived from a primate animal, and to stably obtain a dendritic cell derived from a primate animal. At least one of obtaining a dendritic cell that antigen-specifically controls an individual's immune response (for example, a dendritic cell that strongly activates a cytotoxic T cell response to a specific antigen). The present invention relates to a method for producing dendritic cells from primate embryonic stem cells.

  Still another aspect of the present invention is to control an individual's immune response in an antigen-specific manner (eg, to strongly activate a cytotoxic T cell response to a specific antigen, etc.), antigen-specificity of the immune response The present invention relates to providing a dendritic cell that enables at least one of obtaining a therapeutic effect on a disease for which a therapeutic effect is expected by simple control.

  In another aspect of the present invention, there is provided use of the dendritic cells 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. About that.

  In still another aspect of the present invention, an immune response that enables at least one of antigen-specific control of the immune response, for example, treatment of diseases such as autoimmune diseases and allergic diseases, etc. It relates to providing a control agent. Other problems of the present invention are also apparent from the description of the present specification.

That is, the gist of the present invention is as follows.
[1] (A) a step of co-culturing primate embryonic stem cells and cells having the property of inducing differentiation and proliferation of blood cells to obtain cell group A;
(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,
(C) The cell group B obtained in the step (B) and a newly prepared cell having the property of inducing differentiation and proliferation of blood cells are co-presented in the presence of granulocyte macrophage colony-stimulating factor. Culturing to obtain cell group C, and (D) culturing cell group C obtained in step (C) in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4.
A method of differentiating primate embryonic stem cells into dendritic cells, comprising:
[2] In step (A), from a co-culture of primate embryonic stem cells and cells having the property of inducing differentiation and proliferation of blood cells, derived from primate embryonic stem cells; And the cell group A is isolated as a cell group containing cells differentiated into mesodermal cells, the differentiation method according to the above [1],
[3] The 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 cells obtained are OP9 cells, and the newly prepared cells having the property of inducing differentiation and proliferation of blood cells in step (C) are OP9 cells. Differentiation method,
[4] After step (D), (E) adding the tumor necrosis factor α and lipopolysaccharide to the culture obtained in step (D) and further culturing, [1] -[3] The differentiation method according to any one of the above,
[5] (A) a step of co-culturing primate embryonic stem cells and cells having the property of inducing differentiation and proliferation of blood cells to obtain cell group A;
(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,
(C) The cell group B obtained in the step (B) and a newly prepared cell having the property of inducing differentiation and proliferation of blood cells are co-presented in the presence of granulocyte macrophage colony-stimulating factor. Culturing to obtain cell group C, and (D) culturing cell group C obtained in step (C) in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, and E ′) separating dendritic cells derived from primate embryonic stem cells from the culture obtained in step (D),
A method of producing dendritic cells from primate embryonic stem cells,
[6] Instead of step (E ′),
(E) adding tumor necrosis factor α and lipopolysaccharide to the culture obtained in step (D) and further culturing; and (F) from the culture obtained in step (E), Isolating dendritic cells derived from primate embryonic stem cells;
Performing the method according to [5] above,
[7] The production method according to [5] or [6] above, wherein a nucleic acid containing a gene to be introduced is introduced into embryonic stem cells of a primate prior to performing step (A),
[8] The production method according to any one of [5] to [7] above, wherein the gene to be introduced is a gene for immunosuppression,
[9] The production method according to any one of [5] to [7] above, wherein the gene to be introduced is a gene for immunostimulation.
[10] The production method according to any one of [7] to [9] above, wherein the nucleic acid is introduced by a non-viral vector-mediated nucleic acid introduction method,
[11] Dendritic cells obtained by the production method according to any one of [5] to [10],
[12] The production according to any one of [5] to [10] above for the production of a medicament for the treatment of a disease capable of obtaining a therapeutic effect by specifically controlling an immune response. Use of dendritic cells obtained by the method and [13] Dendritic cells obtained by the production method of any one of [5] to [10] as an active ingredient A cell medicine for the treatment of a disease capable of obtaining a therapeutic effect by being specifically controlled,
About.

  According to the method for differentiating primate embryonic stem cells from dendritic cells according to the present invention, it is possible to efficiently and stably supply dendritic cells derived from primate animals. In addition, according to the differentiation method of the present invention, means for specifically controlling the immune response of an individual (for example, dendritic cells of primates are strongly activated by cytotoxic T cell responses to specific antigens). And the like, and the like, and the like, and the like, can provide a therapeutic means for diseases (for example, autoimmune diseases, allergic diseases, etc.) for which therapeutic effects are expected by antigen-specific control of the immune response.

  Furthermore, according to the method for producing dendritic cells from primate embryonic stem cells of the present invention, the dendritic cells derived from primates can be efficiently and stably supplied in large quantities. There is an effect. In addition, according to the production method of the present invention, 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) are obtained. I can do this.

  According to the dendritic cell of the present invention, it is possible to antigen-specifically control an individual's immune response (for example, to strongly activate a cytotoxic T cell reaction to a specific antigen) Thus, it is possible to obtain a therapeutic effect on a disease for which a therapeutic effect is expected by controlling antigen-specificity or antigen-specific control of immune response.

  According to the use of the dendritic cells for producing the medicament of the present invention, it is possible to provide a medicament capable of treating a disease for which a therapeutic effect is expected by antigen-specific control of the immune response.

  In addition, according to the immune response control agent of the present invention, the immune response can be antigen-specifically controlled, and for example, there is an excellent effect that it is possible to treat diseases such as autoimmune diseases and allergic diseases. .

FIG. 1 shows a schematic diagram of an example of a method for differentiating primate embryonic stem cells into dendritic cells.

FIG. 2 shows micrographs of cells over time when primate embryonic stem cells were differentiated into dendritic cells. In the figure, 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, and panel G is the sixteenth day. Panel H shows day 22 cells, Panel I shows day 26, and Panels J, K and L show day 28 cells. The scale bar indicates 100 μm.

FIG. 3 is a diagram showing the results of analyzing the expression of cell surface molecules DR, CD40 and CD86 in floating cells in a culture medium when primate embryonic stem cells were differentiated into dendritic cells. In the figure, “Day 16” is the end of step (B) in FIG. 1, “Day 22” is the end of step (C), “Day 26” is the end of step (D), and “Day 28” is the end of step E. The analysis result of the cell at the time is shown. In each panel, a thick solid line histogram shows the fluorescence intensity of the antibody stained for the molecule to be analyzed, and a thin dotted line histogram is a negative control and a non-specific antibody. The fluorescence intensity in the case of staining is shown.

FIG. 4 is a diagram showing the results of analysis of CD80 and IL-12p40 mRNA expression by RT-PCR. In the figure, HPRT mRNA indicates the relative amount of RNA sample used for analysis.

FIG. 5 is a diagram showing the results of analysis of T cell stimulation activity by cells induced to differentiate from primate embryonic stem cells. In the figure, diamonds indicate floating cells in the culture solution after completion of step (D), white circles indicate suspension cells in the culture solution after completion of step (E), and squares indicate 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 is a structural diagram of a plasmid DNA vector encoding the HLA-DR53 molecule β chain. Panel B shows the results of detecting transgene-derived mRNA by RT-PCR. In the figure, “cES” is an undifferentiated embryonic stem cell, “cES-53-23” is an undifferentiated embryonic stem cell into which a gene has been introduced, and “cES-DC” is a tree whose differentiation has been induced from an embryonic stem cell. The dendritic cell, “cES-DC-53-23”, represents a dendritic cell that has been induced to differentiate from a gene-introduced embryonic stem cell. HPRT mRNA indicates the relative amount of RNA sample used for analysis.

FIG. 7 is a diagram showing the results of examining the antigen-presenting ability of dendritic cells differentiated from embryonic stem cells introduced with the DR53β gene. Panel A shows that dendritic cells differentiated from DR53β gene-introduced embryonic stem cells were loaded with a GAD65 antigen-derived peptide (concentration 1.25, 2.5, 5.0 μM), and irradiated with X-rays. The proliferation response of SA32.5 when cultured with the T cell line SA32.5 that recognizes the GAD65 antigen-derived peptide presented on the DR53 molecule is shown. In panel A, the value obtained by measuring the incorporation of [ ³ H] -thymidine into chromosomal DNA by SA32.5 using a scintillation counter for β-ray measurement is shown. As negative controls, dendritic cells induced to differentiate from embryonic stem cells into which no gene was introduced were loaded with GAD65 antigen-derived peptides, and DR53β gene-introduced dendritic cells were not loaded with GAD65 antigen-derived peptides The result when each was cultured with SA32.5 is shown. Panel B shows that dendritic cells differentiated from embryonic stem cells introduced with DR53β gene were loaded with GST-fused GAD65 antigen (GST-GAD) prepared by genetic recombination, irradiated with X-rays, and thereby proliferated The proliferation response of SA32.5 is shown when the ability is deficient and cultured with T cell line SA32.5. In panel B, as in panel A, the value obtained by quantifying the proliferation response by [ ³ H] -thymidine incorporation is shown. As negative controls, GST-GAD-loaded dendritic cells that had been induced to differentiate from embryonic stem cells into which no gene had been introduced and those that had been induced to differentiate from embryonic stem cells into which the DR53β gene had been introduced were treated with GST. The result when each loaded one is cultured with SA32.5 is shown.

In one aspect, the present invention provides a cell group A by co-culturing primate embryonic stem cells and cells having the property of inducing differentiation and proliferation of blood cells,
(B) co-culturing the cell group A obtained in the step (A) and newly prepared cells having a property of inducing differentiation of blood cells to obtain a cell group B;
(C) The cell group B obtained in the step (B) and a newly prepared cell having the property of inducing differentiation and proliferation of blood cells are co-presented in the presence of granulocyte macrophage colony-stimulating factor. Culturing to obtain cell group C, and (D) culturing cell group C obtained in step (C) in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4.
And a method for differentiating primate embryonic stem cells into dendritic cells.

  The differentiation method of the present invention has one major feature in that embryonic stem cells (ES cells) of primates are used. Therefore, according to the differentiation method of the present invention, an excellent effect that it can be supplied in a large amount is exhibited as compared with the case of isolating dendritic cells from a living body or the case of differentiation from hematopoietic stem cells. In addition, according to the differentiation method of the present invention, since the ES cell is used, if necessary, a gene expected to show a therapeutic effect (hereinafter also referred to as “therapeutic gene”) can be easily introduced. , Exhibit an excellent effect of being able to be expressed. Furthermore, according to the present invention, since primate ES cells are used, it is suitable for application to so-called cell therapy.

  The differentiation method of the present invention is different from the differentiation from mouse ES cells to dendritic cells. First, in step (A), primate ES cells are differentiated from blood cells and proliferated. There is one major feature in co-culture with cells having the property of inducing. According to the differentiation method of the present invention, ES cells can be differentiated into mesodermal cells by performing the step (A).

  In this specification, “primate animals” refers to humans, monkeys, and the like. Examples of the monkey include cynomolgus monkey, rhesus monkey, Japanese monkey, marmoset, chimpanzee and the like.

  The primate ES cells used in the present invention are not particularly limited, and examples thereof include cynomolgus monkey ES cell line CMK6 cells and human ES cells. The ES cells can be appropriately selected depending on the purpose of use of the obtained dendritic cells.

  Examples of the “cells having the property of inducing the differentiation and proliferation of blood cells” (feeder cells) include, for example, OP9 cells (RIKEN BIORESOURCE CENTER: 3-1-1 Takanodai, Tsukuba City, Ibaraki 305-0074, Japan) 1) cell number: RCB1124), ST2 cell (RIKEN BIORESOURCE CENTER cell number: RCB0224), PA6 cell (RIKEN BIORESOURCE CENTER cell number: RCB1127) and the like. Among these, ST2 cells are preferable from the viewpoint of improving the efficiency of inducing differentiation into blood cells.

  In the present invention, when the “primate animal ES cell” and the “feeder cell” are co-cultured, the “feeder cell” corresponds to the feeder cell in a culture container containing an appropriate medium. It can be used by culturing under culture conditions and growing it to an extent that substantially covers the bottom of the culture vessel, thereby forming a feeder cell layer.

  The medium that can be used for the production of the feeder cells may be any medium that is suitable for the culture of adherent mammalian cells, and may be appropriately selected depending on the type of cells used as the feeder cells, such as αMEM, DMEM [Dulbecco modified Eagle medium (culture solution)] and the like.

  The culture vessel used in the step (A) is coated with a coating suitable for tissue culture from the viewpoint of promoting differentiation from ES cells to mesodermal cells and from the viewpoint of stably adhering feeder cells for a long period of time. Any culture vessel may be used. The coating can be performed with, for example, gelatin, fibronectin or the like.

The culture conditions for the feeder cells can be appropriately set according to the type of cells used as the feeder cells. For example, in the case of ST2 cells, OP9 cells, etc., conditions for culturing at 37 ° C. and 5% by volume CO ₂ on a culture container coated with 0.1% by weight gelatin solution or the like in a medium supplemented with 10% by volume fetal calf serum Etc.

In the step (A), the cell density of the ES cells is preferably from the viewpoint of fully exerting the differentiation ability, from the viewpoint of maximizing the number of mesodermal cells obtained per medium (culture solution) to be used, etc. It is desirable that the culture vessel is 5 × 10 ⁴ to 1 × 10 ⁵ cells / diameter 100 mm.

  The medium used for co-culture of ES cells and feeder cells in the step (A) may be any culture solution suitable for culturing mammalian cells, and is appropriately selected according to the type of ES cells used. Examples thereof include αMEM, 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 ES cells and 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. Conditions such as around 5 ° C. (particularly 37 ° C.), 5% by volume CO _2, etc.

  The time for co-culture of ES cells and feeder cells in step (A) is such that ES cells are fully differentiated into cells that have differentiated into mesodermal system, and the number of cells that have differentiated into mesodermal system is maximized. Therefore, about 9 to 11 days is desirable.

  In 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 the properties of mesodermal cells and may vary depending on the type of animal from which the ES cells are used. Can be obtained as a group of cells containing.

  In addition, after performing the step (A), from the viewpoint of selectively collecting cells differentiated into the mesoderm system and improving the efficiency of differentiation into dendritic cells, primate embryonic stem cells, It is preferable to separate the cell group A as a cell group containing cells derived from primate embryonic stem cells and differentiated into mesodermal cells from a co-culture with feeder cells.

  Isolation of cell group A from the co-culture includes, for example, adding phosphate buffered saline containing 0.25 wt% trypsin / 1 mM ethylenediaminetetraacetic acid (EDTA) to the co-culture at 37 ° C. The treatment can be performed by treating the cells differentiated into mesodermal cells for an appropriate time, for example, 8 to 15 minutes.

  In the differentiation method of the present invention, the cell group A obtained in the step (A) is co-cultured with newly prepared cells (feeder cells) that induce differentiation and proliferation of blood cells [step (B). There is one big feature. Therefore, according to the differentiation method of the present invention, in this step (B), the ratio of mesoderm cells, in particular, blood cells can be further improved, so that dendritic cells derived from primates are more It can be obtained efficiently and exhibits an excellent effect that it can be stably supplied.

  As the “newly prepared cells that induce differentiation and proliferation of blood cells” (feeder cells), the feeder cells mentioned in the step (A) can be used, and for example, OP9 cells are desirable.

  In the step (B), when the cell group A and the “feeder cell” are co-cultured, the “feeder cell” is the same as the “feeder cell” used in the step (A). It can be used by growing to the extent that it almost covers the bottom surface, thereby forming a feeder cell layer.

  The culture conditions used for the culture vessel used in the step (B) and the “feeder cells” are the same as those in the step (A).

  In the step (B), the cell group A recovered from the step (A) is used in the step (A) from the viewpoint of sufficiently exerting the differentiation ability, the viewpoint of maximizing the number of produced cells per culture medium to be used, and the like. ) In a larger area than in the case of step (A), specifically about 4 to 8 times the culture vessel in step (A) [eg, the area of the feeder cell layer is about 4 to 8 times that in step (A)]. It is desirable to do.

  The conditions of the culture gas phase at the time of co-culture of the cell group A and the feeder cells in the step (B) depend on the type of ES cell used, the composition of the culture solution, and the like in the step (A). Similarly, it can be set as appropriate.

  The medium used for the step (B) is the same as the medium used for the co-culture of the step (A).

  The time for co-culture of ES cells and feeder cells in the step (B) is such that ES cells are sufficiently differentiated into mesodermal cells, particularly blood cells, and mesodermal cells, particularly blood cells. From the viewpoint of sufficiently improving the ratio of the cells, the viewpoint of maximizing the number of produced cells per culture medium to be used, and the viewpoint of finally producing dendritic cells having strong T cell stimulating activity, preferably 4 to 6 days. It is desirable that

  In step (B), medium replacement or medium addition may be performed as appropriate during the culture period.

  The cell group B obtained in the step (B) shows properties of mesodermal cells, particularly blood cells, and may vary depending on the type of animal that is the source of the ES cells used. 2 can be obtained as a group of cells containing floating cells having a relatively uniform, small and round shape as shown in panel G of FIG. In such a step (B), by collecting the floating cells by pipetting operation or the like, from cells that are insufficiently differentiated and cells differentiated in a direction other than blood cells (for example, including epithelial cells), A group of cells differentiated in the direction of blood cells can be efficiently collected.

  In the differentiation method of the present invention, the cell group B obtained in the step (B) and the newly prepared cells (feeder cells) having the property of inducing differentiation and proliferation of blood cells are granulocytes. One major feature of co-culture in the presence of macrophage colony-stimulating factor [Step (C)]. Therefore, according to the differentiation method of the present invention, by performing such step (C), differentiation into a myeloid (myelocyte) system can be induced, and differentiation into dendritic cell-like cells is performed more efficiently. Therefore, it is possible to obtain dendritic cells derived from primates more efficiently and to exhibit an excellent effect that they can be stably supplied.

  The culture vessel used in the step (C) is preferably coated with gelatin, fibronectin or the like from the viewpoint of stably adhering feeder cells for a long period of time.

  As the “newly prepared cells that induce differentiation and proliferation of blood cells” (feeder cells), the feeder cells mentioned in the above step (A) can be used, and for example, OP9 cells are desirable.

  The conditions of the culture gas phase during the co-culture in the step (C) are appropriately set in the same manner as in the steps (A) and (B) according to the type of ES cells used, the composition of the medium, and the like. sell.

  The medium used for the step (C) is the same as the medium used for the co-culture of the steps (A) and (B) except that it contains a granulocyte macrophage colony stimulating factor.

  The content of granulocyte macrophage colony-stimulating factor in the medium in the step (C) is preferably in the range of 50 to 200 ng / ml from the viewpoint of maximizing the number of production cells per culture medium to be used and the amount of stimulating factor.

In the step (C), the cell density of the cell group B is (3 × 10 ⁵ from the viewpoint of fully exerting the differentiation ability, and from the viewpoint of maximizing the number of production cells per culture medium and the amount of stimulating factor used. it is desirable that to 6 × ¹⁰ 5/8 ml medium / 100mm diameter culture dish).

  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 such dendritic cells in the finally produced cells From the viewpoint of sufficiently improving the proportion of the like cells, the viewpoint of maximizing the number of cells produced per the amount of the culture medium and stimulating factor to be used, 4 to 6 days is desirable.

  In the step (C), 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 ng / ml from the viewpoint of producing dendritic cells with strong T cell stimulating activity.

  In step (C), granulocyte-macrophage colony stimulation is preferably performed on the third or fourth day from the viewpoint of sufficiently improving the proportion of such dendritic cell-like cells in the finally produced cells. It is desirable to add a medium containing factors and no macrophage colony-stimulating factor to a culture of half of the original medium (4 ml / 100 mm diameter culture dish).

  In the step (C), unlike the case of mouse ES cells, the differentiation induction efficiency of dendritic cells is increased by temporarily adding a macrophage colony stimulating factor during the induction of differentiation into dendritic cells. It can be improved further. In the 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 containing granulocyte macrophage colony stimulating factor. It is also possible to add a medium that does not contain macrophage colony stimulating factor.

  Part of the cell group C obtained in the step (C) shows the properties of dendritic cells, for example, the expression of DR, CD40, CD86, CD80, etc. Although it may differ depending on the type, for example, as shown in panel H of FIG. 2, it can be obtained as a cell group containing floating cells that are morphologically non-uniform and have a form having protrusions.

  In the differentiation method of the present invention, the cell group C obtained in the step (C) is cultured in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) [step (D). There is one big feature. Therefore, according to the differentiation method of the present invention, by performing such step (D), the ratio of dendritic cell-like cells is further improved, and dendritic cells having high stimulating activity and antigen-presenting activity for T cells are produced. Therefore, it is possible to obtain dendritic cells derived from primate animals more efficiently and to provide a stable effect. In addition, according to the differentiation method of the present invention, since step (D) is performed, it can be induced more selectively into dendritic cells than in the step (C).

  The culture vessel used in step (D) is preferably a cell with low cell adhesion, for example, an uncoated polystyrene vessel, but a culture vessel with a cell culture coating can also be used.

  The conditions of the culture gas phase during the culture in the step (D) are appropriately set in the same manner as in the steps (A) and (B) according to the type of ES cells used, the composition of the culture solution, and the like. sell.

  The medium used for the step (D) is the same as the medium used for the co-culture of the steps (A) and (B) except that it contains granulocyte macrophage colony stimulating factor and interleukin-4. .

In the step (D), the cell density of the cell group C is 3 × 10 ⁵ from the viewpoint of sufficiently exerting the differentiation ability, the viewpoint of maximizing the yield of dendritic cells per culture medium and stimulating factor to be used, and the like. Desirably, −6 × 10 ⁵ cells / 2.5 ml culture solution / 60 mm diameter culture vessel.

  The culture time in the step (D) is 2 to 5 days from the viewpoint of further improving the proportion of dendritic cell-like cells in the cell group C and producing dendritic cells with stronger T cell stimulating activity. It is desirable to evaluate and appropriately determine the degree of differentiation according to the cell morphology.

  The content of granulocyte macrophage colony stimulating factor in the medium in the step (D) is preferably in the range of 50 to 200 ng / ml from the viewpoint of maximizing the yield of dendritic cells per stimulating factor used.

  The content of interleukin-4 in the medium in the step (D) is preferably 5 to 20 ng / ml from the viewpoint of producing stronger dendritic cells having stronger T cell stimulating activity.

  In addition, in the step (D), in addition to the granulocyte-macrophage colony-stimulating factor and interleukin-4, from the viewpoint of increasing the expression of MHC class II (DR) of the dendritic cells finally produced, Preferably uses the fms-like tyrosine kinase 3 ligand (FLT-3L).

  In the case of mouse ES cells, IL-4 is not essential for inducing differentiation of dendritic cells, and when IL-4 is added to immature dendritic cells, the final maturation of dendritic cells In step (D), IL-4 is essential in the process of inducing differentiation into dendritic cells.

  By the step (D), dendritic cells exhibiting dendritic cell properties, for example, expression of DR, CD40, CD86, etc., morphologically heterogeneous, and forms having protrusions can be obtained.

  Dendritic cells obtained by the differentiation method of the present invention have an 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. When Th cells are activated by presenting antigens from dendritic cells, they produce various cytokines, which can activate B cells and macrophages.

  In addition, the dendritic cell obtained by performing said step (A)-(D) adds tumor necrosis factor alpha and lipopolysaccharide, and also performs the step which culture | cultivates [step (E)], Dendritic cells (mature dendritic cells) with improved T cell stimulation can be produced.

  In step (E), in addition to interleukin-4 and any fms-like tyrosine kinase 3 ligand in step (D), tumor necrosis factor α and lipopolysaccharide are used. It exhibits an excellent effect of being able to produce improved dendritic cells (mature dendritic cells).

  The conditions of the culture vessel and the culture gas phase used in the step (E) are appropriately set in the same manner as in the steps (A) and (B) according to the type of ES cells used, the composition of the culture solution, and the like. Can be done.

  The medium used in the step (E) is the above step except that GM-CSF, interleukin-4, tumor necrosis factor α (TNF-α), lipopolysaccharide and optionally FLT-3L are added. The same as the medium used for the co-culture of (A) and (B).

  In addition, the culture | cultivation time in the said step (E) should just be 1 to 2 days from a viewpoint of producing a dendritic cell with stronger T cell stimulating activity.

  In step (E), a CD40 ligand or the like may be used instead of TNF-α as long as it is a factor that promotes dendritic cell maturation.

  In the step (E), the amount of TNF-α added is preferably 5 to 20 ng per 1 ml of the culture solution from the viewpoint of preparing dendritic cells having stronger T cell stimulating activity.

  In step (E), a bacterial extract, a fungal extract, a mycoplasma extract, a double-stranded RNA, or the like may be used instead of lipopolysaccharide as long as it is a factor that promotes dendritic cell maturation. Good.

  In the step (E), the addition amount of lipopolysaccharide is desirably 3 to 5 μg per 1 ml of the culture solution from the viewpoint of producing dendritic cells having stronger T cell stimulating activity.

  According to the differentiation method of the present invention, means for antigen-specific control of an individual's immune response (for example, the use of primate dendritic cells to strongly activate the response of cytotoxic T cells to a specific antigen) A means for treating a disease (eg, autoimmune disease, allergic disease, etc.) for which a therapeutic effect is expected by antigen-specific control of the immune response, a means for preventing and treating rejection associated with organ transplantation, etc. Can be supplied.

  In the present specification, “control” of an immune reaction intends a concept including both suppression and activation of the immune reaction.

In another aspect, the present invention is derived from a primate embryonic stem cell from the culture obtained in steps (A) to (D) and (E ′) in the differentiation method. Separating dendritic cells;
A method for producing dendritic cells from primate embryonic stem cells.

  According to the production method of the present invention, since the steps (A) to (D) are performed, dendritic cells derived from primates are efficiently stabilized in a large amount from the same viewpoint as the differentiation method of the present invention. The excellent effect of being able to be supplied is demonstrated.

Moreover, as a manufacturing method of this invention, in another embodiment, it replaces with the said step (E '),
(E) adding interleukin-4, tumor necrosis factor α, lipopolysaccharide and optionally a fms-like tyrosine kinase 3 ligand to the culture obtained in step (D), and further culturing; and (F ) Separating dendritic cells derived from primate embryonic stem cells from the culture obtained in step (E);
The method of performing is mentioned. Here, the step (E) is the same as the step (E) in the differentiation method of the present invention. According to such an embodiment, it is advantageous in that dendritic cells can be supplied with higher efficiency and more stably.

  In each of the steps (E ′) and (F), as means for separating dendritic cells derived from primate embryonic stem cells from the culture, for example, an antibody against a marker specific to dendritic cells An affinity column using the above, a cell sorting by flow cytometry using the antibody, a cell sorting using magnetic microbeads coated with the antibody, and the like.

  Examples of the marker specific to the dendritic cell include HLA-DR, CD40 and CD86, CD1a, CD11c, CD83 and the like.

  The antibody can be easily prepared by a conventional method using a polypeptide that is a marker specific for dendritic cells or a fragment thereof. Such an antibody may be a commercially available antibody.

  In the production method of the present invention, a dendritic cell can also exhibit a desired property by introducing a gene such as a therapeutic gene into an ES cell as a raw material in advance. 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).

  Examples of the gene to be introduced include the therapeutic gene, specifically, for example, a gene for immunosuppression, a gene for immunostimulation, and the like. Gene, a gene of a factor that induces T cell migration, a gene of a factor that enhances a T cell reaction, a gene of a factor that suppresses a T cell reaction, and the like.

  By expressing such a gene to be introduced into a dendritic cell, the function of the dendritic cell that plays a central role in controlling the immune response in vivo can be enhanced.

  In the present invention, for example, when an ES cell into which a nucleic acid containing a gene encoding an antigen is introduced, the obtained dendritic cell has an extremely high antigen-specific T cell stimulating activity or antigen-specific T cell reaction. This is advantageous in that it exhibits the activity of suppressing the above.

  In the present specification, 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, proteins specifically expressed in cancer cells (tumor antigen proteins) ), Peptides that are part of tumor antigen proteins, molecules that are targets of recognition by the immune system in autoimmune and allergic diseases, and the like.

  Nucleic acid can be introduced by a conventional method such as electroporation, lipofection, or the like. The electroporation method is desirable from the viewpoint of sufficiently exerting the differentiation performance of ES cells.

  The nucleic acid may be a so-called naked nucleic acid, or may be linked to 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, if necessary.

  In the present invention, it is desirable to introduce a non-viral vector such as a plasmid vector by electroporation from the viewpoint of ease of operation, therapeutic use, and the like.

The production method of the present invention is a dendritic tree prepared by inducing differentiation from conventional peripheral blood monocytes or hematopoietic stem cells in order to differentiate into dendritic cells by introducing a plasmid vector by electroporation at the ES cell stage. Compared with the method of introducing a gene into a cell using a viral vector, the method is superior in the following points.
1) In the case of a conventional method of gene introduction into a dendritic cell obtained by induction of differentiation from peripheral blood monocytes or hematopoietic stem cells using a viral vector, it is necessary to introduce the gene each time the dendritic cell is used. . In addition, gene transfer using a viral vector is unstable in transfer efficiency, and it is extremely difficult to obtain a gene-transferred dendritic cell as a clone. On the other hand, ES cells used in the present invention can be easily isolated, expanded and cryopreserved as clones. Therefore, if a stock of ES cells clones that have undergone appropriate genetic modification is isolated, expanded, and cryopreserved, a genetically uniform tree can be differentiated by differentiation into dendritic cells when necessary. It is excellent in that the dendritic cells can be stably used for treatment.
2) When a viral vector is used, there are limitations on the number (type) and size (size) of genes that can be introduced. On the other hand, in the case of ES cells, a normal plasmid vector can be used, which is excellent in that a plurality of genes can be introduced by gene co-transfection or stepwise introduction using a plurality of drug resistance genes. Furthermore, the present invention is excellent in that the targeted destruction, modification, and introduction of genes can be performed. These characteristics are particularly useful when more complicated genetic modification is required for the purpose of not only enhancing the immune effect but also for suppressing or qualitatively controlling the immune response.
3) In the method using ES cells, when genetically modified ES cell clones are established, the presence or absence of pathogenic microorganisms or activation of oncogenes is examined, and the characteristics at the stage of differentiation into dendritic cells are fully demonstrated. It is excellent in that it can be used for treatment after examination. Therefore, it is more useful for therapeutic use than the method using a viral vector.

  In another aspect, the present invention relates to dendritic cells obtained by the production method. According to the dendritic cell of the present invention, the immune response of an individual is controlled in an antigen-specific manner (for example, a strong activation of a cytotoxic T cell response to a specific antigen), and the antigen-specificity of the immune response It is possible to obtain a therapeutic effect on a disease for which a therapeutic effect is expected by the effective control.

  Therefore, according to the present invention, the use of dendritic cells obtained by said production method 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 Is also provided.

  In the production of the medicament, an auxiliary agent capable of stably holding the dendritic cells of the present invention, such as a medium, may be used as appropriate.

  Moreover, according to the present invention, a therapeutic effect is obtained by antigen-specifically controlling an immune response in a subject, which comprises administering to the subject a therapeutically effective amount of dendritic cells obtained by the production method. A method of treating a disease that can be obtained is provided.

  Examples of the “disease whose therapeutic effect can be obtained by specifically controlling the immune response by antigen” include, for example, autoimmune diseases, tumors, allergic diseases, infectious diseases, rejection associated with organ transplantation and graft-versus-host Disease (GVHD) and the like.

  The “subject” is preferably a human who needs treatment for a disease that can obtain a therapeutic effect by specifically controlling the immune response as described above. It may be an organism that requires treatment of a disease that can obtain a therapeutic effect by controlling a specific immune response in an antigen-specific manner, for example, a pet animal such as a dog or a cat.

  In addition, the “therapeutically effective amount” means that when a dendritic cell obtained by the production method is administered to the subject, the immune response is expressed as an antigen compared to a subject not administered the dendritic cell. This is the amount of dendritic cells that can be specifically controlled and thus can have a therapeutic effect on such diseases. The specific therapeutically effective amount may be appropriately set depending on the administration form of the dendritic cells, the administration method, the purpose of use and the age, weight, symptom, etc. of the subject and is not generally determined. The number of dendritic cells is preferably 200,000 to 1,000,000 / kg body weight per treatment for a human (for example, an adult).

  In the treatment method of the present invention, a method for administering a therapeutically effective amount of dendritic cells obtained by the production method to a subject includes, for example, subcutaneous injection, intralymphatic injection, intravenous injection, or local malignant tumor. Although direct injection etc. are mentioned, it is not limited to these.

  Moreover, according to this invention, the immune response control agent containing the dendritic cell obtained by the said manufacturing method as an active ingredient is also provided.

  According to the immune response control agent of the present invention, since the dendritic cell of the present invention is contained, the immune 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 that are active ingredients.

  The pharmacological evaluation of the immune response control agent of the present invention can be evaluated using T cell stimulation activity measured by a T cell proliferation assay as an index.

  EXAMPLES Hereinafter, although an Example etc. demonstrate this invention in detail, this invention is not limited by this Example etc.

  We tried to generate dendritic cells from cynomolgus monkey embryonic stem cells by in vitro differentiation induction. FIG. 1 shows an outline of differentiation-inducing culture from ES cells to dendritic cells. Note that some of the cells collected in the following steps were stored frozen. In addition, the cells in each step were analyzed using an inverted microscope (trade name: IX70, manufactured by Olympus).

(1) Step (A)
CMK6 cells, which are ES cell lines established from cynomolgus monkeys, were used as embryonic stem cells (ES cells) of primates.

First, ST2 cells (supplied by RIKEN) were cultured at 37 ° C. in DMEM supplemented with 10% by volume fetal calf serum on a culture dish (diameter: 10 cm, manufactured by Falcon) previously coated with a 0.1 wt% gelatin solution. They were cultured in a 5 vol% CO _2. The ST2 cells were grown until they almost covered the bottom of the culture dish.

The ES cells were suspended in αMEM (α essential medium) supplemented with 20% by volume fetal calf serum (FCS) at a density of 8 × 10 ⁴ cells / 8 ml. The obtained cell suspension was seeded on ST2 cells in the culture dish and cultured at 37 ° C. and 5% by volume CO ₂ to initiate differentiation induction of the ES cells. 4, 6, 8, and 9 days after the initiation of differentiation induction, the culture medium was removed and replaced with a new culture medium. Panels A to F in FIG. 2 show the results of observing the morphology over time of cells produced by induction of ES cell differentiation.

Panel A in FIG. 2 is an undifferentiated ES cell, and panels BF show the morphology of ES cell-derived cells that are differentiating 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. As shown in FIG. 2, the undifferentiated ES cells proceeded to differentiate into mesodermal cells as the number of culture days passed, and the density of the cells differentiated into mesodermal cells increased. In addition, as a result of starting differentiation culture with 8 × 10 ⁴ ES cells per 10 cm diameter culture dish, the surface of the feeder layer was almost ES cell-derived epithelial cell-like by 6 days after the start of differentiation induction. Covered with cells. As shown in panel B, at the third day after the initiation of differentiation induction, many cells formed a large flat cell mass like an epithelial cell surrounded by round cells. Further, as shown in panels C and D, on the 4th day after the initiation of differentiation induction, a round cell mass that appeared to be a cell differentiated into a mesoderm appeared around the epithelial cell-like cell mass. When the medium was changed every other day, the mesoderm-like round cell mass gradually became larger and fused. As shown in panels E and F, on the 10th day after the start of differentiation induction, 40-60% of the surface of the feeder layer was covered with a mesoderm-like round cell mass.

  Ten days after the start of culture, 1.5 ml of phosphate buffered saline containing 0.25 wt% trypsin (manufactured by Gibco-Invitrogen) / 1 mM EDTA (ethylenediaminetetraacetic acid) was added to the culture dish. The cells were added and maintained at 37 ° C. for 10 minutes to separate ES cell-derived cells from the ST2 cell layer. Thereafter, a new medium was added to the culture dish, and ES cell-derived cells were transferred to a centrifuge tube. As a result, cells that differentiated into mesoderm separated and became single cells, and most of the cells that did not differentiate and cells that differentiated into epithelium became clumps and settled to the bottom of the tube within a few minutes. Therefore, the supernatant single cell suspension was transferred to a new tube.

(2) Step (B)
In step (A) of the above (1), ES cell-derived cells recovered from one culture dish are respectively transplanted on OP9 cell layers prepared in 4 to 8 other culture dishes, The cells were further cultured in αMEM containing 20% by volume fetal calf serum at 37 ° C. and 5% by volume CO ₂ . The OP cell layer was obtained by growing until the bottom surface of the culture dish was almost covered, similar to the ST2 cells.

  On the first or second day of transplantation, a small round cell mass was formed on the feeder cell layer. Such cell clumps were initially sparse but gradually increased. On the 4th day after transplantation, 4 ml of a new culture solution was added to the culture dish. On the 6th day after transplantation, spherical floating cells derived from ES cells were collected by pipetting. Panel G in FIG. 2 shows the morphology of cells differentiated from the ES cells obtained in step (B) (6 days after transplantation, 16 days after initiation of differentiation). As shown in the panel (G) of FIG. 2, most of the cells derived from ES cells were small and round.

(3) Step (C)
ES cell-derived cells collected from the four culture dishes in the step (B) of (2) were subjected to a final concentration of 100 ng / ml granulocyte macrophage colony-stimulating factor (GM-CSF; manufactured by Peprotec) and a final concentration of 100 ng. / Ml macrophage colony stimulating factor (M-CSF; manufactured by Peprotec) was suspended in αMEM containing 20% by volume fetal calf serum. The obtained cells were transplanted on an OP9 cell layer prepared in a separate culture dish (diameter 10 cm) and cultured at 37 ° C. and 5% by volume CO ₂ .

  On the 4th day after transplantation, 4 ml of a new culture solution [composition: αMEM containing 20% by volume fetal calf serum] containing 100 ng / ml GM-CSF and not containing M-CSF was added to the culture dish.

  On the sixth day after transplantation, spherical floating cells derived from ES cells were collected by pipetting. Panel H in FIG. 2 shows the morphology of cells differentiated from ES cells (6 days after transplantation, 22 days after initiation of differentiation induction).

  As a result, as shown in panel H, the number of floating cells was further increased. Further, as shown in panel H, the cultured cells contained irregularly shaped dendritic cell-like cells that became morphologically heterogeneous and had protrusions. The total number of floating cells was around 20 times the number of undifferentiated ES cells. In this step (C), the same cells as described above can be obtained even when a medium containing GM-CSF is used from the beginning without M-CSF.

(4) Step (D)
The ES cell-derived cells collected in step (C) of (3) were suspended at a density of 5 × 10 ⁵ cells / 2.5 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% by volume CO ₂ . As the culture solution, a final concentration of 100 ng / ml GM-CSF (manufactured by Peprotec), a final concentration of 10 ng / ml interleukin-4 (IL-4; manufactured by Peprotec) and a final concentration of 10 ng / ml FLT-3L (fms) ΑMEM containing tyrosine kinase 3 ligand (manufactured by Peprotec) and 20% by volume fetal bovine serum was used.

  As a result, some (<30%) cells adhered to the surface of the culture dish like macrophages. In this stage, the floating cells are divided into two types of cell groups, a group of spherical cells and a group of cells (dendritic cell-like cells) having an irregular shape with protrusions shown in panel I. It was. In this step (D), cells similar to those described above can be obtained even when a culture solution that does not contain FLT-3L is used from the beginning.

(5) Step (E)
On the 3rd to 4th day after transplantation in the step (D) of the above (4), a final concentration of 10 ng / ml tumor necrosis factor α (TNF-α; manufactured by Peprotec) and a final concentration of 3 μg / ml lipopolysaccharide are contained in the culture. (LPS, E. coli-derived; manufactured by SIGMA) was added and cultured at 37 ° C. and 5% by volume CO ₂ to stimulate floating cells in the culture obtained in step (D) of (4) above. .

  As a result, the ratio of dendritic cell-like cells contained in the floating cells increased, and some of the attached cells became floating cells.

  From 1 to 3 days after stimulation, cells were collected by pipetting. The number of ES cell-derived dendritic cell-like cells in this culture stage was about 20 times the number of undifferentiated ES cells at the start of differentiation induction. Moreover, the form of the cell differentiated from the ES cell (26th day after the start of differentiation induction) is shown in panels J to L of FIG.

  As a result, as shown in panels J to L of FIG. 2, it can be seen that irregularly shaped cells having protrusions can be obtained.

  In Example 1 (1) Step (A), 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.

  As a result, more epithelial cells appeared and fewer round cell masses were observed when OP9 cells or PA6 cells were used than when ST2 cells of Example 1 were used.

  In Example 1 (1) Step (A), bone morphogenetic protein 4 (BMP-4), thrombopoietin (TPO), stem cell stimulating factor (SCF), Flt-3L, vascular endothelial growth factor (VEGF), etc. The effects of using these cytokines alone or in various combinations were examined.

  As a result, in step (A), even when the cytokine was used, a remarkable effect of promoting differentiation from ES cells to mesodermal cells was not observed.

  In each of steps (B) and (C) of Example 1 (2), ST2 cells or PA6 cells are used instead of OP9 cells, and in the same manner as in the case of OP9 cells, step (B ) And step (C).

  As a result, there were fewer floating cells obtained after step (C) when ST2 cells or PA6 cells were used than when OP9 cells of Example 1 were used.

Cell surface molecule analysis The ES cell in Example 1 was obtained by using a flow cytometer (trade name: FACScan, manufactured by Becton-Dickinson) to express cell surface molecules expressed in physiologically present dendritic cells. Whether it was also expressed in the cells of origin was examined.

  The ES cell-derived dendritic cell-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. Staining was performed using (clone FUN-1, mouse IgG1) and anti-human CD40 (clone 5C3, mouse IgG1). The anti-human mAb cross-reacts with cynomolgus monkey cells. In addition, staining was performed using mouse IgG2a (clone G155-178) and mouse IgG1 (clone MOPC-21) as isotype-matched controls. Together with incubation at 4 ° C. for 30 minutes.

  The cells were then washed 3 times with PBS / 2 vol% FCS.

  The washed cells were analyzed using a cell analyzer equipped with CellQuest software (trade name: FACScan, manufactured by Becton Dickinson).

  As a result, as shown in FIG. 3, in the cells after step (B) (“day 16” in FIG. 3) and the cells after step (C) (“day 22” in FIG. 3), these molecules Almost no expression. In addition, as shown in “day 26” in FIG. 3, the ES cell-derived dendritic cell-like cells recovered in step (D) expressed HLA-DR, CD40, and CD86 at low levels. . On the other hand, as shown by “day 28” in FIG. 3, the cells stimulated with TNF-α and LPS in step (E) expressed these molecules at a high level.

Gene expression analysis by RT-PCR analysis The cells were homogenized with a trade name: Qiashredder (Qiagen), and total RNA was extracted using a trade name: RNeasy kit (Qiagen).

Using the obtained total RNA equivalent to 1 μg, a random hexamer primer (manufactured by Gibco-BRL) and Superscript ^™ II reverse transcriptase (manufactured by Gibco-BRL) under the conditions of incubation at 42 ° C. for 50 minutes. CDNA was synthesized.

  Using the obtained cDNA as a template, using the following primer pairs, the presence or absence of expression in cells derived from the ES cells in Example 1 was examined for the molecules expressed in physiologically existing dendritic cells. The relative amount of the cDNA was normalized with the hypoxanthine phosphoribosyltransferase (HPRT) gene by PCR.

As PCR primers, the following primer pairs:
Nucleic acid encoding HPRT:
5′-gctggattacacatcaagcactgaa-3 ′ (SEQ ID NO: 1), and 5′-caacaaagtctggcttattatccaa-3 ′ (SEQ ID NO: 2),
Nucleic acid encoding CD80:
5'-ctctccattgtgatcctggctctg-3 '(SEQ ID NO: 3), and 5'-accaggagggtgaggctctgaga-3' (SEQ ID NO: 4),
Nucleic acid encoding IL-12p40 subunit:
5′-gctcaagttagaaaaactacaccag-3 ′ (SEQ ID NO: 5), and 5′-cagtgaccgtgggctgaggctttgt-3 ′ (SEQ ID NO: 6),
Nucleic acid encoding cynomolgus monkey CD80: SEQ ID NOs: 3 and 4
Nucleic acid encoding cynomolgus monkey IL-12p40 (40 kD subunit): SEQ ID NOs: 5 and 6
Was used.

  The thermal profile in PCR was such that after incubation at 95 ° C for 5 minutes, 35 cycles of 95 ° C for 1 minute, 56 ° C for 1 minute and 72 ° C for 1 minute were performed, and 72 ° C for 5 minutes. is there.

  The obtained PCR product was subjected to visualization by ethidium bromide-containing 1.25 wt% agarose gel electrophoresis. The results are shown in FIG.

  As a result, as shown in FIG. 4, expression of CD80 was observed in the cells after step (C) (day 22). Further, as shown in FIG. 4, cells stimulated with TNF-α and LPS in step (E) are interleukin-12 (IL-12), which is a cytokine having an action of activating T cells. ) Was also expressed. Therefore, it was suggested that the cells obtained in Example 1 were dendritic cells.

Analysis of T cell stimulation activity by mixed lymphocyte reaction (MLR) Whether dendritic cells derived from ES cells have the activity of stimulating T cells and causing proliferative responses, allo (allogeneic) T cells This was investigated by quantifying T cell proliferation when co-cultured with T. As reactive T cells, cynomolgus monkey peripheral blood T cells that were separate from those used for the establishment of ES cells were used. In addition, as stimulating cells, X-ray irradiation (40 Gy) was performed on ES cells in an undifferentiated state (negative control), ES cell-derived dendritic cells after step (D), and ES cell-derived dendritic cells after step (E), respectively. ) Was used.

  Mononuclear cells were isolated from cynomolgus monkey heparinized blood bred at the Institute of Chemotherapy and Serum Therapy using trade name: Ficoll-Paque PLUS (manufactured by Amersham Biosciences).

Subsequently, CD14 ^- cells were isolated from mononuclear cells using magnetic beads coated with an anti-human CD14 antibody (trade name, supermagneticMicroBeads, manufactured by Miltenyi Biotec), and this was isolated on a tissue culture dish on a medium [composition: 2 In a volume% heat-inactivated fetal bovine serum-containing RPMI-1640 medium], the cells were cultured at 37 ° C. for 1 hour.

Thereafter, cells that do not adhere to the culture dish were collected and allowed to stand at 37 ° C. and 5 vol% CO ₂ for 1 hour in a nylon wool column (made by filling a 10 ml plastic syringe with nylon wool manufactured by Wako Pure Chemical Industries, Ltd.). I put it. Cells that did not adhere to the nylon wool column were used as killer T cells.

The stimulated cells of various numbers of cells were placed in a 96-well round bottom culture dish (Falcon) at 37 ° C. and 5% by volume CO ₂ in RPMI-1640 medium supplemented with 10% heat-inactivated human plasma. Co-cultured with killer T cells for 5 days. [ ³ H] -thymidine (247.9 Gbq / mmol) was added to the medium to a final concentration of 0.037 Mbq / well for the last 8 hours. After the culture, the cells were taken on a glass fiber filter (Wallac), and [ ³ H] -thymidine incorporation in the cells was measured by scintillation. The results are shown in FIG.

  As a result, as shown in FIG. 5, a strong proliferation reaction of reactive T cells was observed only when ES cell-derived dendritic cells were used as stimulating cells. Moreover, as FIG. 5 shows, it turns out that the dendritic cell after adding the maturation stimulus after step (E) has stronger T cell stimulation activity.

(1) Genetic modification of ES cell-derived dendritic cells using a plasmid vector HLA-DRB4 ^* 0103 cDNA, which is a type of human histocompatibility leukocyte antigen (HLA) class II β chain gene, is activated by the CAG promoter Cloning into the mammalian expression vector pCAG-IRES-Neo containing the ribosome entry site (IRES) -neomycin resistance gene cassette yielded pCAG-DRB4-IN. Panel A in FIG. 6 shows a schematic diagram of the pCAG-DRB4-IN.

1.0 × 10 ⁷ undifferentiated cynomolgus monkey ES cells were suspended in 0.4 ml of Dulbecco's modified Eagle medium (DMEM; manufactured by Gibco-BRL). The obtained suspension was mixed with 50 μg of the pCAG-DRB4-IN. The obtained mixture was put in a 4 mm gap cuvette (trade name: BM6400, manufactured by BTX), and the cuvette was set in a trade name: Gene Pulser (manufactured by Bio-Rad). Thereafter, gene transfer by electroporation was performed under conditions of 250 V and 500 μF.

The ES cell into which the gene has been introduced is placed on a PEF (mouse fetal fibroblast) layer on a 10 cm culture dish, in a DMEM containing a medium (composition: 20% by volume KSR (Gibco-BRL)), 37 ° C., 5% by volume. They were cultured in CO _2. From the second day after gene introduction, drug-resistant cells, ie, gene-introduced cells, were selected by adding G418 (150 μg / ml), which is a selective drug, to the medium.

On day 11 after gene introduction, gene-transfected cell (transfectant) clones were collected as G418-resistant cell colonies 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 μg / ml), and a part thereof was stored frozen. The remaining part was cultured at 37 ° C. and 5% by volume CO ₂ in 3 mg / ml G418-containing medium [composition: 20% by volume KSR (manufactured by Gibco-BRL) -containing DMEM]. Transfectant clones that can grow even in the presence of G418, that is, clones that were predicted to have high transgene expression levels were selected.

In addition, the expression of the transgene was examined by RT-PCR. Total RNA was extracted from the obtained cells using a trade name: RNeasy kit (manufactured by Qiagen). CDNA was obtained under the conditions of incubation at 42 ° C. for 50 minutes using 1 μg of the total RNA obtained, random hexamer primer (Gibco-BRL) and Superscript ^™ II reverse transcriptase (Gibco-BRL). Synthesized.

  Using the obtained cDNA as a template, PCR was carried out using 5′-ctgactgaccgcgttactcccaca-3 ′ (SEQ ID NO: 7) and 5′-ttggttattagattagttatgagtgt-3 ′ (SEQ ID NO: 8) to examine the expression of the transgene. It was. As a control, the expression of HPRT gene was examined. The thermal profile in PCR was such that after incubation at 95 ° C for 5 minutes, 35 cycles of 95 ° C for 1 minute, 56 ° C for 1 minute and 72 ° C for 1 minute were performed, and 72 ° C for 5 minutes. is there.

  Based on the G418 resistance and the results of RT-PCR, 12 transfectant clones of ES cells with relatively high transgene expression were selected.

  Next, in the same manner as in Example 1, dendritic cells were induced to differentiate from the obtained ES cells.

(2) Transgene expression analysis by RT-PCR analysis In the same manner as in (1) above, transgene expression was confirmed by RT-PCR analysis. The results are shown in panel B of FIG.

  As a result, as shown in panel B of FIG. 6, it can be seen that the transgene-derived DR53β mRNA is expressed in the cells after gene introduction and drug selection by G418. It can also be seen that the transgene-derived DR53β mRNA is also expressed in dendritic cells induced to differentiate from the transgenic ES cells.

Evaluation of antigen-presenting ability The human T-cell line SA32.h which shows the proliferation reaction by specifically recognizing the GAD65 antigen-derived peptide presented on the DR53 molecule with respect to the DR53β gene-introduced dendritic cells obtained in Example 7 above. Antigen presentation ability to 5 was examined.

(1) Preparation of synthetic peptide An oligopeptide corresponding to human glutamate decarboxylase 65 (GAD65) p111-131 (LQDVMMNILLQYVVKSFDRSTK; SEQ ID NO: 9) was synthesized and purified.

(2) Antigen presentation assay In the assay using a synthetic peptide, the dendritic cells obtained in Example 1 were used in the presence of the synthetic peptide obtained in (1) (0 to 5 μM) at 37 ° C, 5% by volume. Incubated for 3 hours with CO ₂ , washed 4 times with medium and X-irradiated (40 Gy) to lose growth ability.

Loaded with SA32.5 (5 × 10 ⁴ / well), a human CD4 ⁺ T cell clone that recognizes GAD65 p111-131 bound to HLA-DR53 molecule (DRA ^* 0101 + DRB4 ^* 0103), and antigen (the synthetic peptide) The cultured dendritic cells (2 × 10 ⁴ / well) are cultured on a 96-well flat-bottom culture plate in RPMI-1640 medium supplemented with 10% by volume human plasma at 37 ° C. and 5% by volume CO _2. A T cell proliferation assay was performed.

After 48 hours of culturing, [ ³ H] -thymidine was added to the medium and further cultured for 16 hours. Thereafter, the cells were collected, and the incorporation of [ ³ H] -thymidine into the chromosomal DNA accompanying SA32.5 proliferation was measured using a scintillation counter for β-ray measurement. In addition, as a negative control, a cell loaded with a peptide on a dendritic cell derived from an ES cell not transfected with a gene and a DR53β gene-introduced dendritic cell loaded with no peptide were used together with SA32.5 as described above. Cultured. The results are shown in panel A of FIG.

  As a result, as shown in panel A, only DR3β gene-introduced dendritic cells loaded with peptides induced SA32.5 proliferation. From this result, it was confirmed that the DR53β derived from the gene introduced at the ES cell stage functions after dendritic cell differentiation, and that the ES cell-derived dendritic cell into which the DR53β chain gene has been introduced is a GAD65 antigen-derived peptide. On the DR53 molecule and found to stimulate the SA32.5T cell line to induce a proliferative response. In addition, in dendritic cells derived from ES cells into which the DR53β gene has been introduced, the cynomolgus monkey DRα chain encoded by the cynomolgus monkey gene and the transgene-derived DR53β chain associate to form a DR53 molecule that is expressed on the cell surface. It is suggested.

Limited degradation and presentation of antigens by ES cell-derived dendritic cells The DR53β gene-introduced dendritic cells obtained in Example 7 take up protein antigens into the cells and produce these peptides by limited degradation to produce cell surfaces. It was examined whether it has the activity presented on the above DR molecule.

(1) Preparation of recombinant protein A DNA fragment encoding the GAD65 p96-174 protein fragment was ligated to a prokaryotic expression vector pGEX-4T-3 (manufactured by Amersham Biosciences), and then the obtained vector was used. , E.C. E. coli DH5α is transformed, whereby the E. coli DH5α is transformed. In E. coli DH5α, glutathione-S-transferase fusion GAD65 protein (GST-GAD) was expressed. The method reported by Fragioni and Neel [Anal. Biochem. 210, 179-187 (1993)]. Recombinant protein production in E. coli DH5α was induced and the recombinant protein was extracted from bacterial inclusion bodies. The recombinant protein was purified using glutathione-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.

(2) Antigen presentation assay In an assay using a recombinant protein, the DR53β gene-introduced dendritic cell obtained in Example 7 was treated in the presence of GST or the GST-GAD protein obtained in (1) above ( 18 μM), final concentration of 10 ng / ml TNF-α and final concentration of 3 μg / ml LPS, incubated at 37 ° C., 5 vol% CO ₂ for 20 hours, washed 3 times with medium, X-irradiated (40 Gy), proliferative ability Was lost.

The SA32.5 (5 × 10 ⁴ / well) and the dendritic cells (2 × 10 ⁴ / well) loaded with the antigen (the recombinant protein) were placed on a 96-well flat-bottom culture plate at 10% by volume human. T cell proliferation assay was performed by culturing in RPMI-1640 medium supplemented with plasma at 37 ° C., 5% by volume CO ₂ .

After 48 hours, [ ³ H] -thymidine was added to the culture and cultured for another 16 hours. The incorporation of [ ³ H] -thymidine into chromosomal DNA accompanying SA32.5 growth was measured using a scintillation counter for β-ray measurement. As negative controls, ES cell-derived dendritic cells not transfected with GAD65 protein and DR53β-transfected dendritic cells loaded with GST protein were also cultured with SA32.5. The results are shown in panel B of FIG.

  As a result, as shown in panel B, only those in which DR53β gene-introduced dendritic cells were loaded with GST-fused GAD65 protein induced proliferation of SA32.5. These results indicate that ES cell-derived dendritic cells have the ability to take up the dissolved antigen protein into the cell, degrade it, and present it as a peptide on the DR molecule.

  According to the present invention, dendritic cells derived from primates can be efficiently and stably supplied in large quantities, and a normal plasmid vector can be introduced by electroporation without using a viral vector. Can be genetically modified. According to the present invention, genetic modification of dendritic cells can be carried out easily, efficiently and safely. By administering such a genetically modified dendritic cell 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 an 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.

Claims (14)

(A) a step of co-culturing primate embryonic stem cells and cells having the property of inducing differentiation and proliferation of blood cells to obtain cell group A;
(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,
(C) The cell group B obtained in the step (B) and a newly prepared cell having the property of inducing differentiation and proliferation of blood cells are co-presented in the presence of granulocyte macrophage colony-stimulating factor. Culturing to obtain cell group C, and (D) culturing cell group C obtained in step (C) in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4.
A method for differentiating primate embryonic stem cells into dendritic cells, comprising:   In step (A), from a co-culture of a primate embryonic stem cell and a cell having the property of inducing differentiation and proliferation of blood cells, derived from a primate embryonic stem cell and mesoderm The differentiation method of Claim 1 which isolate | separates the cell group A as a cell group containing the cell differentiated into the systemic cell.   The cells having the property of inducing differentiation and proliferation of blood cells in step (A) are ST2 cells, and the newly prepared cells having the property of inducing differentiation and proliferation of blood cells in step (B) are The differentiation method according to claim 1 or 2, 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.   After step (D), (E) adding tumor necrosis factor α and lipopolysaccharide to the culture obtained in step (D), and further performing a step of culturing. 2. The differentiation method according to item 1. (A) a step of co-culturing primate embryonic stem cells and cells having the property of inducing differentiation and proliferation of blood cells to obtain cell group A;
(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,
(C) The cell group B obtained in the step (B) and a newly prepared cell having the property of inducing differentiation and proliferation of blood cells are co-presented in the presence of granulocyte macrophage colony-stimulating factor. Culturing to obtain cell group C, and (D) culturing cell group C obtained in step (C) in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, and E ′) separating dendritic cells derived from primate embryonic stem cells from the culture obtained in step (D),
A method for producing dendritic cells from primate embryonic stem cells. Instead of step (E ')
(E) adding tumor necrosis factor α and lipopolysaccharide to the culture obtained in step (D) and further culturing; and (F) from the culture obtained in step (E), Isolating dendritic cells derived from primate embryonic stem cells;
The manufacturing method of Claim 5 which performs.   The production method according to claim 5 or 6, wherein a nucleic acid containing a gene to be introduced is introduced into an embryonic stem cell of a primate animal prior to performing step (A).   The production method according to any one of claims 5 to 7, wherein the gene to be introduced is a gene for immunosuppression.   The production method according to any one of claims 5 to 7, wherein the gene to be introduced is a gene for immunostimulation.   The production method according to any one of claims 7 to 9, wherein the nucleic acid is introduced by a non-viral vector-mediated nucleic acid introduction method.   The dendritic cell obtained by the manufacturing method of any one of Claims 5-10.   A dendritic tree obtained by the production method according to any one of claims 5 to 10 for the production of a medicament for the treatment of a disease capable of obtaining a therapeutic effect by antigen-specific control of the immune response. Use of cells.   The treatment of the disease which can obtain a therapeutic effect by containing the dendritic cell obtained by the manufacturing method of any one of Claims 5-10 as an active ingredient as an active ingredient, and antigen-specifically controlling it. Cell medicine for.   An antigen-specific control of an immune response in a subject, comprising administering to the subject a therapeutically effective amount of dendritic cells obtained by the production method according to any one of claims 5 to 10. The therapeutic method of the disease which can acquire a therapeutic effect by this.

Images