WO2021161997A1

WO2021161997A1 - Modified human interleukin-4 receptor polypeptide and induction agent for differentiation or polarization into m2 macrophages

Info

Publication number: WO2021161997A1
Application number: PCT/JP2021/004780
Authority: WO
Inventors: 昭彦田口
Original assignee: 国立大学法人山口大学; 昭彦田口
Priority date: 2020-02-10
Filing date: 2021-02-09
Publication date: 2021-08-19

Abstract

The present invention addresses the problem of providing: a polypeptide that contributes to polarization into M2 macrophages independent of IL-4; and an induction agent for differentiation or polarization into M2 macrophages.　A polypeptide derived from the human interleukin-4 receptor, the polypeptide being the result of modifying the amino acid sequence given by SEQ ID NO:1 by adding, deleting, or substituting one or more amino acids, preferably by substituting the 242nd isoleucine of the amino acid sequence given by SEQ ID NO:1 with asparagine.

Description

Mutant polypeptide of human interleukin-4 receptor and differentiation or polarization inducer to M2 macrophages

The present invention includes a mutant polypeptide of IL-4 receptor derived from human interleukin 4 receptor, a polynucleotide encoding the mutant polypeptide of the IL-4 receptor, an expression vector containing the polynucleotide, and the like. , Macrophages or macrophage precursor cells isolated from the living body containing the polynucleotide or expression vector, macrophages or macrophage precursor cells isolated from the living body containing the polypeptide, or pharmacologically with the macrophages or macrophage precursor cells. From macrophages or macrophage precursor cells isolated from living organisms, including prophylactic or therapeutic agents for inflammatory diseases, neurodegenerative diseases or ischemic diseases, and macrophages or macrophage precursor cells containing the polynucleotide or the expression vector as an active ingredient. The present invention relates to an agent for inducing differentiation or polarization into M2 macrophages, and a method for inducing differentiation or polarization of macrophages or macrophage precursor cells isolated from a living body into M2 macrophages.

There are various diseases such as inflammatory diseases, neurodegenerative diseases, and ischemic diseases, and drugs have been developed according to each, but there are still many diseases for which sufficient effects cannot be expected. Macrophages, which are immune cells, are present in the lesions of these inflammatory diseases, divine neurodegenerative diseases, and ischemic diseases, and are considered to be involved in pathogenesis.

In recent years, research on macrophages has progressed, and it has become clear that macrophages include M1 macrophages that induce inflammation and M2 macrophages that are responsible for inflammation suppression and tissue repair. It has been found that M2 macrophages have beneficial functions for living organisms, such as sinking excessive inflammation and phagocytosing β-amyloid protein and the like found in Alzheimer's disease (see Non-Patent Documents 1 and 2). In addition, the ratio of M1 and M2 macrophages becomes predominant in M1 with aging, and it is considered that the M1 / M2 imbalance promotes aging (see Non-Patent Document 3). Against this background, research is underway to activate M2 macrophages by various methods. Specifically, the main method is to systemically administer a humoral factor or a small molecule compound to activate M2 macrophages, but there is currently no trend toward clinical application (see Non-Patent Document 4). The reason is that, for example, a humoral factor for activating to M2 macrophages acts on B lymphocytes and increases IgE production, which causes exacerbation of allergic diseases, and there is a risk of giving to cells other than macrophages. Are known. Furthermore, since humoral factors cannot cross the blood-brain barrier, they have problems such as not being able to exert sufficient effects on central inflammatory and neurodegenerative diseases such as Alzheimer's disease. rice field.

Furthermore, since the inflamed site is rich in bioactive substances that activate M1 macrophages, M2 macrophages that were once activated by systemic administration of humoral factors are repolarized into M1 macrophages in the actual inflamed site. It is suggested that there is a possibility.

From the above, it is required to develop a substance that does not rely on humoral factors, is less likely to polarize to M1 macrophages at the inflamed site, and activates M2 macrophages.

By the way, variants of IL-4 receptor have been reported in hematological malignant lymphoma, bladder cancer or brain tumor (see Non-Patent Documents 5 to 7). However, the relationship between such IL-4 receptor mutations and macrophage differentiation or polarization is unknown.

Until now, as an attempt to increase M2 macrophages, a method of systemically administering a humoral factor or compound has been attempted. However, as a problem of systemic administration, in the actual inflammatory site, the local concentration of the humoral factor that induces M1 macrophages is high, the effect is low for the dose, and it acts on other cells to cause asthma, etc. There was a risk of exacerbating immune disorders. In addition, M2 macrophages are also expected to be effective for degenerative diseases of the central nervous system such as Alzheimer's disease, but since humoral factors and compounds cannot cross the blood-brain barrier, these methods are used to treat the central nervous system such as Alzheimer's disease. The problem was that it could not be used for. Furthermore, when IL-4 is used to activate M2 macrophages, it exerts many actions such as proliferation of immature T cells and enhancement of expression of mast cell proliferation due to the action of IL-4 on other than macrophages. Since it has, there is also a problem that it brings about an action other than activation of M2 macrophages. Therefore, an object of the present invention is to provide a polypeptide involved in differentiation or polarization into M2 macrophages without depending on IL-4, or to provide a differentiation or polarization inducer into M2 macrophages.

The inventor focused on the human interleukin-4 receptor, and proceeded with the study, thinking that if a mutant of the human interleukin-4 receptor was introduced into the macrophage, it would be activated as an M2 macrophage. As a result, the present invention was completed by succeeding in differentiation or polarization into M2 macrophages by expressing a mutant of human interleukin-4 receptor in macrophages or macrophage progenitor cells.

That is, the present invention is as follows.
[1] A polypeptide derived from the human interleukin-4 receptor, wherein one or several amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1.
[2] The polypeptide according to the above [1], which is the following (1-1) or (1-2).
(1-1) A polypeptide derived from the human interleucine 4 receptor, in the amino acid sequence shown in SEQ ID NO: 1, aspartic acid at position 37, tyrosine at position 62, asparagin at position 78, and phenylalanine at position 115. Polypeptide substituted with aspartic acid at position 176, arginine at position 200, isoleucine at position 242, cysteine at position 251 and / or lysine at position 308;
(1-2) In the amino acid sequence shown in SEQ ID NO: 1, the 37th, 62nd, 78th, 115th, 176th, 200th, 242nd, 251st and / or 308 substituted in the above (1-1). It was expressed in THP-1 cells or RAW264.7 cells containing a polypeptide in which one or several amino acids were added, deleted or substituted in a non-th amino acid and stimulated with Holbol 12-millistart 13-acetate. A polypeptide that, in some cases, increases the expression of human M2 macrophage markers as compared to the case where the polypeptide is not expressed.
[3] In the amino acid sequence shown in SEQ ID NO: 1, aspartic acid at position 37, tyrosine at position 62, asparagin at position 78, phenylalanine at position 115, arginine at position 176, arginine at position 200, isoleucine at position 242, and isoleucine at position 251. The polypeptide according to [2] above, wherein the polypeptide substituted with cysteine and / or lysine at position 308 is any of the following (2-1) to (2-9).
(2-1) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which aspartic acid at position 37 is replaced with a neutral polar amino acid;
(2-2) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which tyrosine at position 62 is replaced with a neutral polar amino acid;
(2-3) In the amino acid sequence shown in SEQ ID NO: 1, the polypeptide in which asparagine at position 78 is replaced with a neutral polar amino acid;
(2-4) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which phenylalanine at position 115 is replaced with a neutral non-polar amino acid;
(2-5) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which asparagine at position 176 is replaced with a neutral polar amino acid;
(2-6) In the amino acid sequence shown in SEQ ID NO: 1, the polypeptide in which arginine at position 200 is replaced with a neutral non-polar amino acid;
(2-7) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which isoleucine at position 242 is replaced with an acidic amino acid or a neutral non-polar amino acid;
(2-8) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which cysteine at position 251 is replaced with a neutral non-polar amino acid;
(2-9) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which lysine at position 308 is replaced with a neutral polar amino acid;
[4] The polypeptide according to the above [2], which is a polypeptide derived from the human interleukin-4 receptor and is a polypeptide substituted with isoleucine at position 242 in the amino acid sequence shown in SEQ ID NO: 1. peptide.
[5] The above-mentioned [4], which is a polypeptide derived from the human interleukin-4 receptor, wherein the isoleucine at position 242 is replaced with asparagine in the amino acid sequence shown in SEQ ID NO: 1. Polypeptide.
[6] A polynucleotide encoding the polypeptide according to any one of the above [1] to [5].
[7] An expression vector containing the polynucleotide according to [6] above.
[8] Macrophages or macrophage progenitor cells isolated from a living body containing the polynucleotide described in [6] above or the expression vector described in [7] above.
[9] Macrophage or macrophage progenitor cell isolated from a living body containing the polypeptide according to any one of the above [1] to [5].
[10] A prophylactic or therapeutic agent for an inflammatory disease, a neurodegenerative disease or an ischemic disease, which comprises the macrophage or macrophage progenitor cell according to the above [8] or [9] and a pharmacologically acceptable additive.
[11] An agent for inducing differentiation or polarization of macrophages or macrophage progenitor cells isolated from a living body into M2 macrophages, which comprises the polynucleotide described in [6] above or the expression vector described in [7] above as an active ingredient.
[12] Macrophages or macrophage progenitor cells isolated from living organisms, characterized in that the polynucleotide described in [6] above or the expression vector described in [7] above is introduced into macrophages or macrophage progenitor cells isolated from living organisms. A method for inducing differentiation or polarization from M2 macrophages.

The differentiation or polarization inducing agent for M2 macrophages of the present invention makes it possible to differentiate or polarize macrophages or macrophage progenitor cells isolated from living organisms into M2 macrophages.

In Example 1, a polynucleotide encoding an I242N mutation (isoleucine at position 242 replaced with asparagine) human IL-4 receptor was introduced into RAW264.7 cells or THP-1 cells to express IL-4 receptor. This is the result of the investigation. In Example 2, the polynucleotide encoding the I242N mutant human IL-4 receptor was introduced into RAW264.7 cells or THP-1 cells, and the expression of Arg1 or CD206 was examined. In Example 3, the polynucleotide encoding the I242N mutant human IL-4 receptor was introduced into RAW264.7 cells, and LPS was allowed to act to examine the expression of TNFα or Arg1. In Example 4, a polynucleotide encoding a mutant human IL-4 receptor other than I242N was introduced into RAW264.7 cells, and the expression of Arg1 was examined. In Example 5, the polynucleotide encoding the I242N mutant human IL-4 receptor was introduced into a bone marrow-derived macrophage (BMDM), and the expression of Arg1 was examined. This is the result of introducing a polynucleotide encoding the I242N mutant human IL-4 receptor into RAW264.7 cells and examining the expression of IL-10 in Example 6.

A polypeptide derived from the "human interleukin-4 receptor" of the present invention in which one or several amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1 (hereinafter, "mutation of the present invention". The human interleukin-4 receptor (human IL-4 receptor) in "IL-4R polypeptide") is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and in NCBI, Reference No. It is registered as NP_000409.1.

The "polypeptide in which one or several amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1" in the above-mentioned mutant IL-4R polypeptide is preferably "in the amino acid sequence shown in SEQ ID NO: 1." "Amino acids constituting the extracellular region or transmembrane region, in which one or several amino acids are added, deleted or substituted", more preferably "amino acids constituting the transmembrane region in the amino acid sequence shown in SEQ ID NO: 1". In, a polypeptide to which one or several amino acids have been added, deleted or substituted ”can be mentioned. The extracellular space or transmembrane region can be predicted using a known extracellular space or transmembrane region prediction algorithm. In the amino acid sequence shown in SEQ ID NO: 1, the 1st to 232nd regions are considered to be extracellular regions, and the 233rd to 256th regions are considered to be transmembrane regions. The positions where one or several amino acids are added, deleted or substituted in the above "amino acids constituting the extracellular region" are aspartic acid at position 37, tyrosine at position 62, and position 78 in the amino acid sequence shown in SEQ ID NO: 1. Aspartic acid, 115th phenylalanine, 176th aspartic acid, and 200th arginine. As the position where one or several amino acids are added, deleted or substituted in the above "amino acid constituting the transmembrane region", isoleucine at position 242 and cysteine at position 251 can be mentioned, and isoleucine at position 242 is preferable. Can be listed in.

In the present specification, the neutral non-polar amino acids include glycine (G), alanine (A), valine (V), proline (P), leucine (L), isoleucine (I), phenylalanine (F), and methionine ( It means M) or tryptophan (W). The neutral polar amino acid means asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), or cysteine (C). Further, the acidic amino acid means aspartic acid (D) and glutamic acid (E).

As the above-mentioned "polypeptide in which one or several amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1", preferably "in the amino acid sequence shown in SEQ ID NO: 1, one or several amino acids are substituted". Polypeptides ". As the "polypeptide in which one or several amino acids are substituted in the amino acid sequence shown in SEQ ID NO: 1", preferably any of the following (2-1) to (2-9) can be mentioned. More preferably, any of the following (2-1') to (2-9') can be mentioned, and more preferably, a polypeptide in which isoleucine at the 242nd position is replaced with asparagine can be preferably mentioned. Not limited to.

(2-1) Substitution of aspartic acid at position 37 with a neutral polar amino acid;
(2-2) Substitution of tyrosine with neutral polar amino acid at position 62;
(2-3) Substitution of asparagine at position 78 with neutral polar amino acid;
(2-4) Substitution of phenylalanine at position 115 with a neutral non-polar amino acid;
(2-5) Substitution of asparagine at position 176 with a neutral polar amino acid;
(2-6) Substitution of arginine at position 200 with a neutral non-polar amino acid;
(2-7) Substitution of isoleucine at position 242 with an acidic or neutral non-polar amino acid;
(2-8) Substitution of cysteine at position 251 with a neutral non-polar amino acid;
(2-9) Substitution of lysine at position 308 with a neutral polar amino acid;

(2-1') Substitution of aspartic acid for asparagine at position 37;
(2-2') Substitution of tyrosine with cysteine at position 62;
(2-3') Substitution of asparagine to tyrosine at position 78;
(2-4') Replacement of phenylalanine with leucine at position 115;
(2-5') Substitution of asparagine with serine at position 176;
(2-6') Substitution of arginine for tryptophan at position 200;
(2-7') Substitution of isoleucine for asparagine or aspartic acid at position 242;
(2-8') Replacement of cysteine with tryptophan at position 251;
(2-9') Substitution of lysine for asparagine at position 308;

In the present specification, the "1 or several amino acids" is, for example, in the range of 1 to 30, preferably in the range of 1 to 20, more preferably in the range of 1 to 15, and further preferably in the range of 1. It means an amino acid in the range of ~ 10, more preferably in the range of 1 to 5, still more preferably in the range of 1 to 3, more preferably 1 to 2, and most preferably 1 amino acid.

In the present specification, "when expressed in THP-1 cells stimulated with Holball 12-millistart 13-acetate (PMA) or RAW264.7 cells, humans are compared with the case where the polypeptide is not expressed. As "polypeptides that increase the expression of M2 macrophage markers", THP-1 cells or RAW264.7 cells stimulated with PMA expressing the mutant IL-4R polypeptide and the mutant IL-4R polypeptide are expressed. The expression of M2 macrophage markers was examined by culturing THP-1 cells or RAW264.7 cells stimulated with non-PMA, and compared with the case where the mutant IL-4R polypeptide was not expressed, the mutant IL-4R. The expression of M2 macrophage markers in THP-1 cells or RAW264.7 cells stimulated with PMA expressing the polypeptide is increased, preferably 2 times or more, more preferably 5 times or more, still more preferably 10 times or more. Polypeptides can be mentioned. Here, examples of the human M2 macrophage marker include CD206, CD163, IL-27RA, CD367, F13A1, and CCL22. In addition, THP-1 cells differentiate into macrophages by stimulating with PMA.

The "polynucleotide encoding the mutant IL-4R polypeptide (hereinafter, also referred to as the" polynucleotide ")" of the present invention is not particularly limited as long as it is a polynucleotide encoding the mutant IL-4R polypeptide. The codon selected to encode the amino acid may be appropriately optimized depending on the type of host cell to be expressed.

The expression vector in the "expression vector containing the Polynucleotide of the present invention (hereinafter, also referred to as the" expression vector ") may be linear or cyclic, and may be a viral vector or a non-viral vector such as a plasmid, depending on a transposon. It may be a vector. In addition, such a vector may contain a control sequence such as a promoter or a terminator, or a selectable marker sequence such as a drug resistance gene or a reporter gene.

Examples of the virus vector include a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated virus vector, and a lentiviral vector can be preferably mentioned. If a lentiviral vector or a retroviral vector is used, the transgene is incorporated into the genome of the host cell, so that it can be stably expressed for a long period of time. When expression in a short period of time is preferable, an adenovirus vector or an adeno-associated virus vector that does not integrate into the genome is preferable.

Examples of the promoter include a lentivirus LTR promoter, an SV40 initial promoter, a cytomegalovirus promoter, a virus-derived promoter such as the thymidine kinase promoter of simple herpesvirus, a phosphoglycerate kinase (PGK) promoter, an Xist promoter, and a β-actin promoter. Mammalian-derived promoters such as RNA polymerase II promoter, CAG promoter, and EF1A can be mentioned. Further, a tetracycline-responsive promoter induced by tetracycline, an Mx1 promoter induced by interleukin, a CD68 promoter having a promoter activity in macrophages, an M-CSF promoter and the like may be used.

The "macrophages" in the "macrophages or macrophage progenitor cells isolated from the living body containing the polynucleotide or expression vector of the present invention" (hereinafter, also referred to as "mutant IL-4R macrophages A") are referred to as M1 macrophages. Two phenotypes of M2 macrophages are included. In addition, "macrophage progenitor cells" include cells capable of differentiating into at least M1 macrophages or M2 macrophages, and include CD14-positive (CD14 ⁺ ) cells, inactive macrophages (M0 macrophages), monocytes, monocytes, and bone marrow. Examples thereof include nuclear spheres, peripheral blood mononuclear cells, ES cells, and iPS cells. The M2 macrophages are also called anti-inflammatory macrophages, and among macrophages that are CD14-positive cells, cells expressing CD206, CD163, IL-27RA, CD367, F13A1, or CCL22 in humans, and arginase 1 in mice. Cells expressing (Arg1), CD206, iNOS, CD163 or CD204 can be mentioned. The mutant IL-4R macrophage A and the mutant IL-4R macrophage B described later also include cultures obtained by culturing them.

The monocytes are blood cells in the circulating blood and can differentiate into macrophages when they leave the bloodstream and enter connective tissue. Examples of the origin of the monocytes include mammals such as humans, monkeys, and mice. Peripheral blood mononuclear cells (PBMCs) are separated from peripheral blood whole blood collected from a patient by a known method such as centrifugation, and the monocytes are purified by immunomagnetic bead method or the like as necessary. Can be used. Such monocytes can be activated and differentiated into macrophages by culturing in the presence of cytokines such as M-CSF. The monocytes can also be produced from bone marrow fluid, ES cells, iPS cells and the like using a known method.

The above-mentioned "containing the polynucleotide" may mean that the polynucleotide is contained in a macrophage or a macrophage precursor cell separated from a living body, and for example, the polynucleotide is contained in a state of being incorporated into an expression vector. In addition, it can be mentioned that the polynucleotide is contained in a state of being integrated into the genome of a macrophage or a macrophage precursor cell isolated from a living body.

Examples of the method for producing the mutant IL-4R macrophage A include a method of introducing the polynucleotide or the expression vector into macrophages or macrophage progenitor cells isolated from a living body. Alternatively, it can also be produced by introducing the polynucleotide or the present expression vector into somatic cells such as fibroblasts isolated from the living body, and then establishing iPS cells from such somatic cells. The method for introducing the polynucleotide or the expression vector into macrophages or macrophage progenitor cells isolated from the living body is not particularly limited, but is limited to a virus infection method, a calcium phosphate method, a lipofection method, a microinjection method, an electroporation method, etc. A known method of the above can be mentioned, and a method of introducing by a virus infection method can be preferably mentioned.

In addition, the mutant IL-4R macrophage A is a genome of a macrophage or a macrophage progenitor cell isolated from a living body so that the polynucleotide can be expressed using a known gene editing technique under the control of an appropriate promoter. It may be produced by incorporating it into. Known gene editing techniques include endonucleases such as zinc finger nucleases, TALENs (transcriptional activation-like effector nucleases), CRISPR (Crustered Regularly Interspaced Short Palindromic Repeat) -Cas9 system, and CRISPR-Cas3 systems. .. As a method of incorporating a polynucleotide into the cell genome so that it can be expressed under the control of an appropriate promoter, the polynucleotide in question functionally linked downstream of the appropriate promoter (that is, can be expressed under the control of the promoter). Examples thereof include a method of incorporating a polynucleotide having a coding sequence linked thereto into a non-coding region of the cell genome, a method of incorporating the polynucleotide into the downstream of an endogenous promoter of the cell genome, and the like.

The "macrophages or macrophage progenitor cells isolated from the living body containing the mutant IL-4R polypeptide" (hereinafter, also referred to as "mutated IL-4R macrophages B") of the present invention include macrophages isolated from the living body. Alternatively, it is sufficient that the macrophage progenitor cell contains the mutant IL-4R polypeptide, and the extracellular region of the mutant IL-4R polypeptide is arranged outside the macrophage or the macrophage progenitor cell. preferable.

As a method for producing the mutant IL-4R macrophage B, the polynucleotide or the expression vector of the present invention is introduced into the macrophage or macrophage progenitor cell isolated from the living body as described above, and the macrophage or macrophage progenitor cell isolated from the living body is introduced. Examples thereof include a method for expressing the mutant IL-4R polypeptide. In addition, a method of directly editing an endogenous IL-4 receptor gene into a mutant IL-4 receptor gene sequence encoding the mutant IL-4R polypeptide by using the above-mentioned known gene editing technique will be mentioned. be able to.

The macrophage or macrophage progenitor cell obtained by the above method may be subsequently subjected to cell recovery, separation and purification by a known method. When the polynucleotide or the expression vector is introduced into the macrophage progenitor cell, the cells may be cultured after the polynucleotide or the expression vector is introduced to separate or purify the macrophage. In addition, when macrophage progenitor cells are cultured using a petri dish, the macrophage progenitor cells and macrophages are separated or purified by utilizing the property that when they differentiate into macrophages, they adhere to the petri dish and other cells are contained in the supernatant. can do.

A prophylactic or therapeutic agent for an inflammatory disease, a neurodegenerative disease, or an ischemic disease, which comprises the "mutated IL-4R macrophages and a pharmacologically acceptable additive" of the present invention (hereinafter, "the inflammatory disease, the neurodegenerative disease". Or, also referred to as a "preventive or therapeutic agent for ischemic diseases"), the present invention is not particularly limited as long as the mutant IL-4R macrophage contains a pharmacologically acceptable additive. Such pharmacologically acceptable additives include saline, buffered saline, cell culture medium, dextrose, water for injection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers and surfactants. Buffers and preservatives, isotonic agents, fillers, and lubricants can be mentioned.

Examples of the inflammatory disease, neurodegenerative disease, or ischemic disease include diseases in which macrophages are involved in pathogenesis. The inflammatory diseases include autoimmune diseases, autoimmune diseases, collagen diseases, ulcerative colitis, Crohn's diseases, autoimmune hepatitis, encephalitis, myocarditis, nephritis, and chronic inflammation predominantly M1 macrophages that increases with aging. Dermatitis, photoaging of the skin, ophthalmitis, pancreatitis, enteritis, diabetes, arteriosclerosis such as atherosclerosis, vasculitis, parasite infection, allergic disease, implant-to-host disease (GVHD) can. Examples of the neurodegenerative diseases include Alzheimer's disease, Creutzfeldt-Jakob disease, multiple sclerosis, amyloidosis, Parkinson's disease, amyotrophic lateral sclerosis, and spinocerebellar degeneration. Further, as the ischemic disease, ischemia-reperfusion disorder can be mentioned, and stroke or myocardial infarction can be mentioned.

The prophylactic or therapeutic agent for inflammatory disease, neurodegenerative disease, or ischemic disease is administered to a subject in need of treatment for inflammatory disease, neurodegenerative disease, or ischemic disease by a method known to those skilled in the art. Intravenous, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intramedullary, intracardiac, intra-articular, intrasacral, intracranial, intrathecal, And submucosal (medullary fluid) injections can be mentioned.

The amount of the mutant IL-4R macrophages contained in the prophylactic or therapeutic agent for the inflammatory disease, neurodegenerative disease, or ischemic disease to be administered is the type, location, and severity of the inflammatory disease, neurodegenerative disease, or ischemic disease. It can be appropriately adjusted according to the age, weight, condition, etc. of the subject to be treated, but preferably 1 × 10 ⁴ to 1 × 10 ¹⁰ pieces, preferably 1 × 10 ⁵ to 1 × 10 per administration. ^Nine , more preferably 1 × 10 ⁶ to 1 × 10 ⁸ can be mentioned.

The prophylactic or therapeutic agent for the inflammatory disease, neurodegenerative disease, or ischemic disease to be administered is 4 times a day, 3 times, 2 times or 1 time, every 1 day, every 2 days, every 3 days, every 4 days. Independent administration every 5, 5 days, once a week, every 7 days, every 8 days, every 9 days, twice a week, twice a month, once a month, every 1 month, or every 2 months be able to.

It should be noted that the target for administration of the prophylactic or therapeutic agent for the inflammatory disease, neurodegenerative disease, or ischemic disease, and the macrophages used for producing the prophylactic or therapeutic agent for the inflammatory disease, neurodegenerative disease, or ischemic disease. Alternatively, it may be the same as or different from the donor of macrophage precursor cells. That is, the donor and the administration subject may be the same or disagreeable. Furthermore, when the administration target is a human, the macrophages or macrophage precursor cells used for producing a prophylactic or therapeutic agent for the inflammatory disease, neurodegenerative disease, or ischemic disease are selected from the patient himself / herself as the administration target. The collected autologous cells may be used, or the allogeneic cells collected from another person may be used. When allogeneic cells are used, for example, β2 microglobulin (β2M), human leukocyte antigen (HLA), etc. may be modified by a known method such as genome editing technology in order to reduce immune rejection. ..

"Differentiation or polarization inducer from macrophage or macrophage progenitor cell isolated from living body to M2 macrophage containing the polynucleotide or expression vector of the present invention as an active ingredient" (hereinafter, "differentiation or induction of polarization to M2 macrophage" The agent (also referred to as) is not particularly limited as long as the polynucleotide or the expression vector is used as an active ingredient, and is introduced into macrophages or macrophage progenitor cells isolated from a living body by the above-mentioned known method, and is determined as necessary. By culturing in the medium of, macrophages or macrophage progenitor cells can be differentiated or polarized into M2 macrophages. In the present specification, the acquisition of different properties or functions in cells from macrophage precursor cells other than M0 macrophages to becoming M0 macrophages is differentiated, that M0 macrophages become M1 macrophages or M2 macrophages, and that M1 macrophages become M2. Becoming a macrophage or changing from an M2 macrophage to an M1 macrophage is called polarization. For example, when bone marrow stem cells become M2 macrophages via monocytes and M0 macrophages, differentiation is called until they become M0 macrophages, and polarization is called from M0 macrophages to M2 macrophages. The present M2 macrophage differentiation or polarization inducer may contain the above-mentioned pharmacologically acceptable additive. In the differentiation or polarization inducer for the M2 macrophages, the polynucleotide may be incorporated into the expression vector.

The "method for inducing differentiation or polarization of macrophages or macrophage progenitor cells isolated from living organisms into M2 macrophages, which comprises introducing the polynucleotide into macrophages or macrophage progenitor cells isolated from living organisms" of the present invention. , The present polynucleotide or the present expression vector may be introduced into macrophages or macrophage progenitor cells isolated from the living body and cultured in a predetermined medium as needed. The present polynucleotide or the present expression vector may be the present mutant IL-4R macrophages. Can be introduced into macrophages or macrophage progenitor cells by the same method as described in the method for producing.

As another aspect 1 of the present invention, 1) macrophages or macrophage precursor cells isolated from a living body containing the polynucleotide or the expression vector can be used for the prevention or treatment of inflammatory diseases, neurodegenerative diseases, or ischemic diseases. Used as a prophylactic or therapeutic agent for inflammatory, neurodegenerative, or ischemic diseases, and 2) prophylactic or therapeutic agents for inflammatory, neurodegenerative, or ischemic diseases, characterized by administration to patients in need. Macrophages or macrophages precursor cells isolated from the living body containing the polynucleotide or the expression vector for the purpose of 3) macrophages or macrophages precursor cells isolated from the living body containing the polynucleotide or the expression vector. Can be mentioned for use in the preparation of prophylactic or therapeutic agents for inflammatory diseases, neurodegenerative diseases, or ischemic diseases.

As another aspect 2 of the present invention, 1) the polynucleotide or the present expression vector for use as a differentiation or polarization inducing agent for M2 macrophages, and 2) the present polynucleotide or the present expression vector to M2 macrophages. Use in the preparation of differentiation or polarization inducers can be mentioned.

As another aspect 3 of the present invention, a kit for producing a differentiation or polarization inducing agent for M2 macrophages, which comprises the above-mentioned polynucleotide or the present expression vector, can be mentioned, and the above-mentioned kit includes the above-mentioned present invention. The present invention is not particularly limited as long as it has a polynucleotide or the present expression vector, and a description for producing a differentiation or polarization inducer into M2 macrophages and an expression vector containing the present polynucleotide are introduced into macrophages or macrophage progenitor cells. It may contain a reagent used to do so.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.

[Example 1] (Preparation of cell line expressing human IL-4 receptor)
In the amino acid sequence of the human IL-4 receptor shown in SEQ ID NO: 1, the mutant amino acid sequence of the human IL-4 receptor in which isoleucine at position 242 is replaced with asparagine (I242N) (I242N mutant human IL-4 receptor: SEQ ID NO: The polynucleotide encoding 2) (SEQ ID NO: 3) was produced by outsourcing to Vector Builder. Next, the prepared polynucleotide encoding the I242N mutant human IL-4 receptor was subjected to RAW264.7 cells (n = 4: KAC), which is a mouse macrophage immortalized cell line, using lentivirus. ) Or a human monocyte-derived cell line, which was introduced into THP-1 cells (n = 3 purchased from JCRB Cell Bank, National Institute of Pharmaceutical Sciences, Health and Nutrition) that differentiate into macrophages when activated. As control cells, cells were prepared in which a polynucleotide encoding the red fluorescent protein mCherry or green fluorescent protein (GFP) was introduced in the same manner as above in place of the I242N mutant human IL-4 receptor.

RAW264.7 cells are DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (Fetal Bovine Serum), and THP-1 cells are THP medium (2 mM Glutamine, 10). the% FBS-containing RPMI1640 (Roswell Park Memorial Institute medium, Inc.)), 37 ℃, were cultured in _CO 2 5%. THP-1 cells were cultured in THP medium containing 250 nM phorbol 12-myristate 13-acetylate (PMA) for 24 hours, differentiated into macrophages, and then in THP medium without PMA. The cells were cultured for another 2 days. Then, each cell is collected, total RNA is isolated by Trizol reagent (Invitrogen), cDNA is synthesized, and expression of IL-4 receptor and human GAPDH or mouse Gapdh (internal standard) is confirmed by real-time PCR method. bottom. In the real-time PCR method, the primers shown in Table 1 below were used. The hIL-4R forward primer set forth in SEQ ID NO: 4 and the hIL-4R reverse primer set forth in SEQ ID NO: 5 are used to obtain the amino acid sequence of the human IL-4 receptor shown in wild-type SEQ ID NO: 1 and the human shown in SEQ ID NO: 2. Any of the mutated amino acid sequences of the IL-4 receptor (I242N mutated human IL-4) can be amplified.

The results are shown in Fig. 1. In FIG. 1, the upper row shows RAW264.7 cells (mCherry RAW264.7 cells) into which a polynucleotide encoding mCherry was introduced and RAW264.7 cells (mut_IL-) into which a polynucleotide encoding an I242N mutant human IL-4 receptor was introduced. This is the result of 4R RAW264.7 cells). In addition, in FIG. 1, the lower row shows THP-1 cells (GFP THP-1 cells) into which a polynucleotide encoding a GFP was introduced and THP-1 cells (GFP THP-1 cells) into which a polynucleotide encoding an I242N mutant human IL-4 receptor was introduced. This is the result of mut_IL-4R THP-1 cells). The vertical axis is the relative mutant human IL-4 receptor expression level when the IL-4 receptor expression level in the control mCherry RAW264.7 cells or GFP THP-1 cells is 1. The expression level was corrected by human GAPDH or mouse Gapdh, and tested by Unpaired T-test.

As shown in the upper part of FIG. 1, it was confirmed that human IL-4 receptor was expressed in mut_IL-4R RAW264.7 cells, and I242N mutant human IL-4 receptor introduced by lentivirus was expressed. .. In addition, as shown in the lower part of FIG. 1, in mut_IL-4R THP-1 cells, the expression of human IL-4 receptor was increased as compared with the control, and the I242N mutant human IL-4 receptor introduced by lentivirus was increased. It was confirmed that the body was expressed.

[Example 2] (Expression of M2 macrophage marker Arg1 or CD206 in cells expressing the I242N mutant human IL-4 receptor)
The differentiation or polarization of the cells prepared in Example 1 into M2 macrophages was examined by the expression of Arg1 which is a mouse M2 macrophage marker and CD206 which is a human M2 macrophage marker. The specific method is as follows.

Similar to Example 1, a polynucleotide encoding a wild-type human IL-4 receptor was introduced into RAW264.7 cells to prepare WT_IL-4R RAW264.7 cells. Next, mCherry RAW264.7 cells (n = 4), mut_IL-4R RAW264.7 cells (n = 4), mCherry THP-1 cells (n = 3), mut_IL-4R THP-1 prepared in Example 1 four and WT_IL-4R RAW264.7 cells (n = 4) a 37 ° C. cell (n = 3), were cultured for 3 days in _CO 2 5%, the cells were harvested and extracts the RNA. Next, the expression of Arg1 and the expression of CD206 were examined by real-time PCR. In addition, mCherry RAW264.7 cells, mut_IL-4R RAW264.7 cells and WT_IL-4R RAW264.7 cells were cultured in RAW medium, and mCherry THP-1 cells and mut_IL-4R THP-1 cells were cultured in THP cells ( Contains PMA for the first 24 hours of the 3 days). In the real-time PCR method, the primers shown in Table 2 below were used.

The results are shown in Fig. 2. The expression level was corrected by human GAPDH or mouse Gapdh, RAW264.7 cells were tested by 1-way ANOVA, and THP-1 cells were tested by Unpaired T-test.

As shown in FIG. 2, the expression of Arg1 was low in the control mCherry RAW264.7 cells and WT_IL-4R RAW264.7 cells, but in the mut_IL-4R RAW264.7 cells, Arg1 was the control mCherry RAW264.7. It was 59 times more expressed than the cells and 28 times more than the WT_IL-4R RAW264.7 cells. Similarly, the expression of CD206 was low in the control GFP THP-1 cells, but the expression of CD206 in the mut_IL-4R THP-1 cells was 13 times higher than that in the control cells. Therefore, it was revealed that macrophage progenitor cells differentiate and / or polarize into M2 macrophages by expressing the I242N mutant human IL-4 receptor.

[Example 3] (Suppression of M1 macrophage induction in cells expressing the I242N mutant human IL-4 receptor)
Whether M2 macrophages polarized from mut_IL-4R RAW264.7 cells are maintained as M2 macrophages without being polarized to M1 macrophages when lipopolysaccharide (LPS), which is a humoral factor that induces M1 macrophages, is allowed to act. Whether or not it was examined by the expression of tumor necrosis factor (TNFα), which is an Arg1 and M1 macrophage marker. The specific method is as follows.

mut_IL-4R RAW264.7 cells (n = 4) and control mCherry RAW264.7 cells (n = 4) were mixed with LPS (1 ng / mL-containing RAW medium or control LPS-free (Basal)) RAW medium at 37 ° C. , and cultured for 3 days in _CO 2 5%, the cells were harvested and extracts the RNA. Next, the expression of TNFα and the expression of Arg1 were examined by a real-time PCR method. In the real-time PCR method, the primers shown in Table 3 below were used.

The results are shown in Fig. 3. The expression level was corrected by human GAPDH or mouse Gapdh, and tested by Unpaired T-test.

As is clear from FIG. 3, the expression of the M1 macrophage marker TNFα is increased by acting on LPS in the control mCherry RAW264.7 cells, but even if it is allowed to act on LPS in mut_IL-4R RAW264.7 cells. The expression of TNFα was hardly increased. In addition, in mut_IL-4R RAW264.7 cells, the expression of Arg1 of the M2 macrophage marker was not significantly different from that of Basal (control without LPS), which did not act on LPS, and no decrease in Arg1 expression was observed. rice field.

From the results of FIGS. 2 and 3, expression of the I242N mutant human IL-4 receptor differentiates and / or polarizes macrophage progenitor cells into M2 macrophages, or M1 macrophages are polarized into M2 macrophages. It was also clarified that it can be maintained as M2 macrophages without being polarized to M1 macrophages even in the presence of a humoral factor that induces M1 macrophages.

[Example 4] (Polarization to M2 macrophages in human IL-4 receptors other than I242N)
In Examples 1 to 3, the mutation at position 242 shown in SEQ ID NO: 2 was used, but from macrophage progenitor cells to M2 macrophages in control GFP and mutations other than positions 242 of (1) to (10) below. The differentiation and / or polarization effect of Agr1 was examined by the expression of Agr1. Expression of Agr1 was carried out in the same manner as in Example 2. That is, GFP or polynucleotides encoding the following (1) to (10) were prepared by PrimeSTAR (registered trademark) Mutagenesis Basal Kit (TAKARA) and introduced into RAW264.7 cells (n = 3), respectively, to RAW. 37 ° C. the culture medium, and cultured for 3 days in _CO 2 5%, the cells were harvested and extracts the RNA, expression was examined Arg1 by real-time PCR method.

(1) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which the 37th aspartic acid was replaced with asparagine (D37N mutant human IL-4 receptor: SEQ ID NO: 16).
(2) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which the 62nd tyrosine was replaced with cysteine (Y62C mutant human IL-4 receptor: SEQ ID NO: 17).
(3) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which the 78th asparagine was replaced with tyrosine (N78Y mutant human IL-4 receptor: SEQ ID NO: 18).
(4) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which phenylalanine at position 115 is replaced with leucine (F115L mutant human IL-4 receptor: SEQ ID NO: 19).
(5) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which asparagine at position 176 was replaced with serine (N176S mutant human IL-4 receptor: SEQ ID NO: 20).
(6) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which the 200th arginine was replaced with tryptophan (R200W mutant human IL-4 receptor: SEQ ID NO: 21).
(7) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which isoleucine at position 242 is replaced with aspartic acid (I242D mutant human IL-4 receptor: SEQ ID NO: 22).
(8) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which the 251st cysteine is replaced with tryptophan (C251W mutant human IL-4 receptor: SEQ ID NO: 23).
(9) In the amino acid sequence of the human IL-4 receptor, the mutant amino acid sequence of the human IL-4 receptor in which lysine at position 308 was replaced with asparagine (K308N mutant human IL-4 receptor: SEQ ID NO: 24).

The results are shown in Fig. 4. The expression level was corrected for human GAPDH or mouse Gapdh, and tested by Unpaired T-test.

As shown in FIG. 4, D37N mutant human IL-4 receptor, Y62C mutant human IL-4 receptor, N78Y mutant human IL-4 receptor, F115L mutant human IL-4 receptor, N176S mutant human IL-4. Expression of the receptor, R200W mutant human IL-4 receptor, I242D mutant human IL-4 receptor, C251W mutant human IL-4 receptor, and K308N mutant human IL-4 receptor increased the expression of Arg1. rice field. That is, it was clarified that even if the mutant IL-4R polypeptide other than the I242N mutant human IL-4 receptor is contained, it can be differentiated or polarized into M2 macrophages.

[Example 5] (Persistence of M2 macrophages)
In cells expressing the I242N mutant human IL-4 receptor, the extent to which the characteristics as M2 macrophages could be maintained was investigated using the expression of Arg1 as an index.

Mouse bone marrow cells were collected by a known method and then cultured in a culture medium containing M-CSF (20 ng / ml) for 5 days to differentiate into bone marrow-derived macrophages (BMDM). Then, in the presence of M-CSF (20 ng / ml), as in Example 1, a polynucleotide or control (polynucleotide encoding GFP) encoding the I242N mutant human IL-4 receptor was used with a lentivirus. The BMDM was introduced into the above BMDM, and after culturing for 48 hours, the BMDM was collected and stored. Each BMDM was stimulated with IL-4 (100 pg / ml) for 24 hours to polarize it into M2 macrophages, and then cultured as it was for 1 week, and the expression of Arg1 which is an M2 macrophage marker was analyzed by real-time PCR. The results are shown in FIG. The horizontal axis is the number of days of culture (Day 0 is defined after stimulation with IL-4 for 24 hours), and the vertical axis is the relative expression level of Arg1 when the expression level of Arg1 on the control Day 0 is 1. The expression level was corrected with mouse Gapdh (N = 3).

BMDM (cells) polarized into M2 macrophages only by control IL-4 stimulation reduced the expression of Arg1 on Day 3, whereas cells expressing the I242N mutant human IL-4 receptor were Day 7 (1 week). Later), the expression of Arg1 continued. Therefore, it was clarified that if the mutant IL-4R polypeptide is contained, it can be differentiated or polarized into M2 macrophages and then maintained as M2 macrophages for at least one week. In the conventional method, when M2 macrophages enter the body, they lose their function within a few days, so that the therapeutic effect is limited. On the other hand, since the M2 macrophage produced in the present invention can maintain its function as an M2 macrophage for a long period of time, it is expected to have a high therapeutic effect on inflammatory diseases, myoneurodegenerative diseases, and ischemic diseases.

[Example 6] (secretion of IL-10)
In order to confirm the anti-inflammatory effect on M2 macrophages expressing the mutant IL-4R polypeptide, the secretion of IL-10, which is a potent anti-inflammatory cytokine, was examined. Similar to Example 1, a polynucleotide encoding an I242N mutant human IL-4 receptor or control (polynucleotide encoding GFP) was introduced into RAW264.7 cells using a lentivirus to obtain a stable expression cell line. Made. Next, each cell line was seeded in a culture dish and cultured for 24 hours. The supernatant after culturing was collected, and IL-10 contained in the supernatant was analyzed by the ELISA method. Tested by Unpaired T-test (N = 3). The results are shown in FIG.

As is clear from FIG. 6, when the mutant IL-4R polypeptide was contained, the expression of IL-10 was increased more than 4-fold as compared with the control. Therefore, it was confirmed that the M2 macrophage containing the mutant IL-4R polypeptide has an anti-inflammatory effect.

The present invention can be used as a biopharmacy for inflammatory diseases, wound healing, neurodegenerative diseases, and ischemic diseases.

Claims

A polypeptide derived from the human interleukin 4 (human IL-4) receptor, wherein one or several amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1.
The polypeptide according to claim 1, which is the following (1-1) or (1-2).
(1-1) A polypeptide derived from the human interleucine 4 receptor, in the amino acid sequence shown in SEQ ID NO: 1, aspartic acid at position 37, tyrosine at position 62, asparagin at position 78, and phenylalanine at position 115. Polypeptide substituted with aspartic acid at position 176, arginine at position 200, isoleucine at position 242, cysteine at position 251 and / or lysine at position 308;
(1-2) In the amino acid sequence shown in SEQ ID NO: 1, the 37th, 62nd, 78th, 115th, 176th, 200th, 242nd, 251st and / or 308 substituted in the above (1-1). It was expressed in THP-1 cells or RAW264.7 cells containing a polypeptide in which one or several amino acids were added, deleted or substituted in a non-th amino acid and stimulated with Holbol 12-millistart 13-acetate. A polypeptide that, in some cases, increases the expression of human M2 macrophage markers as compared to the case where the polypeptide is not expressed.
In the amino acid sequence shown in SEQ ID NO: 1, aspartic acid at position 37, tyrosine at position 62, asparagin at position 78, phenylalanine at position 115, asparaginine at position 176, arginine at position 200, isoleucine at position 242, and cysteine at position 251 ,. The polypeptide according to claim 2, wherein the polypeptide substituted with lysine at position 308 and / or is any of the following (2-1) to (2-9).
(2-1) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which aspartic acid at position 37 is replaced with a neutral polar amino acid;
(2-2) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which tyrosine at position 62 is replaced with a neutral polar amino acid;
(2-3) In the amino acid sequence shown in SEQ ID NO: 1, the polypeptide in which asparagine at position 78 is replaced with a neutral polar amino acid;
(2-4) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which phenylalanine at position 115 is replaced with a neutral non-polar amino acid;
(2-5) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which asparagine at position 176 is replaced with a neutral polar amino acid;
(2-6) In the amino acid sequence shown in SEQ ID NO: 1, the polypeptide in which arginine at position 200 is replaced with a neutral non-polar amino acid;
(2-7) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which isoleucine at position 242 is replaced with an acidic amino acid or a neutral non-polar amino acid;
(2-8) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which cysteine at position 251 is replaced with a neutral non-polar amino acid;
(2-9) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide in which lysine at position 308 is replaced with a neutral polar amino acid;
The polypeptide according to claim 2, which is a polypeptide derived from the human interleukin-4 receptor and is a polypeptide substituted with isoleucine at position 242 in the amino acid sequence shown in SEQ ID NO: 1.
The polypeptide according to claim 4, which is a polypeptide derived from the human interleukin-4 receptor and in which isoleucine at position 242 is replaced with asparagine in the amino acid sequence shown in SEQ ID NO: 1.
A polynucleotide encoding the polypeptide according to any one of claims 1 to 5.
An expression vector containing the polynucleotide according to claim 6.
A macrophage or macrophage progenitor cell isolated from a living body containing the polynucleotide according to claim 6 or the expression vector according to claim 7.
A macrophage or macrophage progenitor cell isolated from a living body containing the polypeptide according to any one of claims 1 to 5.
A prophylactic or therapeutic agent for an inflammatory disease, a neurodegenerative disease or an ischemic disease, which comprises the macrophage or macrophage progenitor cell according to claim 8 or 9 and a pharmacologically acceptable additive.
An agent for inducing differentiation or polarization of macrophages or macrophage progenitor cells isolated from a living body into M2 macrophages, which comprises the polynucleotide according to claim 6 or the expression vector according to claim 7 as an active ingredient.
From a macrophage or macrophage progenitor cell isolated from a living body to an M2 macrophage, which comprises introducing the polynucleotide according to claim 6 or the expression vector according to claim 7 into a macrophage or macrophage progenitor cell isolated from the living body. Differentiation or polarization induction method.