JP7464956B2 - Mesenchymal stem cells, immune disease treatments and anti-inflammatory agents - Google Patents

Description

The present invention relates to mesenchymal stem cells, therapeutic agents for immune diseases, and anti-inflammatory agents.

Immunodeficiency disorders cause infections, frequent recurrences, and severe or prolonged symptoms due to the immune system not functioning normally. Immunodeficiency disorders impair the immune system's ability to defend the body against foreign invaders such as bacteria, viruses, and fungi, as well as attacks from abnormal cells such as cancer cells. As a result, people with normal immune function become susceptible to bacterial, viral, and fungal infections and cancers that would not occur if the immune system was functioning normally.

There are two types of immunodeficiency diseases: congenital (primary) immunodeficiency diseases that are present at birth, and acquired (secondary) immunodeficiency diseases that develop later in life as a result of some kind of illness. Acquired immunodeficiency diseases often develop as a result of a long-term serious illness, such as cancer, aplastic anemia, leukemia, myelofibrosis, renal failure, diabetes, liver disease, and splenic disease.

Furthermore, as we age, our immune system weakens and we gradually lose the ability to distinguish between self and foreign substances, making us more susceptible to autoimmune diseases. There are a variety of autoimmune diseases, in which various cells and tissues are targeted for attack. Treatment for these autoimmune diseases involves suppressing the immune system to control the autoimmune response. However, many of the drugs used to control the autoimmune response also interfere with the body's ability to fight infections, and so there is a need for the development of new therapeutic agents that only have the effect of controlling the autoimmune response.

Mesenchymal stem cells are precursor cells with multipotency that were first isolated from bone marrow by Friedenstein in 1982 (see Non-Patent Document 1). It has been revealed that mesenchymal stem cells exist in various tissues such as bone marrow, umbilical cord, and fat, and mesenchymal stem cell transplantation is expected to be a new treatment method for various intractable diseases (see Patent Documents 1 and 2). Recently, it has been found that cells with equivalent functions exist in interstitial cells of adipose tissue, placenta, umbilical cord, fetal membrane, etc. For this reason, mesenchymal stem cells are sometimes called stromal cells.

JP 2012-157263 A JP 2012-508733 A

Pittenger F. M. et al. Science, (1999), 284, pp. 143-147

In the circumstances described above, the present invention aims to provide a novel therapeutic agent for immune diseases and a novel anti-inflammatory agent.

As a result of intensive research to solve the above problems, the present inventors discovered that mesenchymal stem (stromal) cells (MSCs), which are characterized by high kynurenine production or secretion rates, are effective in treating immune diseases and inflammatory diseases, and completed the present invention. According to the present invention, it is possible to provide a therapeutic agent that is effective in treating immune diseases and inflammatory diseases. In other words, the gist of the present invention is as follows.

[1] A mesenchymal stem cell characterized by a high amount of kynurenine production or secretion.
[2] A mesenchymal stem cell characterized by a high production amount of kynurenine synthesized from tryptophan or a high secretion amount of kynurenine due to IDO induced in the cells by IFNγ treatment.
[3] A mesenchymal stem cell characterized in that the JAK/STAT pathway is activated at a steady state.
[4] A mesenchymal stem cell according to any one of [1] to [3], which is derived from umbilical cord tissue.
[5] A therapeutic agent for immune diseases, comprising the mesenchymal stem cells according to any one of [1] to [4].
[6] An anti-inflammatory agent comprising the mesenchymal stem cells according to any one of [1] to [4].
[7] A therapeutic agent for GVHD, comprising the mesenchymal stem cells according to any one of [1] to [4].
[8] A method for treating an immune disease, characterized by using mesenchymal stem cells having a high amount of kynurenine production or secretion.
[9] A method for treating an immune disease, comprising using mesenchymal stem cells that produce a high amount of kynurenine synthesized from tryptophan by IDO induced in the cells by IFNγ treatment, or that secrete a high amount of kynurenine.
[10] A method for treating an immune disease, characterized by using mesenchymal stem cells in which the JAK/STAT pathway is activated at a steady state.
[11] The method for treating an immune disease described in any one of [8] to [10], wherein the mesenchymal stem cells are derived from umbilical cord tissue.
[12] A method for treating an inflammatory disease, characterized by using mesenchymal stem cells having a high amount of kynurenine production or secretion.
[13] A method for treating an inflammatory disease, comprising using mesenchymal stem cells that produce a high amount of kynurenine synthesized from tryptophan by IDO induced in the cells by IFNγ treatment, or that secrete a high amount of kynurenine.
[14] A method for treating an inflammatory disease, characterized by using mesenchymal stem cells in which the JAK/STAT pathway is activated at a steady state.
[15] The method for treating an inflammatory disease described in any one of [12] to [14], wherein the mesenchymal stem cells are derived from umbilical cord tissue.
[16] A method for treating GVHD, comprising using mesenchymal stem cells having a high level of kynurenine production or secretion.
[17] A method for treating GVHD, comprising using mesenchymal stem cells that produce a high amount of kynurenine synthesized from tryptophan by IDO induced in the cells by IFNγ treatment, or that secrete a high amount of kynurenine.
[18] A method for treating GVHD, characterized by using mesenchymal stem cells in which the JAK/STAT pathway is activated at a steady state.
[19] The method for treating GVHD described in any one of [16] to [18], wherein the mesenchymal stem cells are derived from umbilical cord tissue.

The present invention makes it possible to provide novel therapeutic agents for immune diseases and anti-inflammatory agents, as well as novel methods for treating immune diseases and methods for treating inflammatory diseases.

FIG. 1 shows a comparison of intracellular kynurenine levels between mesenchymal stem cells cultured on a plate (ADH) and cells cultured in suspension (SUS). FIG. 2 is a diagram showing a comparison of the kynurenine levels in the culture supernatants of mesenchymal stem cells cultured on a flat surface (ADH) and cells cultured in suspension (SUS). FIG. 3 shows a comparison of the kynurenine amount (IDO activity) in the culture supernatant of mesenchymal stem cells cultured on a flat surface (ADH) and cells cultured in suspension (SUS). FIG. 4 shows a comparison of the kynurenine amount (IDO activity) in the culture supernatant of mesenchymal stem cells cultured on a plate (ADH) and cells cultured in suspension (SUS). FIG. 5 shows a comparison of the kynurenine amount (IDO activity) in the culture supernatant of mesenchymal stem cells cultured on a flat surface (ADH) and cells cultured in suspension (SUS). FIG. 6 shows a comparison of the kynurenine amount (IDO activity) in the culture supernatant of mesenchymal stem cells cultured on a flat surface (ADH) and cells cultured in suspension (SUS). FIG. 7 shows a comparison of the kynurenine amount (IDO activity) in the culture supernatant of mesenchymal stem cells cultured on a flat surface (ADH) and cells cultured in suspension (SUS). FIG. 8 shows a comparison of kynurenine amounts (IDO activity) in culture supernatants of plate-cultured cells. FIG. 9 shows the results of gene expression analysis using microarrays of mesenchymal stem cell plate-cultured cells (ADH) and suspension-cultured cells (SUS) (the ratio of the expression level in SUS to the expression level in ADH). FIG. 10 shows the effect of administration of UC-MSC (EXP-SUS) to GVHD model mice. FIG. 11 shows the effect of administration of UC-MSC (EXP-SUS) to GVHD model mice. FIG. 12 is a graph showing the anti-inflammatory effects of mesenchymal stem cells cultured on a flat surface (ADH) and suspension cultured (SUS). FIG. 13 is a diagram showing the JAK/STAT signal pathway.

The mesenchymal stem cells, immune disease therapeutic agent, and anti-inflammatory agent of the present invention are described in detail below.

[Mesenchymal stem cells]
The mesenchymal stem cells of the present invention are characterized by a high amount of kynurenine production or secretion.

Kynurenine is a metabolite produced by the enzyme indolamine 2,3-dioxygenase (IDO) during the intracellular synthesis of niacin from tryptophan. Tryptophan is important for T cell proliferation, and it is known that tryptophan is converted to kynurenine by IDO activity, and its depletion inhibits T cell proliferation and induces immune tolerance.

Kynurenine production amount, or kynurenine secretion amount, refers to the amount of kynurenine that is synthesized intracellularly from tryptophan by indolamine 2,3-dioxygenase (IDO) induced by TNFα or IFNγ treatment under inflammatory conditions and then released extracellularly.

The mesenchymal stem cells of the present invention may have a higher expression of kynurenine than other cells, and more specifically, the mesenchymal stem cells of the present invention may have a higher production amount of kynurenine synthesized from tryptophan by IDO induced in the cells by IFNγ treatment, or a higher amount of kynurenine secretion.

The mesenchymal stem cells of the present invention need only express kynurenine at a higher level than mesenchymal stem cells obtained by culture using a conventional medium (e.g., 10% serum-containing MEMα medium), and preferably have a kynurenine production or secretion level that is at least 1.1 times, more preferably at least 1.2 times, and even more preferably at least 1.5 times higher than mesenchymal stem cells obtained under culture conditions using 10% serum-containing MEMα medium.

Furthermore, the mesenchymal stem cells of the present invention may express kynurenine at a higher level than other cells, for example, at a higher level than MRC-5 cells, human neonatal dermal fibroblasts (HDF), or human umbilical vein endothelial cells (HUVEC), and preferably have a kynurenine production or secretion level that is at least 5 times, and more preferably at least 10 times, higher than MRC-5 cells, HDF, or HUVEC.

For example, it is sufficient that kynurenine is highly expressed compared to mesenchymal stem cells obtained by conventional culture methods (e.g., culture by plate culture method), and the kynurenine production or secretion amount is preferably at least 2 times, more preferably at least 5 times, even more preferably at least 10 times, and particularly preferably at least 100 times higher than mesenchymal stem cells obtained under conventional culture conditions. Note that the plate culture method is a static culture in which cells are attached to a flat surface such as a flask or a petri dish, and there is no particular restriction on the container, medium, etc.

Note that the above-mentioned high kynurenine production amount or high kynurenine secretion amount includes high expression or high activity of IDO, which is involved in kynurenine synthesis. High expression or high activity of IDO is defined in the same manner as above, and the mesenchymal stem cells of the present invention may express IDO at a high level and/or have a high activity of IDO compared to mesenchymal stem cells obtained by culture in a conventional medium (e.g., MEM-α medium containing 10% FBS). The expression and/or activity of IDO is preferably enhanced by 1.1 times or more, more preferably 1.2 times or more, and even more preferably 1.5 times or more, compared to mesenchymal stem cells obtained under conventional culture conditions.

The mesenchymal stem cells of the present invention have high IDO expression and/or enhanced activity compared to other cells, such as MRC-5 cells, human neonatal dermal fibroblasts (HDF), and human umbilical vein endothelial cells (HUVEC). Compared to MRC-5 cells, HDF, or HUVEC, IDO is highly expressed or highly active, preferably 5-fold or more, and more preferably 10-fold or more, higher in IDO expression and/or activity compared to MRC-5 cells, HDF, or HUVEC.

The mesenchymal stem cells of the present invention have high IDO expression and/or activity enhanced compared to mesenchymal stem cells obtained by conventional culture methods (e.g., culture by plate culture method). The IDO expression and/or activity is preferably at least 2-fold higher, more preferably at least 5-fold higher, even more preferably at least 10-fold higher, and particularly preferably at least 100-fold higher, compared to mesenchymal stem cells obtained under conventional culture conditions.

IDO is known to be induced by the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway. This JAK/STAT signaling pathway is induced by various cytokines and growth factors, and is known to be involved in various functions such as cell proliferation, differentiation, migration, apoptosis, and cell survival.

The mesenchymal stem cells of the present invention are characterized by high expression of the following factors in addition to kynurenine.

The mesenchymal stem cells of the present invention highly express CSF3, CSF2, LIF, IL6, IL6R, or STAT4. Colony stimulating factor 3 (CSF3) is a cytokine involved in the production and differentiation of granulocytes. Colony stimulating factor 2 (CSF2) is a cytokine involved in the production and differentiation of granulocytes and macrophages. Leukemia inhibitory factor (LIF) is a cytokine that plays an important role in inducing cell differentiation in the hematopoietic system and nervous system, controlling mesenchymal to epithelial conversion in renal development, and maternal-fetal immune tolerance. Interleukin 6 (IL6) is a cytokine that has various functions under various inflammatory conditions and in the maturation of B cells. Interleukin 6 receptor (IL6R) is a receptor for IL6. Signal transducer and activator of transcription 4 (STAT4) functions as a transcription factor in response to cytokines and growth factors. It is known to play an important role in the response to IL12 in lymphocytes and in the differentiation of helper T cells.

Furthermore, the mesenchymal stem cells of the present invention highly express the following factors:
ciliary neurotrophic factor receptor, growth hormone receptor, Cbl proto-oncogene B, E3 ubiquitin protein ligase, interleukin 15, interleukin 6 signal transducer, interleukin 10, cardiotrophin-like cytokine factor 1, cyclin D2, interleukin 11 receptor, alpha, interleukin 15 receptor, alpha, Pim-1 proto-oncogene, serine/threonine kinase, suppressor of cytokine signaling 1, signal transducer and activator of transcription 2, 113 kDa, interleukin 19, interleukin 7, interleukin 24, interleukin 22 receptor, alpha 1, suppressor of cytokine signaling 3, Janus kinase 2, interferon (alpha, beta and omega) receptor 1, interferon (alpha, beta and omega) receptor 2, interferon gamma receptor 2 (interferon gamma transducer 1), prolactin, leukemia inhibitory factor receptor alpha, sprouty homolog 2 (Drosophila), v-akt murine thymoma viral oncogene homolog 3, interleukin 4 receptor, interleukin 2 receptor, alpha, son of sevenless homolog 1 (Drosophila), leptin receptor, v-akt murine thymoma viral oncogene homolog 3, interleukin 24, growth hormone 1, interleukin 7 receptor, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit delta, interleukin 21, and protein inhibitor of activated STAT 4; these factors are known to be important in the formation and induction of the JAK/STAT pathway. In the mesenchymal stem cells of the present invention, the JAK/STAT pathway is activated in a steady state, resulting in high expression of these factors. These factors generally take the positional relationship shown in FIG. 13 in the JAK/STAT pathway.

In the present invention, mesenchymal stem cells refer to cells that have the ability to differentiate into one or more types of cells belonging to the mesenchymal system (such as bone cells, cardiac muscle cells, chondrocytes, tendon cells, and adipocytes) and can proliferate while maintaining this ability. The term mesenchymal stem cells used in the present invention refers to the same cells as interstitial cells and does not particularly distinguish between the two. They may also be simply referred to as mesenchymal cells. Examples of tissues that contain mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amniotic membrane, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, and tooth germ. For example, adipose tissue-derived mesenchymal stem cells refer to mesenchymal stem cells contained in adipose tissue, and may also be referred to as adipose tissue-derived stromal cells. Among these, from the standpoint of effectiveness in treating neurological disorders and ease of availability, adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells are preferred, adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells are more preferred, and umbilical cord-derived mesenchymal stem cells are most preferred.

The mesenchymal stem cells in the present invention may be derived from the same species as the subject (specimen) to be treated, or may be derived from a different species. Examples of species of mesenchymal stem cells in the present invention include humans, horses, cows, sheep, pigs, dogs, cats, rabbits, mice, and rats, and are preferably derived from the same species as the subject (specimen) to be treated. The mesenchymal stem cells in the present invention may be derived from the subject (specimen) to be treated, i.e., autologous cells, or may be derived from another subject of the same species, i.e., allogeneic cells. Allogeneic cells are preferred.

Because mesenchymal stem cells are unlikely to cause rejection even in allogeneic subjects, donor cells prepared in advance, expanded and frozen, can be used as the mesenchymal stem cells in the disease treatment agent of the present invention. Therefore, compared to preparing and using autologous mesenchymal stem cells, from the viewpoints of ease of commercialization and ease of obtaining a stable and consistent effect, it is more preferable that the mesenchymal stem cells in the present invention are allogeneic.

In the present invention, mesenchymal stem cells refer to any cell population that contains mesenchymal stem cells. The cell population is at least 20%, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98% or 99% mesenchymal stem cells.

In the present invention, the umbilical cord is a white tubular tissue that connects the fetus and the placenta, and is composed of the umbilical vein, umbilical artery, gelatinous tissue (Wharton's jelly), umbilical cord matrix itself, etc., and contains a large amount of mesenchymal stem cells. The umbilical cord is preferably obtained from an animal of the same species as the subject (administration subject) who will use the disease treatment agent of the present invention, and when considering administration of the disease treatment agent of the present invention to humans, a human umbilical cord is more preferable.

In the present invention, adipose tissue means tissue containing adipocytes and stromal cells including microvascular cells, and is, for example, tissue obtained by surgically removing or aspirating subcutaneous fat of a mammal. Adipose tissue can be obtained from subcutaneous fat. It is preferable to obtain it from the same animal species as the subject of administration of adipose tissue-derived mesenchymal stem cells described later, and considering administration to humans, it is more preferable to obtain it from human subcutaneous fat. The individual that supplies the subcutaneous fat may be alive or dead, but the adipose tissue used in the present invention is preferably tissue collected from a living individual. When collecting from an individual, liposuction can be, for example, PAL (power-assisted) liposuction, Erchoria laser liposuction, or body jet liposuction, and it is preferable not to use ultrasound from the viewpoint of maintaining the state of the cells.

In the present invention, bone marrow refers to the soft tissue that fills the cavity of bones and is a hematopoietic organ. Bone marrow contains bone marrow fluid, and the cells present therein are called bone marrow cells. Bone marrow cells include red blood cells, granulocytes, megakaryocytes, lymphocytes, adipocytes, and the like, as well as mesenchymal stem cells, hematopoietic stem cells, and vascular endothelial precursor cells. Bone marrow cells can be collected, for example, from human ilium, long bones, or other bones.

In the present invention, mesenchymal stem cells derived from various tissues, such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells, refer to any cell population containing mesenchymal stem cells derived from various tissues, such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells. At least 20%, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98% or 99% of the cell population are mesenchymal stem cells derived from various tissues, such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells.

In addition to high kynurenine production or secretion, the mesenchymal stem cells of the present invention may be characterized by, for example, growth characteristics (e.g., population doubling ability from passage to senescence, doubling time), karyotype analysis (e.g., normal karyotype, maternal lineage or neonatal lineage), surface marker expression by flow cytometry (e.g., FACS analysis), immunohistochemistry and/or immunocytochemistry (e.g., epitope detection), gene expression profiling (e.g., gene chip arrays; polymerase chain reaction such as reverse transcription PCR, real-time PCR, conventional PCR), miRNA expression profiling, protein arrays, protein secretion such as cytokines (e.g., plasma coagulation analysis, ELISA, cytokine arrays), metabolites (metabolomic analysis), and other methods known in the art.

(Method of preparing mesenchymal stem cells)
The method for preparing mesenchymal stem cells with high kynurenine production or secretion is not particularly limited, but for example, they can be prepared as follows. That is, mesenchymal stem cells are isolated and cultured from tissues such as fat, umbilical cord, bone marrow, etc. according to a method known to those skilled in the art, and cells with high kynurenine production or secretion can be obtained by separating them using a cell sorter, magnetic beads, etc., using IDO expression as an indicator. In addition, mesenchymal stem cells with high kynurenine production or secretion can also be obtained by culturing them using a specific medium. In the cell population obtained by this induction, it is preferable that 50% or more of the cell population are cells with high kynurenine production or secretion amount, more preferably 70% or more are cells with high kynurenine production or secretion amount, even more preferably 80% or more are cells with high kynurenine production or secretion amount, particularly preferably 90% or more are cells with high kynurenine production or secretion amount, and it is most preferable that the cell population is a homogeneous cell population of cells with substantially high kynurenine production or secretion amount. Below, a method for preparing mesenchymal stem cells with high kynurenine production or secretion amount will be specifically described.

Mesenchymal stem cells can be prepared by methods well known to those skilled in the art. As an example, a method for preparing umbilical cord tissue-derived mesenchymal stem cells and adipose tissue-derived mesenchymal stem cells is described below.

The umbilical cord can be collected by removing the placenta from the postpartum tissues including the placenta and umbilical cord delivered by vaginal delivery and cesarean section. After removing the umbilical cord blood from the collected umbilical cord, it may be subjected to a sterilization or bacteriostatic treatment. The umbilical cord blood is removed by rinsing with an anticoagulant solution such as a heparin-containing solution. The sterilization or bacteriostatic treatment may be, but is not limited to, application of povidone-iodine or immersion in a medium or buffer containing one or more antibiotics and/or antifungals such as penicillin, streptomycin, amphotericin B, gentamicin, and nystatin. If necessary, the process may include a step of selectively lysing red blood cells. Methods for selectively lysing red blood cells may be methods well known in the art, such as incubation in a hypertonic or hypotonic medium by lysis with ammonium chloride.

The umbilical cord-derived cells of the present invention refer to a cell population prepared using umbilical cord as a raw material, and may be obtained by a known production method, for example, by a method including the following steps (i) to (iii):
(i) cutting the umbilical cord;
(ii) culturing the umbilical cord obtained in step (i); and (iii) passaging.

In addition, another method for preparing the cells may include, instead of the step (i) of cutting the umbilical cord, a step (i') of dissociating the tissue by enzymatic treatment of the umbilical cord. Furthermore, in addition to the step (i) of cutting the umbilical cord, a step (i') of dissociating the tissue by enzymatic treatment of the umbilical cord may be included.

In the (i) step of cutting the umbilical cord of the present invention, the umbilical cord obtained by the above-mentioned method may be cut by mechanical force (chopping force or shear force) in a state in which the umbilical cord contains the amniotic membrane, blood vessels, perivascular tissues, and Wharton's jelly. Although not particularly limited, the umbilical cord section obtained by cutting may have a size of 1 to 10 mm ³ , 1 to 5 mm ³ , 1 to 4 mm ³ , 1 to 3 mm ³ , or 1 to 2 mm ³ . In the (i') step of dissociating the tissue from the umbilical cord by enzyme treatment of the present invention, the umbilical cord obtained by the above-mentioned method may be dissociated by enzyme treatment in a state in which the umbilical cord contains the amniotic membrane, blood vessels, perivascular tissues, and Wharton's jelly. Although not particularly limited, examples of the enzyme treatment include enzyme treatment using one or more enzymes such as collagenase, dispase, and hyaluronidase.

In the present invention, the process of culturing the umbilical cord obtained in step (ii)(i) involves culturing the umbilical cord obtained in step (i) at appropriate cell density and culture conditions using an appropriate cell culture medium.

The umbilical cord tissue-derived mesenchymal stem cells of the present invention can be produced using a suspension culture production method. Examples of suspension culture production methods include a method of agitating and culturing a spherical cell mass formed by any method in a culture vessel, and a method of adhering cells to a microcarrier and agitating the microcarrier in a culture tank. The agitation can be performed by rotating the agitating blades in the vessel with a stirrer, or by placing a bag containing the culture solution and cells on a shaker and shaking the bag to suspend the culture solution. The medium used in the suspension culture production method is not particularly limited as long as it is a medium capable of culturing mesenchymal stem cells, and examples thereof include the above-mentioned medium. The microcarrier is not particularly limited as long as it can be used in suspension culture, and examples thereof include polyester, polystyrene, glass, dextran, gelatin, collagen, etc. Specific examples of available microcarriers include Cytodex 1, Cytodex 3, cytopore 1, Cytopore 2, Cultisphere, Cytoline 1, and Cytoline 2 from GE Healthcare, CellBIND, low-concentration Synthemax II, high-concentration Synthemax II, positively charged microcarriers, collagen-coated microcarriers, soluble microcarriers, and SoloHill microcarriers from Pall, etc. The material of the microcarrier preferably has properties such as solubility and biodegradability, and more preferably is xeno-free and animal-free in addition to these.

The adipose tissue-derived mesenchymal stem cells may be obtained by, for example, the production method described in U.S. Pat. No. 6,777,231, and can be produced, for example, by a method including the following steps (i) to (iii):
(i) obtaining a cell suspension by enzymatic digestion of adipose tissue;
(ii) sedimenting the cells and resuspending the cells in an appropriate medium; and (iii) culturing the cells on a solid surface and removing cells that do not exhibit binding to the solid surface.

The adipose tissue used in step (i) is preferably washed. Washing can be performed by vigorous agitation and sedimentation using a physiologically compatible saline solution (e.g., phosphate-buffered saline (PBS)). This is to remove contaminants (also called debris, e.g., damaged tissue, blood, red blood cells, etc.) contained in the adipose tissue from the tissue. Therefore, washing and sedimentation are generally repeated until the debris is totally removed from the supernatant. Since the remaining cells exist as clumps of various sizes, it is preferable to treat the washed cell clumps with an enzyme (e.g., collagenase, dispase, trypsin, etc.) that weakens or destroys intercellular bonds in order to dissociate the cells while minimizing damage to the cells themselves. The amount and duration of such enzymes vary depending on the conditions used, but are known in the art. Alternatively or in combination with such enzyme treatment, a method using cells that naturally ooze out from the cell clumps is also preferred (e.g., improved explant method). Although the cell mass can be decomposed by other treatment methods such as mechanical agitation, ultrasonic energy, and thermal energy, it is preferable to use only enzyme treatment in order to minimize damage to the cells. When using an enzyme, it is desirable to inactivate the enzyme using a medium or the like after an appropriate period of time in order to minimize harmful effects on the cells.

The cell suspension obtained by step (i) contains a slurry or suspension of aggregated cells as well as various contaminating cells, such as red blood cells, smooth muscle cells, endothelial cells, and fibroblasts. The aggregated cells may then be separated and removed from the contaminating cells, but this step may be omitted since they can be removed by adhesion and washing in step (iii) described below. The separation and removal of the contaminating cells may be achieved by centrifugation, which forcibly separates the cells into a supernatant and a precipitate. The resulting precipitate containing the contaminating cells is suspended in a physiologically compatible solvent. The suspended cells may contain red blood cells, but the lysis step is not necessarily required since the red blood cells are excluded by selection by adhesion to solid surfaces, as described below. Methods known in the art can be used to selectively lyse red blood cells, such as lysis with ammonium chloride followed by incubation in a hypertonic or hypotonic medium. After lysis, the lysate may be separated from the desired cells, for example by filtration, centrifugation, or density fractionation.

In step (ii), the cells in suspension may be washed once or multiple times, centrifuged, and resuspended in culture medium to increase the purity of the mesenchymal stem cells. Alternatively, the cells may be separated based on cell surface marker profiles or based on cell size and granularity.

The medium used for resuspension is not particularly limited as long as it is a medium capable of culturing mesenchymal stem cells, but such a medium may be prepared by adding serum to a basal medium and/or adding one or more serum substitutes such as albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. These media may further contain substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc., as necessary.

Next, in step (iii), the cells in the cell suspension obtained in step (ii) are cultured on a solid surface without differentiation, using the appropriate cell culture medium described above, at an appropriate cell density and culture conditions. In the present invention, the term "solid surface" refers to any material that allows the adipose tissue-derived mesenchymal stem cells of the present invention to bind and adhere. In a specific embodiment, such a material is a plastic material that has been treated to promote the binding and adhesion of mammalian cells to its surface. The shape of the culture vessel having a solid surface is not particularly limited, but a petri dish, a flask, or the like is preferably used. To remove unbound cells and cell debris, the cells are washed after incubation.

In the present invention, cells that ultimately remain bound and adhered to the solid surface can be selected as a cell population of adipose tissue-derived mesenchymal stem cells.

The selected cells may be analyzed for surface antigens by conventional methods such as flow cytometry to confirm that they are mesenchymal stem cells according to the present invention. Furthermore, the cells may be examined for their ability to differentiate into each cell lineage, and such differentiation may be performed by conventional methods.

The medium used in culturing the cells of the present invention is not particularly limited as long as it is a medium capable of culturing mesenchymal stem cells, but such a medium may be prepared by adding serum to a basal medium and/or adding one or more serum substitutes such as albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. These media may further contain substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts, as necessary.

Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, MEM-α medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, MCDB201 medium, and mixed media thereof.

Examples of the above serum include, but are not limited to, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, porcine serum, sheep serum, rabbit serum, rat serum, etc. When serum is used, it may be added to the basal medium at 5 v/v% to 15 v/v%, preferably 10 v/v%.

Examples of the fatty acids include, but are not limited to, linoleic acid, oleic acid, linoleic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. Examples of lipids include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. Examples of amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, and L-glycine. Examples of proteins include, but are not limited to, ecotin, reduced glutathione, fibronectin, and β2-microglobulin. Examples of polysaccharides include glycosaminoglycans, and among glycosaminoglycans, in particular, hyaluronic acid, heparan sulfate, and the like, but are not limited to these. Examples of growth factors include, but are not limited to, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), epidermal growth factor (EGF), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), etc. From the viewpoint of using the umbilical cord-derived mesenchymal stem cells obtained in the present invention for cell transplantation, it is preferable to use a xeno-free medium that does not contain xenogeneic components such as serum. Such a medium is provided as a medium prepared in advance for mesenchymal stem cells (stromal cells) by, for example, PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, Corning, and Rohto.

The mesenchymal stem cells of the present invention can be prepared as described above, but may also be defined as cells having the following characteristics:
(1) Under standard culture conditions, it exhibits adhesion to plastic.
(2) They are positive for the surface antigens CD44, CD73, and CD90, and negative for CD31 and CD45, and (3) can differentiate into osteocytes, adipocytes, and chondrocytes under the appropriate culture conditions.

From the mesenchymal stem cells obtained by the above process, cells with high IDO expression levels, kynurenine production levels, or kynurenine secretion levels can be selectively separated by immunological techniques using a cell sorter, magnetic beads, etc., to obtain mesenchymal stem cells with high kynurenine production levels or kynurenine secretion levels. In addition, by performing a specific culture method that can induce kynurenine production or kynurenine secretion, or culturing in a specific medium, etc., it is possible to efficiently obtain mesenchymal stem cells with high kynurenine production levels or kynurenine secretion levels. As an example, a specific method of selective separation by immunological techniques using a cell sorter and a method of adhering cells to a microcarrier and agitating the microcarrier to perform agitation culture in a culture tank are described below.

The above-prepared mesenchymal stem cells are treated with trypsin-EDTA solution or the like to obtain a cell suspension, which is then centrifuged (room temperature, 400G, 5 minutes) to remove the supernatant. Staining buffer (1% BSA-PBS) is added to the cells to prepare a cell suspension concentration of 1 x 10 ⁶ cells/500 μL, and the cell suspension concentration is uniformized by pipetting, and then 50 μL is dispensed into new 1.5 mL microtubes. A primary antibody (anti-IDO antibody, anti-kynurenine antibody, etc.) is added to the dispensed cell suspension at a concentration of 5 to 20 μg/mL, suspended, and then reacted in a dark place and refrigerated for 30 minutes to 1 hour. After washing three times with 1 mL of staining buffer, staining buffer is added to make 50 μL, secondary antibody is added at a concentration of 1 to 10 μg/mL, and then the mixture is reacted for 30 minutes to 1 hour under light-shielded refrigeration. After washing three times with 1 mL of staining buffer, 300 μL of PI buffer (prepared by adding 28.8 μL of propidium iodide solution (SIGMA, P4864) to 14.4 mL of staining buffer) is added and thoroughly suspended, passed through a tube with a cell strainer, and separated by fluorescence activated cell sorting (FACS).

The cell suspension containing mesenchymal stem cells prepared as above is mixed with microcarriers, added to a culture tank, and stirred culture is performed using serum-free medium for mesenchymal stem cells, etc., and suspension culture is performed for a certain period of time, whereby cells with higher IDO expression levels, kynurenine production levels, or kynurenine secretion levels can be obtained compared to conventional plate culture in flasks, etc.

(Cryopreservation of mesenchymal stem cells)
The mesenchymal stem cells in the present invention may be cells that have been repeatedly frozen and thawed as appropriate, so long as they have an immune disease therapeutic effect and an inflammatory disease therapeutic effect. In the present invention, cryopreservation can be performed by suspending the mesenchymal stem cells in a cryopreservation solution known to those skilled in the art and cooling them. Suspension can be performed by detaching the cells with a detaching agent such as trypsin as necessary, transferring them to a cryopreservation container, treating them appropriately, and then adding a cryopreservation solution.

The cryopreservation solution may contain DMSO (dimethyl sulfoxide) as a cryoprotectant, but since DMSO has the property of inducing differentiation of mesenchymal stem cells in addition to cytotoxicity, it is preferable to reduce the DMSO content. Examples of substitutes for DMSO include glycerol, propylene glycol, and polysaccharides. When DMSO is used, it contains 5% to 20%, preferably 5% to 10%, and more preferably 10%. In addition, it may contain additives described in WO2007/058308. As such a cryopreservation solution, for example, cryopreservation solutions provided by Bio Verde, Nippon Genetics, ReproCell, Zenoac, Cosmo Bio, Kohjin Bio, Thermo Fisher Scientific, etc. may be used.

When the above-mentioned suspended cells are cryopreserved, they may be frozen at a temperature between -80°C and -100°C (e.g., -80°C) using any freezer that can achieve that temperature. Although not particularly limited, in order to avoid sudden temperature changes, the cooling rate may be appropriately controlled using a programmable freezer. The cooling rate may be appropriately selected depending on the components of the cryopreservation solution, and may be performed according to the manufacturer's instructions for the cryopreservation solution.

The storage period is not particularly limited as long as the cells cryopreserved under the above conditions retain the same properties as before freezing after thawing, but may be, for example, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 1 year or more, or more. Since cell damage can be suppressed by storing at a lower temperature, the cells may be transferred to the gas phase above liquid nitrogen (approximately -150°C or less to -180°C or more) for storage. When storing in the gas phase above liquid nitrogen, storage containers well known to those skilled in the art can be used. Although not particularly limited, for example, when storing for 2 weeks or more, it is preferable to store in the gas phase above liquid nitrogen.

The thawed mesenchymal stem cells may be appropriately cultured before the next cryopreservation. The mesenchymal stem cells are cultured using a medium capable of culturing the above-mentioned mesenchymal stem cells, and may be cultured at a culture temperature of about 30 to 40°C, preferably about 37°C, under an atmosphere of _CO2 -containing air, although this is not particularly limited. The _CO2 concentration is about 2 to 10%, preferably about 5 to 10%. In the culture, after the cells reach an appropriate confluency for the culture vessel (for example, 50% to 80% of the culture vessel is occupied by the cells), the cells may be detached with a detaching agent such as trypsin, and the culture may be continued by seeding them at an appropriate cell density in a separately prepared culture vessel. When the cells are seeded, typical cell densities include 100 cells/cm ² to 100,000 cells/cm ² , 500 cells/cm ² to 50,000 cells/cm ² , 1,000 to 10,000 cells/cm ² , and 2,000 to 10,000 cells/cm ² . In a specific embodiment, the cell density is 2,000 to 10,000 cells/cm ² . It is preferable to adjust the period until the cells reach an appropriate confluency to 3 to 7 days. During the culture, the medium may be appropriately replaced as necessary.

Cryopreserved cells can be thawed by methods well known to those skilled in the art, such as by leaving the cells in a 37°C incubator or in a water bath and shaking them.

The mesenchymal stem cells of the present invention may be in any state, for example, cells recovered by detaching cells during culture, or cells frozen in a cryopreservation solution. The use of cells obtained by the same lot obtained by expansion culture and divided into small portions and frozen is preferable in terms of the same stable action and effect and excellent ease of handling. The mesenchymal stem cells in a cryopreserved state may be thawed immediately before use and directly mixed with a solution such as an infusion or culture medium while still suspended in the cryopreservation solution. Alternatively, the cryopreservation solution may be removed by a method such as centrifugation and then suspended in a solution such as an infusion or culture medium. Here, the "infusion" in the present invention refers to a solution used in human treatment, and includes, but is not limited to, physiological saline, Japanese Pharmacopoeia physiological saline solution, 5% glucose solution, Japanese Pharmacopoeia glucose injection solution, Ringer's solution, Japanese Pharmacopoeia Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, solution No. 1 (initiation solution), solution No. 2 (dehydration supply solution), solution No. 3 (maintenance solution), solution No. 4 (postoperative recovery solution), etc.

[Therapeutic agents for immune diseases and anti-inflammatory agents]
The immune disease therapeutic agent or anti-inflammatory agent of the present invention contains the above-mentioned mesenchymal stem cells of the present invention having a high kynurenine production or secretion amount. The immune disease therapeutic agent or anti-inflammatory agent of the present invention can be used for prevention, suppression or treatment of immune disease therapeutic agents and anti-inflammatory agents. The explanation in the above mesenchymal stem cell section can be applied to the mesenchymal stem cells containing the immune disease therapeutic agent and anti-inflammatory agent of the present invention.

The immune disease therapeutic agent and anti-inflammatory agent of the present invention may contain, in addition to the above-mentioned mesenchymal stem cells, pharma- ceutically acceptable carriers and additives according to the usual method depending on the application and form, so long as the effects of the present invention are not impaired. Examples of such carriers and additives include, but are not limited to, isotonicity agents, thickeners, sugars, sugar alcohols, preservatives (preservatives), bactericides or antibacterial agents, pH regulators, stabilizers, chelating agents, oily bases, gel bases, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizing agents, dispersants, emulsifiers, buffers, solubilizers, antioxidants, sweeteners, sour agents, colorants, flavoring agents, fragrances, or refreshing agents. Representative components include, for example, the following carriers and additives.

Examples of carriers include aqueous carriers such as water and aqueous ethanol; examples of isotonicity agents (inorganic salts) include sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc.; examples of polyhydric alcohols include glycerin, propylene glycol, polyethylene glycol, etc.; examples of thickeners include carboxyvinyl polymers, hydroxyethyl cellulose, hydroxypropylmethyl cellulose, methyl cellulose, alginic acid, polyvinyl alcohol (completely or partially saponified), polyvinylpyrrolidone, macrogol, etc.; examples of sugars include cyclodextrin, glucose, etc.; examples of sugar alcohols include xylitol, sorbitol, mannitol, etc. (these may be in the d-, l- or dl-form); examples of preservatives, disinfectants or antibacterial agents include dibutylhydroxytoluene, butylhydroxyanisole, alkyldiaminoethyl hydrochloride, etc. Examples of suitable pH adjusters include hydrochloric acid, boric acid, aminoethylsulfonic acid, epsilon-aminocaproic acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, sodium bicarbonate, sodium carbonate, borax, triethanolamine, monoethanolamine, diisopropanol, isopropyl alcohol ... Examples of the stabilizer include dibutylhydroxytoluene, trometamol, sodium formaldehyde sulfoxylate (Rongalite), tocopherol, sodium pyrosulfite, monoethanolamine, aluminum monostearate, glycerin monostearate, sodium hydrogensulfite, sodium sulfite, etc.; examples of the oily base include vegetable oils such as olive oil, corn oil, soybean oil, sesame oil, cottonseed oil, etc., medium-chain fatty acid triglycerides, etc.; examples of the aqueous base include macrogol 400, etc.; examples of the gel base include carboxyvinyl polymer, gum, etc.; examples of the surfactant include polysorbate 80, hydrogenated castor oil, glycerin fatty acid ester, sesquioxane, etc. Examples of suitable suspending agents include beeswax, various surfactants, gum arabic, powdered gum arabic, xanthan gum, soybean lecithin, etc.; examples of suitable binders include hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, etc.; examples of suitable excipients include sucrose, lactose, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, etc.; examples of suitable lubricants include sucrose fatty acid esters, magnesium stearate, talc, etc.; examples of suitable disintegrating agents include low-substituted hydroxypropyl cellulose, crospovidone, croscarmellose sodium, etc.; examples of suitable foaming agents include sodium bicarbonate, etc.; and examples of suitable flow agents include sodium aluminometasilicate, light anhydrous silicic acid, etc.

The immune disease therapeutic agent or anti-inflammatory agent of the present invention can be provided in various forms, such as solid agents, semi-solid agents, and liquid agents, depending on the purpose. For example, it can be used in the form of a solid agent (tablet, powder, powder, granule, capsule, etc.), a semi-solid agent [ointment (hard ointment, soft ointment, etc.), cream, etc.], a liquid agent [lotion, extract, suspension, emulsion, syrup, injection (including infusion, embedded injection, sustained injection, injection prepared at the time of use), dialysis agent, aerosol, soft capsule, drink, etc.], a patch, a cataplasm, etc. The immune disease therapeutic agent or anti-inflammatory agent of the present invention can also be used in the form of a solution or emulsion in an oily or aqueous vehicle. Furthermore, the immune disease therapeutic agent or anti-inflammatory agent of the present invention can be applied to the affected area by spraying, and the immune disease therapeutic agent or anti-inflammatory agent of the present invention can also be used in the form of a gel or sheet at the affected area after spraying. The therapeutic agent for immune diseases or anti-inflammatory agent of the present invention can also be applied to the affected area after forming the above-mentioned mesenchymal stem cells into a sheet-like or three-dimensional structure.

The immune disease therapeutic agent or anti-inflammatory agent of the present invention can be used by suspending or diluting it in an infusion solution such as physiological saline, Japanese Pharmacopoeia physiological saline solution, 5% glucose solution, Japanese Pharmacopoeia glucose injection solution, Ringer's solution, Japanese Pharmacopoeia Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, Solution No. 1 (starting solution), Solution No. 2 (dehydration replenishment solution), Solution No. 3 (maintenance solution), Solution No. 4 (postoperative recovery solution), or a cell culture medium such as DMEM, and can be used by suspending or diluting it in preferably physiological saline solution, 5% glucose solution, Solution No. 1 (starting solution), more preferably 5% glucose solution, Solution No. 1 (starting solution).

When the immune disease therapeutic agent and anti-inflammatory agent of the present invention are liquid preparations, the pH of the immune disease therapeutic agent and anti-inflammatory agent is not particularly limited as long as it is within a medicamentously, pharmacologically (pharmaceutical) or physiologically acceptable range, but an example is a range of 2.5 to 9.0, preferably 3.0 to 8.5, and more preferably 3.5 to 8.0.

When the immune disease therapeutic agent and the anti-inflammatory agent of the present invention are liquid preparations, the osmotic pressure of the immune disease therapeutic agent and the anti-inflammatory agent is not particularly limited as long as it is within a range that is acceptable to the living body. An example of the osmotic pressure ratio of the composition of the present invention is preferably 0.7 to 5.0, more preferably 0.8 to 3.0, and even more preferably 0.9 to 1.4. The osmotic pressure can be adjusted by a method known in the art using inorganic salts, polyhydric alcohols, sugar alcohols, sugars, etc. The osmotic pressure ratio is the ratio of the osmotic pressure of the sample to the osmotic pressure of 286 mOsm (0.9 w/v% sodium chloride aqueous solution) based on the 15th revised Japanese Pharmacopoeia, and the osmotic pressure is measured with reference to the osmotic pressure measurement method (freezing point depression method) described in the Japanese Pharmacopoeia. The standard solution for measuring osmolarity ratios (0.9 w/v % aqueous sodium chloride solution) is prepared by drying sodium chloride (Japanese Pharmacopoeia standard reagent) at 500 to 650°C for 40 to 50 minutes, allowing it to cool in a desiccator (silica gel), and then accurately weighing out 0.900 g of the dried solution and dissolving it in purified water to make exactly 100 mL; alternatively, a commercially available standard solution for measuring osmolarity ratios (0.9 w/v % aqueous sodium chloride solution) is used.

The immune disease therapeutic agent and anti-inflammatory agent of the present invention can be administered to a subject by oral administration, subcutaneous administration, intramuscular administration, intravenous administration, intra-arterial administration, intraumbilical vein administration, intraventricular administration, intrathecal administration, intraperitoneal administration, sublingual administration, rectal administration, vaginal administration, intraocular administration, nasal administration, inhalation, transdermal administration, implantation, spraying onto the surface of an organ, and direct administration by application of a sheet or the like. From the viewpoint of the effectiveness of the immune disease therapeutic agent and anti-inflammatory agent of the present invention, intra-arterial administration, intravenous administration, and intraventricular administration are preferred, and from the viewpoint of reducing the burden on the subject, intravenous administration, intramuscular administration, and intranasal administration are more preferred.

The dose (administration amount) of the immune disease therapeutic agent and anti-inflammatory agent of the present invention may vary depending on the patient's condition (body weight, age, symptoms, physical condition, etc.) and the dosage form of the immune disease therapeutic agent and anti-inflammatory agent of the present invention, but from the viewpoint of exerting a sufficient therapeutic effect of the immune disease therapeutic agent and anti-inflammatory agent, a larger amount tends to be preferable, while from the viewpoint of suppressing the occurrence of side effects, a smaller amount tends to be preferable. Usually, when administered to an adult, the number of cells is 1 x ¹⁰³ to 1 x ¹⁰¹² cells/time, preferably 1 x ¹⁰⁴ to 1 x ¹⁰¹¹ cells/time, more preferably 1 x ¹⁰⁵ to 1 x ¹⁰¹⁰ cells/time, and even more preferably 5 x ¹⁰⁶ to 1 x ¹⁰⁹ cells/time. The dosage per patient body weight is 1×10 to 5× ¹⁰ cells/kg, preferably 1× ¹⁰ to 5×10 cells/kg, more preferably 1× ¹⁰ to ^{5×10 cells} /kg, and even more preferably 1× ¹⁰ to 5× ¹⁰ cells/kg. When administered to a newborn, the number of cells is 1× ¹⁰ to 1× ¹⁰ cells/time, preferably 1× ¹⁰ to 1× ¹⁰ cells/time, more preferably 1× ¹⁰ to 1×10 cells/time, and even more preferably ⁵ × ¹⁰ to 5× ¹⁰ cells/time. The dosage per patient body weight is 1×10 to 5× ¹⁰ cells/kg, preferably 1× ¹⁰ to 5×10 cells/kg, more preferably 1× ¹⁰ to ⁵ × ¹⁰ cells/kg, and even more preferably 1× ¹⁰ to 5× ¹⁰ cells/kg. This dose may be administered multiple times as a single dose, or this dose may be administered in multiple divided doses.

The immune disease therapeutic agent and anti-inflammatory agent of the present invention may be administered together with one or more other drugs. Examples of other drugs include any drug that can be used as an immune disease therapeutic agent or anti-inflammatory agent, such as abciximab, adalimumab, alemtuzumab, basiliximab, bevacizumab, cetuximab, daclizumab, efalizumab, gemtuzumab, ibritumomab, infliximab, mepolizumab, OKT3, omaluzumab, palivizumab, pexelizumab, rituximab, tositumomab, trastuzumab, and the like. Monoclonal antibodies such as mab, fusion proteins such as alefacept, denileukin diftitox, etanercept, soluble cytokine receptors such as anakinra, cytokines such as IFN-α, IFN-β, IFN-γ, IL-2, IL-11, G-CSF, GM-CSF, azatadine maleate, brompheniramine maleate, chlorpheniramine maleate, clemastine fumarate, cyproheptadine hydrochloride, H1 blockers such as d-chlorpheniramine, diphenhydramine hydrochloride, diphenylpyraline hydrochloride, hydroxyzine hydrochloride, methdilazine hydrochloride, promethazine hydrochloride, trimeprazine tartrate, loepelenamine citrate, tripelenamine hydrochloride, triprolidine hydrochloride, acrivastine, cetirizine, desloratadine, ebastine, fexofenadine, levocetirizine, loratadine, and mizolastine; H2 blockers such as cimetidine. Examples of the anti-inflammatory agent include corticosteroids such as car, beclomethasone dipropionate, budesonide, flunisolide, fluticasone, triamcinolone acetonide, dexamethasone, and methylprednisolone, mast cell stabilizers such as cromolyn and nedocromil, β-agonists such as albuterol, cytotoxic agents such as daunomycin, etoposide, and 6-mercaptopurine, antihistamines, NSAIDs, epinephrine, glucagon, and aspirin. Hypothermia can also be performed in conjunction with the administration of the immune disease therapeutic agent or anti-inflammatory agent of the present invention.

The mesenchymal stem cells of the present invention can be used for various autoimmune diseases and inflammatory diseases, including, for example, GVHD, cartilage degradation, rheumatoid arthritis, systemic lupus erythematosus, psoriatic arthritis, spondyloarthritis, osteoarthritis, gout, psoriasis, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, congestive heart failure, stroke, aortic stenosis, renal failure, nephrotic syndrome, uremia, lupus, pancreatitis, allergies, fibrosis, anemia, and atherosclerosis. , restenosis, chemotherapy/radiation-related complications, diabetes mellitus type I, diabetes mellitus type II, liver failure, autoimmune hepatitis, hepatitis C, primary biliary cirrhosis, primary sclerosing cholangitis, fulminant hepatitis, celiac disease, nonspecific colitis, intestinal lymphangiectasia, protein-losing enteropathy, Crohn's disease, allergic conjunctivitis, diabetic retinopathy, Sjogren's syndrome, atopic diseases, uveitis, allergic rhinitis, food allergies, anaphylaxis, autoimmune diseases, drug hypersensitivity, mastocytosis, asthma, stones Cottonsis, silicosis, chronic obstructive pulmonary disease, chronic granulomatous inflammation, cystic fibrosis, histiocytosis, sarcoidosis, glomerulonephritis, vasculitis, dermatitis, HIV-associated cachexia, cerebral malaria, ankylosing spondylitis, leprosy, COPD, pulmonary fibrosis, fibromyalgia, esophageal cancer and other cancers, gastroesophageal reflux disease, Barrett's esophagus, aplastic anemia, graft-versus-host disease, sickle cell disease, CVID, hyper-IgM syndrome, IgA deficiency, transient hypogammaglobulinemia, X-linked agammaglobulinemia, chronic mucocutaneous candidiasis, Examples of such conditions include myelopathy, DiGeorge syndrome, X-linked lymphoproliferative syndrome, ataxia telangiectasia, cartilage-hair hypoplasia, combined immunodeficiency, hyper-IgE syndrome, MHC deficiency, severe combined immunodeficiency, Wiskott-Aldrich syndrome, Chediak-Higashi syndrome, chronic granulomatous disease, leukocyte adhesion deficiency, IFN-γ receptor deficiency, interleukin (IL)-12 deficiency, IL-12 receptor β1 deficiency, ZAP-70 deficiency, and angioedema.

The present invention also includes a method for treating immune diseases, characterized by using mesenchymal stem cells with high kynurenine production or secretion, a method for treating immune diseases, characterized by using mesenchymal stem cells with high kynurenine production or secretion synthesized from tryptophan by IDO induced in cells by IFNγ treatment, and a method for treating immune diseases, characterized by using mesenchymal stem cells in which the JAK/STAT pathway is activated in a steady state. The present invention also includes a method for treating inflammatory diseases, characterized by using mesenchymal stem cells with high kynurenine production or secretion, a method for treating inflammatory diseases, characterized by using mesenchymal stem cells with high kynurenine production or secretion synthesized from tryptophan by IDO induced in cells by IFNγ treatment, and a method for treating inflammatory diseases, characterized by using mesenchymal stem cells in which the JAK/STAT pathway is activated in a steady state. Furthermore, the present invention also includes a method for treating GVHD, characterized by using mesenchymal stem cells with high kynurenine production or secretion, a method for treating GVHD, characterized by using mesenchymal stem cells with high kynurenine production or secretion synthesized from tryptophan by IDO induced in cells by IFNγ treatment, and a method for treating GVHD, characterized by using mesenchymal stem cells in which the JAK/STAT pathway is activated in a steady state. Note that the explanations for each term can be applied to the above-mentioned mesenchymal stem cells, immune disease therapeutic agent, anti-inflammatory agent, and GVHD therapeutic agent.

The present invention will be described in detail below with reference to examples and test examples, but the present invention is not limited to these examples.

[Preparation and culture of umbilical cord-derived mesenchymal stem cells (UCMSC-EXP, UCMSC-CP)]
Umbilical cord-derived cells were collected by the method described in Cytotherapy, 18, 229-241, 2016. Briefly, after obtaining approval from the ethics committee of the Institute of Medical Science, University of Tokyo, and obtaining the consent of the donor, the umbilical cord was chopped into 1 to ² mm3 fragments, seeded on a culture dish, covered with Cell Amigo (Tsubakimoto Chain Co., Ltd.), and cultured in α-minimal essential medium (MEM-α) supplemented with 10% fetal bovine serum (FBS) and antibiotics, to obtain umbilical cord-derived mesenchymal stem cells (hereinafter referred to as "UCMSC-EXP") by the "improved explant method."

Similarly, umbilical cord-derived cells were collected by the method described in Cytotherapy, 18, 229-241, 2016. After obtaining approval from the ethical committee of the Institute of Medical Science, The University of Tokyo, and obtaining the consent of the donor, the umbilical cord was cut into pieces of 1 to ² mm3, treated with collagenase, and centrifuged (400×g for 5 minutes). The obtained cells were cultured in a serum-free medium for mesenchymal stem cells (manufactured by Rohto) in a culture flask as adherent culture, to obtain umbilical cord-derived mesenchymal stem cells (hereinafter referred to as "UCMSC-CP") by the "collagenase method."

Regarding the number of passages, cells obtained from umbilical cord tissue by the above-mentioned improved explant method or collagenase method are designated as first passage cells (P1), and as the number of passages increases with each passage, they are designated as second passage cells (P2), third passage cells (P3), and so on.

The obtained UCMSC-EXP and UCMSC-CP were detached using a cell detachment solution (TrypLE Select (1X)), transferred to a centrifuge tube, and centrifuged at 400 x g for 5 minutes to obtain a cell pellet. After removing the supernatant, an appropriate amount of cell freezing storage solution (STEM-CELLBANKER (Zenoac)) was added and suspended. The cell suspension was dispensed into cryotubes and stored at -80°C in a freezer. It was then transferred to the gas phase above liquid nitrogen and continued to be stored.

[Measurement of intracellular kynurenine levels in plate-cultured cells (ADH) and suspension-cultured cells (SUS)]
The UCMSC-EXP, which is a frozen cell derived from umbilical cord tissue obtained by the improved explant method, was put to sleep, and a cell suspension containing the cells was mixed with a microcarrier, added to a culture vessel, and stirred cultured using a serum-free medium for mesenchymal stem cells (Rohto Co., Ltd.) to obtain suspension cultured cells for 8 days (hereinafter referred to as "EXP-SUS"). Similarly, the UCMSC-EXP was put to sleep, and the cells were seeded in a cell culture flask and plate cultured using a serum-free medium for mesenchymal stem cells (Rohto Co., Ltd.), and cultured for 8 days to obtain plate cultured cells (hereinafter referred to as "EXP-ADH"). The cellular contents of the plate cultured cells (EXP-ADH) or suspension cultured cells (EXP-SUS) obtained by the above method over a culture period of 8 days were extracted, and a comprehensive analysis (metabolome analysis) of metabolites accumulated in the cells was performed by capillary electrophoresis and LC-MS. As a result, it was found that the amount of intracellular kynurenine accumulated was higher in EXP-SUS than in EXP-ADH (Figure 1). Furthermore, the amount of kynurenine in the culture supernatant was quantified by ELISA (Immundiagnostik GmbH, product number K7728). This quantification method also showed that the amount of intracellular kynurenine accumulated after 8 days of culture was higher in EXP-SUS than in EXP-ADH (Figure 2). This demonstrated that intracellular kynurenine production was enhanced by suspension culture in the steady state.

[Measurement of kynurenine production and IDO activity in suspension cultured cells (SUS) and plate cultured cells (ADH) 1]
The UCMSC-CP, which is the umbilical cord tissue-derived frozen cells obtained by the collagenase method described above, was put to sleep, and the cell suspension containing the cells was mixed with a microcarrier, added to a culture tank, and stirred cultured using a serum-free medium for mesenchymal stem cells (Rohto) for 4 days to obtain suspension cultured cells (hereinafter referred to as "CP-SUS"). Similarly, the UCMSC-CP described above was put to sleep, and the cells were seeded in a cell culture flask and plate cultured using a serum-free medium for mesenchymal stem cells (Rohto) for 4 days to obtain plate cultured cells (hereinafter referred to as "CP-ADH"). IFNγ (product number: AF-300-02, manufactured by PeproTeck, Inc.) was added in an amount to give a final concentration of 100 ng/mL and 200 ng/mL, and the amount of kynurenine in the cell supernatant after 48 hours was measured by a colorimetric method using Ehrlich reagent (IFNγ concentration 100 ng/mL group and 200 ng/mL group). In addition, in order to confirm that this reaction is mediated by IDO, a group (inhibitor group) was also set in which IFNγ (100 ng/mL) was added and an IDO inhibitor (30 nM, Epacadostat, CAS No: 1204669-58-8) was added at the same time. As a comparison, the kynurenine in the culture supernatant was also quantified in a control group to which IFNγ was not added. In addition, the cell activity of each mesenchymal stem cell was measured using WST-8 (Cell Counting Kit-8, Dojindo Laboratories), and the measured value of kynurenine was corrected. The results are shown in Figure 3. Since kynurenine production was suppressed by the IDO inhibitor, IDO activity can be evaluated using the kynurenine production ability of each sample as an index.

It was found that CP-SUS released more kynurenine into the culture supernatant in response to IFNγ stimulation under all conditions than CP-ADH. This indicates that CP-SUS is more capable of inducing and activating IDO through IFNγ stimulation, and of producing kynurenine intracellularly and secreting it into the culture supernatant than CP-ADH (Figure 3).

[Measurement of kynurenine production and IDO activity in suspension cultured cells (SUS) and plate cultured cells (ADH) 2]
The above-mentioned umbilical cord tissue-derived frozen cells (UCMSC-EXP) were put to sleep, a cell suspension containing the cells was mixed with a microcarrier, and the mixture was added to a culture vessel, and agitation culture was performed using a serum-free medium for mesenchymal stem cells (Rohto) to obtain suspension cultured cells for 8 days (hereinafter referred to as "EXP-SUS"). Similarly, the above-mentioned umbilical cord tissue-derived frozen cells (UCMSC-EXP) were put to sleep, the cells were seeded in a cell culture flask, and plate culture was performed using a serum-free medium for mesenchymal stem cells (Rohto), and the cells were subcultured after 4 days of culture and further cultured for 4 days to obtain plate cultured cells (hereinafter referred to as "EXP-ADH"). IFNγ (product number: AF-300-02, manufactured by PeproTeck, Inc.) was added in an amount to give a final concentration of 50 ng/mL and 100 ng/mL, and the amount of kynurenine in the cell supernatant after 64 hours was measured by a colorimetric method using Ehrlich reagent (IFNγ concentration 50 ng/mL group and 100 ng/mL group). In addition, kynurenine in the culture supernatant was also quantified for a control group to which IFNγ was not added as a comparison subject. In addition, the cell activity of each mesenchymal stem cell was measured using WST-8 (Cell Counting Kit-8, Dojindo Laboratories), the measured value of kynurenine was corrected, and the IDO activity was evaluated using the kynurenine production ability of each sample as an index. The IDO activity ratio was calculated by setting the IDO/WST8 value of the EXP-ADH control group to 1 (FIG. 4).

It was found that EXP-SUS had higher IDO activity than EXP-ADH under all conditions. This indicates that EXP-SUS is more capable of inducing and activating IDO through IFNγ stimulation, and of producing kynurenine intracellularly and secreting it into the culture supernatant than EXP-ADH.

[Measurement of kynurenine production and IDO activity in suspension cultured cells (SUS) and plate cultured cells (ADH) 3]
The above-mentioned umbilical cord tissue-derived frozen cells (UCMSC-CP) were put to sleep, the cell suspension containing the cells was mixed with a microcarrier, and added to a culture vessel, and agitation culture was performed using a serum-free medium for mesenchymal stem cells (Rohto Co., Ltd.) to obtain suspension cultured cells (hereinafter referred to as "CP-SUS"). Similarly, UCMSC-CP was put to sleep, the cells were seeded in a cell culture flask, and plate culture was performed using a serum-free medium for mesenchymal stem cells (Rohto Co., Ltd.), and cultured for 8 days to obtain plate cultured cells (hereinafter referred to as "CP-ADH"). IFNγ (product number: AF-300-02, manufactured by PeproTeck, Inc.) was added in an amount to give final concentrations of 50 ng/mL and 100 ng/mL, and the amount of kynurenine in the cell supernatant after 64 hours was measured by a colorimetric method using Ehrlich's reagent (IFNγ concentration 50 ng/mL group and 100 ng/mL group). For comparison, IDO activity was also measured for a control group to which IFNγ was not added. The cell activity of each mesenchymal stem cell was measured using WST-8 (Cell Counting Kit-8, Dojindo Laboratories), the measured value of kynurenine was corrected, and the IDO activity was evaluated using the kynurenine production ability of each sample as an index. The IDO activity ratio was calculated by setting the IDO/WST8 value of the CP-ADH control group to 1 (Figure 5).

It was found that CP-SUS had higher IDO activity than CP-ADH under all conditions. This demonstrated that CP-SUS was more capable of inducing and activating IDO through IFNγ stimulation, and thus producing kynurenine intracellularly and secreting it into the culture supernatant, than CP-ADH.

[Measurement of kynurenine production and IDO activity in suspension cultured cells (SUS) and plate cultured cells (ADH) 4]
The above-mentioned umbilical cord tissue-derived frozen cells (UCMSC-CP) were put to sleep, a cell suspension containing the cells was mixed with a microcarrier, and the mixture was added to a culture vessel, and agitation culture was performed using a serum-free medium for mesenchymal stem cells (Rohto) to obtain suspension cultured cells for 8 days (hereinafter referred to as "CP-SUS"). Similarly, the above-mentioned umbilical cord tissue-derived frozen cells (UCMSC-CP) were put to sleep, the cells were seeded in a cell culture flask, and plate culture was performed using a serum-free medium for mesenchymal stem cells (Rohto) to obtain plate cultured cells (hereinafter referred to as "CP-ADH"). IFNγ (product number: AF-300-02, manufactured by PeproTeck, Inc.) was added in an amount to give a final concentration of 50 ng/mL and 100 ng/mL, and the amount of kynurenine in the cell supernatant after 90 hours was measured by a colorimetric method using Ehrlich reagent (IFNγ concentration 50 ng/mL group and 100 ng/mL group). In addition, IDO activity was also measured for a control group to which IFNγ was not added as a comparison object. In addition, the cell activity of each mesenchymal stem cell was measured using WST-8 (Cell Counting Kit-8, Dojindo Laboratories), the measured value of kynurenine was corrected, and the IDO activity was evaluated using the kynurenine production ability of each sample as an index. The IDO activity ratio was calculated by setting the IDO/WST8 value of the CP-ADH control group to 1 (FIG. 6).

It was found that CP-SUS had higher IDO activity than CP-ADH under all conditions. This demonstrated that CP-SUS was more capable of inducing and activating IDO through IFNγ stimulation, and thus producing kynurenine intracellularly and secreting it into the culture supernatant, than CP-ADH.

[Measurement of kynurenine production and IDO activity in suspension cultured cells (SUS) and plate cultured cells (ADH) 5]
The above-mentioned umbilical cord tissue-derived frozen cells (UCMSC-EXP) were put to sleep, a cell suspension containing the cells was mixed with a microcarrier, and the mixture was added to a culture tank, and agitation culture was performed using a serum-free medium for mesenchymal stem cells (Rohto) to obtain suspension cultured cells for 8 days (hereinafter referred to as "EXP-SUS"). Similarly, the above-mentioned umbilical cord tissue-derived frozen cells (UCMSC-EXP) were put to sleep, the cells were seeded in a cell culture flask, and plate culture was performed using a serum-free medium for mesenchymal stem cells (Rohto) and cultured for 8 days to obtain plate cultured cells (hereinafter referred to as "EXP-ADH"). IFNγ (product number: AF-300-02, manufactured by PeproTeck, Inc.) was added in an amount to give a final concentration of 50 ng/mL and 100 ng/mL, and the amount of kynurenine in the cell supernatant after 90 hours was measured by a colorimetric method using Ehrlich reagent (50 ng/mL group and 100 ng/mL group). In addition, IDO activity was also measured for a control group to which IFNγ was not added as a comparison subject. In addition, the cell activity of each mesenchymal stem cell was measured using WST-8 (Cell Counting Kit-8, Dojindo Laboratories), the measured value of kynurenine was corrected, and the IDO activity was evaluated using the kynurenine production ability of each sample as an index. The IDO activity ratio was calculated by setting the IDO/WST8 value of the EXP-ADH control group to 1 (FIG. 7).

It was found that EXP-SUS had higher IDO activity than EXP-ADH under all conditions. This indicates that EXP-SUS is more capable of inducing and activating IDO through IFNγ stimulation, and of producing kynurenine intracellularly and secreting it into the culture supernatant than EXP-ADH.

[Measurement of kynurenine production and IDO activity in umbilical cord tissue-derived mesenchymal stem cells and other cells]
The above-mentioned umbilical cord tissue-derived frozen cells (UCMSC-CP) were put to sleep, seeded in a cell culture flask, and plate cultured using a serum-free medium for mesenchymal stem cells (Rohto) or 10% serum-containing MEMα medium for 8 days to obtain plate cultured cells (hereinafter referred to as "UCMSC-SF" and "UCMSC-MEM", respectively). In addition, MRC-5 cells (ATCC) and human neonatal dermal fibroblasts (HDF, ScienCell Research Laboratories) were thawed and plate cultured using 10% serum-containing MEMα medium for 3 days to obtain plate cultured cells. Furthermore, human umbilical vein endothelial cells (HUVEC, PromoCell) were thawed and plate culture was performed for 3 days using endothelial cell growth medium (PromoCell, product number C-22210) to obtain plate culture cells. UCMSC-SF, UCMSC-MEM, MRC-5, HDF and HUVEC were each cultured at 30,000 cells/ ^cm2 for 4 days, and the culture supernatant was collected. The amount of kynurenine in the culture supernatant was measured by a colorimetric method using Ehrlich's reagent. In addition, the cell activity of each mesenchymal stem cell was measured using WST-8 (Cell Counting Kit-8, Dojindo Laboratories), the measured value of kynurenine was corrected, and the IDO activity was evaluated using the kynurenine production ability of each sample as an index (Figure 8).

It was found that UCMSC-SF had higher IDO activity than UCMSC-MEM. It was also found that UCMSC-SF and UCMSC-MEM had higher IDO activity than MRC-5, HDF and HUVEC. This demonstrated that umbilical cord tissue-derived mesenchymal stem cells cultured in serum-free medium for mesenchymal stem cells have a higher ability to produce kynurenine intracellularly and secrete it into the culture supernatant than umbilical cord tissue-derived mesenchymal stem cells cultured in 10% serum-containing MEMα. It was also found that umbilical cord tissue-derived mesenchymal stem cells have a higher ability to produce kynurenine intracellularly and secrete it into the culture supernatant than MRC-5, HDF and HUVEC.

[Comparison of plate-cultured cells (ADH) and suspension-cultured cells (SUS) by microarray analysis]
Total RNA was extracted from the flat-plate cultured cells (EXP-ADH) or suspension cultured cells (EXP-SUS) obtained by the above method after 8 days of culture, and cDNA was synthesized according to the usual method. The expressed genes were comprehensively analyzed using Agilent's SurePrint G3 Human GE Microarray 8x60K Ver. 3.0. As a result, of the 155 genes related to the JAK-STAT signal pathway registered in Kyoto Encyclopedia of Genes and Genomes (KEGG), 33 were 1.5 times more expressed in EXP-SUS than in EXP-ADH at steady state. The ratio of the expression level in EXP-SUS to that in EXP-ADH (expression level ratio) was ranked in descending order as follows: CSF3, CSF2, LIF, IL6, IL6R, STAT4, CNTFR, GHR, CBLB, IL15, IL6ST, IL10, CLCF1, CCND2, IL11RA, IL15RA, PIM1, SOCS1, STAT2, IL19, IL7, IL24, IL22RA1, SOCS3, JAK2, IFNAR1, IFNAR2, IFNGR2, PRL, LIFR, SPRY2, AKT3, IL4R. The expression level ratios of the top nine genes are shown in FIG. 9.

[Quantitative analysis of IDO expression in IFNγ-stimulated UC-MSCs]
Frozen stocks of plate-cultured cells (EXP-ADH) or suspension-cultured cells (EXP-SUS) obtained by culturing for 7, 14, or 21 days using the above method were thawed and seeded on cell culture plates, to which IFNγ (product number 300-02, manufacturer PEPROTECH) was added, followed by culturing for 2 days. Then, total RNA was extracted using NucleoSpin RNA (product number 740955.50, manufacturer MACHERY-NAGEL), cDNA was synthesized using PrimeScript (registered trademark) RT reagent Kit (PrimeScript (registered trademark) RT reagent Kit product number RR037B, manufacturer Takara), real-time PCR was performed using SYBR (registered trademark) Premix Ex Taq (trademark) II (product number RR081A, manufacturer Takara) with PikoReal96 (manufacturer Thermo), and analyzed with PikoReal Software2.0 (manufacturer Thermo). The IDO expression level of SUS was calculated when the IDO expression level of ADH at each culture day was set to 1 (Table 1). The primer sequences used are as follows:

IDO: GGG ACA CTT TGC TAA AGG CG (F; SEQ ID NO: 1) and GTC TGA TAG CTG GGG GTT GC (R; SEQ ID NO: 2)
GAPDH: AGC CTC AAG ATC ATC AGC AAT G (F; SEQ ID NO: 3) and ATG GAC TGT GGT CAT GAG TCC TT (R; SEQ ID NO: 4)

In cells cultured for any number of days, IDO expression upon IFNγ stimulation was greater in EXP-SUS than in EXP-ADH.

[Evaluation of the effect of UC-MSC administration in GVHD model mice]
Human PBMCs were transplanted into NOG mice (5×10 ⁶ cells/body), and suspension cultured cells (EXP-SUS) were administered via the tail vein once a week from one week for a total of four times. The high dose group (EXP-SUS high) and the low dose group (EXP-SUS low) received 1×10 ⁷ cells/kg and 2×10 ⁶ cells/kg of cells per administration, respectively. As a result of a total of six weeks of follow-up observation, the EXP-SUS administration group showed suppression of weight loss due to GVHD (FIG. 10) and suppression of the decrease in survival rate due to GVHD (FIG. 11) compared to the control group. This demonstrated that EXP-SUS is effective against GVHD.

[Study of anti-inflammatory effect in UC-MSC]
Human-derived monocytic THP1 cells were seeded in a 12-well plate at 3 x 10 ⁴ cells/well, and 100 nM of Phorborl 12-Myristate 13-Acetate (PMA, product number 162-23591, Fujifilm Wako Pure Chemical Industries, Ltd.) was added and cultured for 3 days. Frozen stocks of flat-cultured cells (EXP-ADH) or suspension-cultured cells (EXP-SUS) obtained by culturing for 8 days using the above method were thawed and seeded at 40,000 cells/well on cell culture inserts, and placed in the wells seeded with PMA-stimulated THP1 cells, and 1 μg/mL of Lipopolysaccharide (LPS, product number 14874-24, InvivoGen) was added. After 2 days, total RNA was extracted from the THP1 cells, and the expression level of the CCL2 gene was analyzed by real-time PCR ( FIG. 12 ). The primer sequences used are as follows:

CCL2: AAGAAGCTGTGATCTTCAAGAC (F; SEQ ID NO: 5) and CCATGGAATCCTGAACCCA (R; SEQ ID NO: 6)

Compared to no LPS stimulation (untreated group), LPS stimulation increased CCL2 mRNA expression (LPS group), but the expression was suppressed by co-culture with EXP-SUS and EXP-ADH. Furthermore, the results showed that EXP-SUS had a greater inhibitory effect (Figure 12). From this, it was found that EXP-SUS and EXP-ADH are effective against inflammation, and EXP-SUS in particular has an excellent anti-inflammatory effect.

The present invention makes it possible to provide a therapeutic agent for immune diseases and an anti-inflammatory agent.

Claims (7)

Mesenchymal stem cells characterized by high kynurenine production or secretion , obtained by mixing a mesenchymal stem cell suspension with microcarriers, adding the mixture to a culture tank, and carrying out agitation culture using a serum-free medium for mesenchymal stem cells . The mesenchymal stem cell according to claim 1 , characterized in that the amount of kynurenine produced or the amount of kynurenine secreted is high due to IDO induced in the cells by IFNγ treatment. The mesenchymal stem cell according to claim 1 or 2 , characterized in that the JAK/STAT pathway is activated in a steady state. The mesenchymal stem cell according to any one of claims 1 to 3, which is derived from umbilical cord tissue. A therapeutic agent for immune diseases comprising the mesenchymal stem cells according to any one of claims 1 to 4. An anti-inflammatory agent containing the mesenchymal stem cells according to any one of claims 1 to 4. A GVHD therapeutic agent containing the mesenchymal stem cells according to any one of claims 1 to 4.

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