Treatment for myocarditis

Abstract

A cell preparation for the therapy of myocarditis, the preparation containing multipotent stem cells (Muse cells) that are positive for SSEA-3 and are derived from cultured mesenchymal cells or a mesenchymal tissue of a living body.

Description

[Technical field]

[0001] The present invention relates to a cell preparation for regenerative medicine, more specifically to a cell preparation containing pluripotent stem cells that is effective for treating myocarditis. [Background technology]

[0002] Myocarditis is an inflammatory disease that mainly affects the myocardium. When the inflammation spreads to the pericardium, it is called myocarditis. There are various types of myocarditis, including acute myocarditis, chronic myocarditis, fulminant myocarditis, and dilated cardiomyopathy-like types. The onset period can range from a few hours to one or two weeks or even longer, and the prognosis can range from recovery to complete normalcy to death.

[0003] Inflammation of the myocardium is often triggered by a viral infection. Other causes include bacterial infections, drugs, chemicals, allergies, autoimmune diseases, collagen diseases, Kawasaki disease, sarcoidosis, radiation, heat stroke, and AIDS. However, myocarditis can also develop without a clear cause being identified.

[0004] The incidence of myocarditis is unclear, but one survey has found that it occurs in 115 people per 100,000.

[0005] Myocarditis is often triggered by a viral infection, and its precursor symptoms include cold-like symptoms (chills, fever, headache, muscle pain, general fatigue, etc.) and digestive symptoms such as loss of appetite, nausea, vomiting, and diarrhea. As the condition progresses, (1) signs of heart failure, (2) chest pain due to pericardial irritation, and (3) cardiac symptoms associated with heart block or arrhythmia may appear, and if fatal arrhythmia develops, lightheadedness or fainting may occur, leading to sudden death.

[0006] In cases of eosinophilic myocarditis and giant cell myocarditis, steroids and immunosuppressants are used to treat myocarditis, but since myocarditis is often triggered by a viral infection, it is often not possible to treat the underlying cause. Therefore, in order to relieve myocardial function from the inhibition caused by inflammatory substances, short-term high-dose steroid therapy, high-dose immunoglobulin therapy, plasma exchange therapy, etc. are being considered. Furthermore, if treatment for heart failure or fatal arrhythmia is required during the progression of myocarditis, diuretics and pressor drugs will be used, artificial ventilation will be administered, and auxiliary circulatory devices and pacemaker therapy will be performed. In addition, intra-aortic balloon pumping, percutaneous cardiopulmonary support devices, artificial hearts, etc. may be used to provide auxiliary circulation to help the heart function.

[0007] Thus, symptomatic treatment is being performed for myocarditis, and there is currently no fundamental therapeutic agent for myocarditis, and there is an urgent need to provide a medicine that is effective for the direct treatment of myocarditis.

[0008] Meanwhile, with recent advances in regenerative medicine research, treatments for myocarditis, such as bone marrow stem cell transplantation, are being explored. For example, Non-Patent Document 1 discloses that bone marrow-derived mesenchymal stem cells reduce myocardial damage and dysfunction in a rat model of acute myocarditis. However, the effect is still not sufficient, and the clinical effect is unknown.

[0009] Meanwhile, research by the present inventor, Idezawa et al. has revealed that pluripotent stem cells (Multilineage-differentiating Stress Enduring cells; Muse cells) that are present in the mesenchymal cell fraction and express SSEA-3 (Stage-Specific Embryonic Antigen-3) as a surface antigen, which can be obtained without gene transfer or induction using cytokines, etc., are responsible for the pluripotency of the mesenchymal cell fraction and may be applicable to disease treatment aimed at tissue regeneration (e.g., Patent Document 1; Non-Patent Documents 2-4). Muse cells can be obtained from bone marrow fluid, adipose tissue (Non-Patent Document 5), dermal connective tissue of the skin, etc., and are known to exist widely in the connective tissue of tissues and organs. However, the effect of Muse cells on myocarditis is unknown. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Patent No. 5185443 [Non-patent literature]

[0011] [Non-Patent Document 1] Ohnishi S et al. J Mol Cell Cardio. 2007 Jan;42(1):88-97. [Non-Patent Document 2] Kuroda Y et al. Proc Natl Acad Sci USA 2010; 107: 8639-8643. [Non-Patent Document 3] Wakao S et al. Proc Natl Acad Sci USA 2011; 108: 9875-9880. [Non-Patent Document 4] Kuroda Y et al. Nat Protc 2013; 8: 1391-1415. [Non-Patent Document 5] Ogura,F.,et al.,Stem Cells Dev, 2013 Nov; 23;7 Summary of the Invention [Problem to be solved by the invention]

[0012] An object of the present invention is to provide a cell preparation for the treatment of myocarditis. [Means for solving the problem]

[0013] As a result of intensive research to achieve the above object, the inventors discovered that Muse cells accumulate in myocardial tissue damaged by myocarditis, differentiate into cardiomyocytes within the damaged myocardial tissue, and bring about the repair of the myocardial tissue, the reduction of the damaged myocardial tissue, and the improvement or recovery of cardiac function, thereby completing the present invention.

[0014] That is, the present invention is as follows. [1] A cell preparation for treating myocarditis, containing SSEA-3-positive pluripotent stem cells derived from living mesenchymal tissue or cultured mesenchymal cells. [2] The cell preparation described in [1], which contains a cell fraction enriched in the pluripotent stem cells. [3] The cell preparation described in claim 1 or 2, wherein the pluripotent stem cells are CD105 positive.

[0015] [4] The cell preparation described in any of [1] to [3], wherein the pluripotent stem cells are CD117 negative and CD146 negative. [5] The cell preparation described in any of [1] to [4], wherein the pluripotent stem cells are CD117-negative, CD146-negative, NG2-negative, CD34-negative, vWF-negative and CD271-negative. [6] The cell preparation according to any of [1] to [5], wherein the pluripotent stem cells are CD34-negative, CD117-negative, CD146-negative, CD271-negative, NG2-negative, vWF-negative, Sox10-negative, Snai1-negative, Slug-negative, Tyrp1-negative and Dct-negative.

[0016] [7] The cell preparation according to any one of [1] to [6], wherein the pluripotent stem cells have all of the following properties: (i) low or absent telomerase activity; (ii) have the ability to differentiate into cells of any of the three germ layers; (iii) does not exhibit neoplastic growth; and (iv) It has self-renewal ability. [8] The cell preparation according to [7], wherein the pluripotent stem cells have all of the following properties: (i) SSEA-3 positive; (ii) CD105 positive; (iii) low or absent telomerase activity; (iv) have the ability to differentiate into any of the three germ layers; (v) does not exhibit neoplastic growth; and (vi) Having self-renewal ability. [9] The cell preparation according to any of [1] to [8], wherein the myocarditis is one or more types selected from lymphocytic myocarditis, giant cell myocarditis, eosinophilic myocarditis and granulomatous myocarditis.

[10] Use of SSEA-3-positive pluripotent stem cells derived from biological mesenchymal tissue or cultured mesenchymal cells in the manufacture of a cell preparation for treating or preventing myocarditis.

[11] A method for treating myocarditis, comprising the step of administering an effective amount of a cell preparation containing SSEA-3-positive pluripotent stem cells derived from biological mesenchymal tissue or cultured mesenchymal cells to a myocarditis patient in need of treatment or prevention. Effect of the Invention

[0017] According to the present invention, there is provided a cell preparation for treating myocarditis, comprising Muse cells. Muse cells can efficiently migrate to and engraft at sites of myocardial tissue damage caused by myocarditis, and are thought to spontaneously differentiate at the engrafted site, making it unnecessary to induce differentiation into cells to be treated prior to transplantation. They are also non-tumorigenic and have excellent safety. Furthermore, since Muse cells are not subject to immune rejection, treatment with allogeneic preparations produced from donors is also possible. Therefore, Muse cells with the excellent performance shown above can provide an easily feasible means for the treatment of myocarditis. [Brief description of the drawings]

[0018] [Figure 1] Figure 1 shows the results of cell analysis by FACS. From the top left, the results are for an unstained cell population, a cell population treated with an isotype control, and a cell population treated with an SSEA-3 antibody. The bottom left shows the results for an SSEA-3 positive cell population enriched by MACS. [Diagram 2]Figure 2 shows the results of cardiac function measurements in myocarditis rats. Figure 2A shows an optical microscope image (photograph in place of a drawing) of a paraffin section (HE stained) of cardiac tissue from a myocarditis rat. The right image is an enlarged image of the left image. Figure 2B shows the ejection fraction (EF) obtained from MRI imaging data. [Diagram 3] FIG. 3 shows the results of cardiac function measurements (EF) of cell-transplanted rats with different numbers of cells. [Figure 4] FIG. 4 shows the results of measuring sphingosine-1-phosphate (S1P) concentrations in cell-transplanted rats 0 days after transplantation. [Diagram 5] Figure 5 shows the biodistribution of administered cells in cell transplanted rats. Figure 5A shows an analysis image (photograph in place of a drawing) using an in vivo imaging system. Figure 5B shows the light intensity of each organ obtained by the in vivo imaging system. [Figure 6] Figure 6 shows the results of cardiac function measurements of cell transplanted rats. Figure 6A to Figure 6F show the results of measurement of cardiac function (left ventricular end diastolic volume (EDV), left ventricular end systolic volume (ESV), ejection fraction (EF)) 2 weeks and 8 weeks after transplantation. Figure 6G shows the results of measurement of cardiac function (brain natriuretic peptide (BNP)) 2 weeks after transplantation. [Figure 7] Figure 7 shows the results of histological evaluation of cell transplanted rats (photographs in lieu of drawings). Figure 7, A to F, respectively show the detection results for the immature myocardial marker ANP, the mature myocardial markers sarcomeric α-actinin, troponin-I, and connexin 43, and the vascular component cells CD31 and αSMA. In A to F, the left images show merged images, the center images show GFP, and the right images show the detection results for each myocardial marker. [Figure 8] Figure 8 shows the results of histological evaluation of cell-transplanted rats. Figure 8A shows optical microscopic images (photographs substituting drawings) of Masson-Trichrome stained paraffin sections of cardiac tissue 8 weeks after transplantation in each cell administration group. Figure 8B shows the cardiac fiber area / cardiac cross-sectional area calculated from the optical microscopic images. [Figure 9]Figure 9 shows the results of measuring the number of apoptotic cells in cell-transplanted rat tissues. Figure 9A shows optical microscopic images (photographs in place of drawings) of TdT-mediated dUTP nick end labeling (TUNEL) stained frozen sections of cardiac tissue 3 days after transplantation in each cell administration group. Figure 9B shows the number of TUNEL-positive cells. [Figure 10] Figure 10 shows the results of evaluating angiogenesis by cell transplantation. Figure 10A shows cross-sectional images of blood vessels in transparentized cardiac tissue for each group, taken with a multiphoton microscope. Figure 10B also shows the proportion of blood vessel volume in cardiac tissue. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention will now be described. <1> A cell preparation containing SSEA-3-positive pluripotent stem cells (Muse cells) derived from in vivo mesenchymal tissue or cultured mesenchymal cells The present invention relates to a cell preparation containing Muse cells for treating myocarditis (hereinafter, sometimes referred to as "the cell preparation of the present invention"). In the present invention, "treatment of myocarditis" includes treatment and improvement of myocarditis, such as repair of damaged myocardial tissue caused by myocarditis, reduction of damaged myocardial tissue, and improvement or recovery of cardiac function, as well as cure, alleviation, prevention of recurrence, etc. of symptoms of myocarditis.

[0020] 1.Applicable diseases The cell preparation containing Muse cells of the present invention is used for the treatment of myocarditis. Myocarditis is classified into lymphocytic, giant cell, eosinophilic, and granulomatous myocarditis based on histological characteristics. In terms of etiology, lymphocytic myocarditis is often caused by viral infection, while giant cell, eosinophilic, and granulomatous myocarditis are often considered to be complications of cardiotoxic substances, drug allergies, autoimmune, and systemic diseases.

[0021] On the other hand, myocarditis can be divided into acute and chronic myocarditis depending on the onset mode. Acute myocarditis that leads to cardiopulmonary crisis in the early stages of onset is called fulminant myocarditis. Chronic myocarditis refers to myocarditis that continues for a relatively long period of time, for example, for more than several months, and often leads to heart failure and arrhythmia, and presents with a pathology similar to dilated cardiomyopathy. Chronic cardiomyopathy can be classified into two types: asymptomatic, which develops inapparently and progresses chronically, and chronic, which is acute myocarditis that continues for a long time.

[0022] Pericarditis is an inflammatory disease of the heart, and sometimes pericarditis and myocarditis occur together. In such cases, the condition is called pericarditis-myocarditis. In addition, acute myocarditis may lead to dilated cardiomyopathy. In the present invention, "myocarditis" includes all of the above. Although not limited thereto, preferred examples of myocarditis targeted by the cell preparation of the present invention include autoimmune myocarditis, and more preferred examples include giant cell myocarditis.

[0023] 2. Cell Preparation (1) SSEA-3-positive pluripotent stem cells (Muse cells) derived from in vivo mesenchymal tissue or cultured mesenchymal cells The pluripotent stem cells used in the cell preparation of the present invention are cells that the present inventors, Idezawa et al., found in the human body and named "Muse (Multilineage-differentiating Stress Enduring) cells". Muse cells can be obtained from bone marrow fluid, adipose tissue (Ogura, F., et al., Stem Cells Dev., Nov 20, 2013 (Epub) (published on Jan 17, 2014)), dermal connective tissue of the skin, etc., and are known to exist widely in connective tissues of tissues and organs. In addition, these cells have the properties of both pluripotent stem cells and mesenchymal stem cells, and are identified as, for example, cells positive for the cell surface marker "SSEA-3 (Stage-specific embryonic antigen-3)", preferably double positive cells positive for SSEA-3 and CD-105. Therefore, Muse cells or cell populations containing Muse cells can be isolated from biological tissues, for example, using the expression of SSEA-3 alone or SSEA-3 and CD-105 as an indicator. Furthermore, taking advantage of the high resistance of Muse cells to various external stresses, Muse cells can be selectively enriched by culturing them under various external stress conditions, such as proteolytic enzyme treatment, low oxygen conditions, low phosphate conditions, low serum concentration, low nutrition conditions, exposure to heat shock, in the presence of harmful substances, in the presence of active oxygen, under mechanical stimulation, under pressure treatment, etc. Details of the isolation, identification, characteristics, enrichment, etc. of Muse cells are disclosed in International Publication No. WO2011 / 007900 and can be referred to. In this specification, as a cell preparation for treating myocarditis, pluripotent stem cells (Muse cells) or cell populations containing Muse cells prepared from biological mesenchymal tissue or cultured mesenchymal cells using SSEA-3 as an indicator may be simply referred to as "SSEA-3 positive cells."

[0024] Muse cells or a cell population containing Muse cells can be prepared from biological tissue (eg, mesenchymal tissue) using the cell surface markers SSEA-3 or SSEA-3 and CD-105 as indicators. Here, the term "living body" refers to a living body of a mammal. In the present invention, the living body does not include fertilized eggs or embryos that are earlier than the blastula stage, but includes embryos at developmental stages after the blastula stage, including fetuses and blastulas. Mammals include, but are not limited to, primates such as humans and monkeys; rodents such as mice, rats, rabbits, and guinea pigs; cats, dogs, sheep, pigs, cows, horses, donkeys, goats, and ferrets. The Muse cells used in the cell preparation of the present invention are clearly distinguished from embryonic stem cells (ES cells) and induced pluripotent stem (iPS) cells in that they are directly isolated from living tissues and have markers. Furthermore, the term "mesenchymal tissue" refers to tissues containing mesenchymal cells, such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, umbilical cord, umbilical cord blood, and amniotic membrane, and tissues present in various organs. For example, Muse cells can be obtained from bone marrow, skin, fat tissue, blood, dental pulp, umbilical cord, umbilical cord blood, and amniotic membrane. For example, it is preferable to collect mesenchymal tissue from a living organism, prepare Muse cells from this tissue, and use the same. Furthermore, Muse cells may be prepared from cultured mesenchymal cells, such as fibroblasts and bone marrow mesenchymal stem cells, using the above-mentioned preparation means.

[0025] Furthermore, the cell population containing Muse cells used in the cell preparation of the present invention can also be prepared by a method comprising applying an external stress stimulus to mesenchymal tissue of a living body or cultured mesenchymal cells, selectively proliferating cells resistant to the external stress, and recovering cells with an increased abundance ratio. The external stress may be any one or a combination of protease treatment, culture at a low oxygen concentration, culture under low phosphate conditions, culture at a low serum concentration, culture under low nutrient conditions, culture under exposure to heat shock, culture at low temperature, freezing treatment, culture in the presence of a harmful substance, culture in the presence of active oxygen, culture under mechanical stimulation, culture under shaking treatment, culture under pressure treatment, or physical shock. The treatment time with the protease is preferably 0.5 to 36 hours in total in order to apply external stress to the cells. The protease concentration may be any concentration used when detaching cells attached to a culture vessel, disaggregating cell clumps into single cells, or recovering single cells from tissue. The protease is preferably a serine protease, an aspartic acid protease, a cysteine ​​protease, a metalloprotease, a glutamic acid protease or an N-terminal threonine protease, and more preferably, the protease is trypsin, collagenase or dispase.

[0026] In the cell preparation of the present invention, the Muse cells used may be autologous or allogeneic to the recipient of the cell transplantation.

[0027] As described above, Muse cells or cell populations containing Muse cells can be prepared from biological tissues using, for example, SSEA-3 positivity or double positivity for SSEA-3 and CD-105 as an indicator, but human adult skin is known to contain various types of stem cells and progenitor cells. However, Muse cells are not the same as these cells. Such stem cells and progenitor cells include skin-derived progenitor cells (SKPs), neural crest stem cells (NCSCs), melanoblasts (MBs), perivascular cells (PCs), endothelial progenitor cells (EPs), and adipose-derived stem cells (ADSCs). Muse cells can be prepared using the "non-expression" of markers specific to these cells as an indicator. More specifically, Muse cells can be separated using non-expression of at least one, for example, two, three, four, five, six, seven, eight, nine, ten or eleven of eleven markers selected from the group consisting of CD34 (EP and ADSC marker), CD117 (c-kit) (MB marker), CD146 (PC and ADSC marker), CD271 (NGFR) (NCSC marker), NG2 (PC marker), vWF factor (von Willebrand factor) (EP marker), Sox10 (NCSC marker), Snai1 (SKP marker), Slug (SKP marker), Tyrp1 (MB marker), and Dct (MB marker) as an indicator. For example, but not limited to, the non-expression of CD117 and CD146 can be used as an indicator, and the non-expression of CD117, CD146, NG2, CD34, vWF and CD271 can be used as an indicator, and the non-expression of the above 11 markers can be used as an indicator.

[0028] Furthermore, the Muse cells having the above-mentioned characteristics used in the cell preparation of the present invention are as follows: (i) low or absent telomerase activity; (ii) have the ability to differentiate into cells of any of the three germ layers; (iii) does not exhibit neoplastic growth; and (iv) Self-renewing ability Preferably, the Muse cells used in the cell preparation of the present invention have all of the above properties.

[0029] Regarding the above (i), "low or no telomerase activity" means that the telomerase activity is low or undetectable when it is detected using a known telomerase activity detection method, for example, TRAPEZE XL telomerase detection kit (Millipore). "Low" telomerase activity means, for example, that the telomerase activity is the same as that of human fibroblasts, which are somatic cells, or that the telomerase activity is 1 / 5 or less, preferably 1 / 10 or less, of that of HeLa cells.

[0030] Regarding (ii) above, Muse cells have the ability to differentiate into three germ layers (endodermal, mesodermal, and ectodermal) in vitro and in vivo. For example, by in vitro induction culture, they can differentiate into hepatocytes (including hepatoblasts or cells expressing hepatocyte markers), nerve cells, skeletal muscle cells, smooth muscle cells, bone cells, adipocytes, etc. In addition, when transplanted into the testis in vivo, they may also exhibit the ability to differentiate into the three germ layers. Furthermore, when transplanted into a living body by intravenous injection, they have the ability to migrate and engraft in damaged organs (heart, skin, spinal cord, liver, muscle, etc.) and differentiate into cells according to the tissue. "Having the ability to differentiate into cells of any of the three germ layers" means such a differentiation ability.

[0031] Regarding (iii) above, Muse cells proliferate at a rate of about 1.3 days, but in suspension culture, they proliferate from a single cell, create embryoid-like cell clusters, and when they reach a certain size, proliferation stops in about 14 days. However, when these embryoid-like cell clusters are transferred to adhesion culture, cell proliferation resumes, and the cells proliferating from the cell clusters spread at a proliferation rate of about 1.3 days. Furthermore, when transplanted into the testis, they have the property of not becoming cancerous for at least six months. "Does not exhibit neoplastic proliferation" refers to this non-neoplastic proliferation ability.

[0032] Regarding (iv) above, Muse cells have the ability to self-renew (self-replicate). Here, "having the ability to self-renew" means that differentiation into three germ layer cells can be confirmed from cells contained in an embryoid-like cell mass obtained by culturing a single Muse cell in suspension culture, and at the same time, by bringing a cell from the embryoid-like cell mass into suspension culture once again, an embryoid-like cell mass of the next generation can be formed, from which differentiation into three germ layers and an embryoid-like cell mass in suspension culture can be confirmed again. Self-renewal can be achieved by repeating one or more cycles.

[0033] (2) Preparation and use of cell preparations containing SSEA-3-positive pluripotent stem cells (Muse cells) derived from biological mesenchymal tissue or cultured mesenchymal cells The cell preparation containing Muse cells of the present invention can be obtained by suspending the Muse cells or a cell population containing Muse cells as specified in (1) above in physiological saline or an appropriate buffer solution (e.g., phosphate-buffered saline), although it is not limited thereto. In this case, when the number of Muse cells isolated from autologous or allogeneic tissue is small, the cells may be cultured before cell transplantation and allowed to grow until a predetermined number of cells is obtained. As already reported (International Publication No. WO2011 / 007900), Muse cells do not become tumorigenic, so that even if cells collected from biological tissues are contained in an undifferentiated state, there is a low possibility of canceration and it is safe. In addition, the culture of the collected Muse cells can be performed in a normal growth medium (e.g., α-minimal essential medium (α-MEM) containing 10% calf serum, etc.), although it is not particularly limited thereto. For more details, with reference to the above International Publication No. WO2011 / 007900, a medium, additives (e.g., antibiotics, serum), etc. can be appropriately selected for the culture and growth of Muse cells, and a solution containing Muse cells at a predetermined concentration can be prepared.

[0034] When administering a cell preparation containing the Muse cells of the present invention to a human subject, bone marrow fluid can be collected from the human ilium, and for example, bone marrow mesenchymal stem cells can be cultured as adherent cells from the bone marrow fluid to increase the cell amount to obtain an effective therapeutic amount of Muse cells, and then Muse cells can be isolated using the SSEA-3 antigen marker as an indicator to prepare autologous or allogeneic Muse cells as a cell preparation. Alternatively, for example, bone marrow mesenchymal stem cells obtained from the bone marrow fluid can be cultured under external stress conditions to grow and concentrate the Muse cells until an effective therapeutic amount is obtained, and then autologous or allogeneic Muse cells can be prepared as a cell preparation.

[0035] Furthermore, when Muse cells are used in a cell preparation, the cell preparation may contain dimethyl sulfoxide (DMSO) or serum albumin to protect the cells, and antibiotics to prevent bacterial contamination and proliferation. Furthermore, the cell preparation may contain other components that are acceptable for formulation (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.). Those skilled in the art can add these factors and drugs to the cell preparation at appropriate concentrations. In this way, Muse cells can also be used as a pharmaceutical composition containing various additives (preferably as a pharmaceutical composition for regenerative medicine).

[0036] The number of Muse cells contained in the cell preparation prepared above can be appropriately adjusted in consideration of the sex, age, weight, condition of the affected area, condition of the cells used, etc. of the subject so that the desired effect in the treatment of myocarditis can be obtained. The subject individual is not limited to individuals who have myocarditis, are suspected of having myocarditis, or have had myocarditis, and includes, but is not limited to, mammals such as humans. In addition, the cell preparation containing Muse cells of the present invention may be administered once or multiple times at appropriate intervals (e.g., twice a day, once a day, twice a week, once a week, once every two weeks, once a month, once every two months, once every three months, once every six months) until the desired therapeutic effect is obtained. Therefore, although it depends on the condition of the subject, the therapeutically effective amount is, for example, 1×10 per individual per administration.3 cells ~1×10 10 The preferred dose is 1 to 10 times per year for cells. The total dose per individual is not limited, but is preferably 1×10 3 cells ~1×10 11 Cells, preferably 1 x 10 4 cells ~1×10 10 cells, more preferably 1×10 5 cells ~1×10 9 Examples include cells.

[0037] The dosage and frequency of administration of the cell preparation containing Muse cells of the present invention can be set based on the effect on a myocarditis model. The myocarditis model can be prepared based on a known method, and includes, but is not limited to, an autoimmune myocarditis model in rats, which is considered to be a model of human giant cell myocarditis (Kodama M, Matsumoto Y, Fujiwara M, Masani F, Izumi T, Shibata A. A novel experimental model of giant cell myocarditis induced in rats by immunization with cardiac myosin fraction. Clin Immunol Immunopathol. 1990;57 (2):250-262.), Coxsackie B3 viral myocarditis mice (Silver MA, Kowalczyk D. Coronary microvascular narrowing in acute murine coxsackie B3 myocarditis. Am Heart J. 1989;118:173-174), and the like.

[0038] The Muse cells used in the cell preparation of the present invention have the property of migrating to and engrafting at the site of injury. Therefore, the site and method of administration of the cell preparation are not limited, and examples of the administration include intravascular administration (intravenous or intraarterial administration) and local administration.

[0039] The cell preparation containing Muse cells of the present invention can achieve repair and regeneration of damaged areas in patients with myocarditis.

[0040] In one embodiment of the present invention, the administration of the cell preparation of the present invention can reduce the size of damaged tissue caused by myocarditis in a subject with myocarditis. The damaged tissue is not limited to, but may be a tissue in which one or more of the following are observed: infiltration of inflammatory cells, degeneration of cardiomyocytes, edema of the interstitium, and fibrosis. Here, in the present specification, the "damaged tissue size" is defined as the ratio (%) of damaged tissue to normal cardiac tissue. The damaged tissue size can be measured by ordinary testing, analysis, and measurement techniques. When examining the effect of the cell preparation of the present invention in reducing the size of damaged tissue, it is useful to use the reduction rate relative to the size of damaged tissue in a control (i.e., (damaged tissue size in control - damaged tissue size after cell transplantation) / damaged tissue size in control x 100). According to the present invention, it is preferable that the size of damaged tissue is reduced by 100% compared to a group (control) not administered with the cell preparation. More preferably, the reduction is 10 to 90%, even more preferably, 20 to 70%, and even more preferably, 30 to 50%. As shown in the Examples below, when a rat cardiomyopathy model was used, the size of the damaged tissue in the control group was 12.1±4.5%, whereas in the Muse cell transplantation group, the size of the damaged tissue was 6.0±2.0% or 4.8±3.4%. These figures show that the size of the damaged tissue could be reduced by approximately 50-60% by Muse cell transplantation.

[0041] In one embodiment of the present invention, the cell preparation of the present invention can improve or restore cardiac function to normal (or normal value) after myocarditis. As used herein, "improving" cardiac function means improving cardiac function that has been reduced by myocarditis, and preferably improving cardiac function to a level that does not interfere with daily life. Furthermore, "recovering cardiac function to normal" means that cardiac function that has been reduced by myocarditis returns to the state before myocarditis. In one aspect of the present invention, the cell preparation of the present invention can be used for the prevention and / or treatment of (chronic) heart failure after myocarditis.

[0042] Here, the indices for evaluating cardiac function are not limited, but common ones include the left ventricular end-diastolic volume (EDV), the left ventricular end-systolic volume (ESV), the ejection fraction (EF), and the brain natriuremic peptide (BNP). These indices can be measured by ordinary examination, analysis, and measurement techniques. The improvement or recovery of cardiac function by the cell preparation of the present invention can be judged, for example, by using at least one of the above four indices. For example, as described in the Examples below, 8 weeks after cell transplantation, in the Muse cell transplantation group, the EF was 68.8±4.5% or 70.1±3.4%, and in the control group, it was 59.5±3.9%, and from the measured values ​​of both, it can be seen that the cardiac function is significantly improved in the cell transplantation group compared to the control group. Furthermore, two weeks after cell transplantation, plasma BNP levels were 347.2±188.6 pg / ml or 395.2±283.3 pg / ml in the Muse cell transplantation group and 980±241.1 pg / ml in the control group. Both measurements indicate that cardiac function was significantly improved in the cell transplantation group compared to the control group. EXAMPLES

[0043] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples in any way.

[0044] <Experimental Method> <Creation of myocarditis model> Eight-week-old male Lewis rats (LEW / SsNSlc, SLC Japan, Hamamatsu, Japan) were used. Under sedation by isoflurane inhalation anesthesia, based on past literature (Kodama M, Matsumoto Y, Fujiwara M, Masani F, Izumi T, Shibata A. A novel experimental model of giant cell myocarditis induced in rats by immunization with cardiac myosin fraction. Clin Immunol Immunopathol. 1990;57(2):250-262.), autoimmune myocarditis was created by subcutaneously injecting 0.1 ml each of porcine cardiac myosin (10 mg / ml; Sigma-Aldrich Japan, Tokyo, Japan) and Freund's complete adjuvant (Difco Laboratories, Sparks, MD, USA) containing the same amount of 11 mg / ml Mycobacterium tuberculosis into the plantar surface of the foot.

[0045] <Isolation of Muse cells> Human bone marrow-derived mesenchymal stem cells (MSCs) (Lonza Japan, Tokyo, Japan) were used. MSCs were cultured in a 10 cm dish at 37°C in 5% CO using Dulbecco's Modified Eagle's Medium low-glucose (DMEM; Life Technologies, Carlsbad, CA, USA), 10% fetal bovine serum (FBS; Hyclone; Thermo-Fisher Scientific, Waltham MA, USA), and 0.1 mg / mL kanamycin (Life Technologies) according to previous publications (Kuroda Y, Wakao S, Kitada M, Murakami T, Nojima M, Dezawa M. Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells. Nature protocols. 2013;8:1391-1415.) and International Publication No. WO2011 / 007900 (Patent No. 5185443). 2MSCs from passages 7 to 8 were used to isolate Muse cells as follows: the primary antibody was rat anti-stage-specific embryonic antigen-3 (SSEA-3) IgM antibody (1:1000; BioLegend, San Diego, CA, USA), and the isotype control was goat anti-rat FITC μ-chain IgM antibody (1:1000; Miltenyi Biotec, Bergisch Gladbach, Germany). The secondary antibody was fluorescein isothiocyanate (FITC)-labeled anti-rat IgM antibody (1:100; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA), and the tertiary antibody was anti-FITC microbeads (1:50; Miltenyi). SSEA-3 positive cells were isolated using an autoMACS® Pro Separator (Miltenyi). After cell isolation, the percentage of SSEA-3 positive cells in the cells enriched by magnetic cell sorting (MACS) was analyzed using a BD FACS Aria (BD Biosciences, Franklin Lakes, USA) (FACS; fluorescence activated cell sorting). Cells containing 70% or more SSEA-3 positive cells were defined as Muse cells and used as transplant cells. Some MSCs were transfected with green fluorescent protein (GFP) or Nano-lantern / pcDNA3 using lentivirus, referring to a previous publication (Hayase M, Kitada M, Wakao S, et al. Committed neural progenitor cells derived from genetically modified bone marrow stromal cells ameliorate deficits in a rat model of stroke. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 2009;29:1409-1420.), and incubated with rat anti-SSEA-3 IgM antibody (1:1000; BioLegend) as the primary antibody and allophycocyanin (APC)-labeled anti-rat IgM antibody (1:100; Jackson ImmunoResearch) as the secondary antibody, and SSEA-3-positive and SSEA-3-negative cells were separated using FACS. To mimic Muse cells isolated by MACS, isolated cells were mixed so that the proportion of SSEA-3 positive cells was 70%.

[0046] <Consideration of cell transplantation timing and number of transplanted cells> As a preliminary experiment to determine the timing of cell transplantation, cardiac function was evaluated by magnetic resonance imaging (MRI) (1T ICON, Bruker, Billerica, MA, USA) 2, 3, 4, 6, and 8 weeks after the creation of the myocarditis model, and the changes in cardiac function were compared. In addition, to investigate the optimal number of transplanted cells, we established groups receiving 100,000 Muse cells, 200,000 Muse cells, and 400,000 Muse cells (n=5 per group). Cell transplantation was performed at the optimal time, and cardiac function was compared by MRI.

[0047] <Cell transplantation by intravenous administration> Three weeks after the myocarditis was created, the mice were randomly divided into groups and cell transplantation was performed: a vehicle group (Vehicle group) in which normal saline (NS) was administered to the myocarditis model, a group (MSC group) in which 200,000 MSC cells were administered (Muse group), a group (Muse group) in which 200,000 Muse cells were administered (Muse group), and a sham group (S group) in which myocarditis was not induced. In addition, a group (Muse' group) in which 200,000 Muse cells were administered 3 and 4 weeks after the creation of myocarditis was created to examine the therapeutic effect of multiple administrations, and a group (MSC' group) in which 3,000,000 MSCs were administered to examine the therapeutic effect of MSCs containing SSEA-3 positive cells equivalent to those in the Muse cell administration group. All transplanted cells were suspended in 1 ml of normal saline and administered via the tail vein. In the experiments to measure cardiac function and evaluate histological fibrosis, cells concentrated by MACS were administered. In the immunohistochemistry experiment, GFP-Muse cells isolated by FACS were transplanted, and in the biodistribution evaluation experiment, nano-lantern-Muse cells were transplanted. No immunosuppressants were administered to either cell administration group.

[0048] <Cardiac function measurement in cell transplanted rats> During weeks 2 and 8, MRI (1T ICON, Bruker, Billerica, MA, US) was performed under 1.5% isoflurane inhalation anesthesia. Left ventricular short-axis images from the apex to the left ventricular outflow tract were obtained with the following parameters: repetition time 17 ms, echo time 1.72 ms, flip angle 30°, field of view 40 × 40 mm, slice thickness 1.25 mm, averages 8. The following items were measured from the obtained image data: left ventricular end-diastolic volume (EDV), left ventricular end-systolic volume (ESV), and ejection fraction (EF). Week 2 included n=9 patients per group, and week 8 included n=6 patients per group. Plasma brain natriuretic peptide (BNP) levels during Week 2 were measured using enzyme-linked immunosorbent assay (ELISA) (BNP-32 Rat RIA Kit, Peninsula Laboratories, San Carlos, CA, USA).

[0049] <Heart histological evaluation of cell transplanted rats> On day 3, week 2, and week 8, rats were sacrificed by isoflurane overdose and then perfused with 4% paraformaldehyde (PFA). Hearts were removed and divided into four sections 3 mm thick along the left ventricular short axis, then embedded in paraffin or frozen tissue embedding material (OCT Compound; Sakura Finetek, Tokyo, Japan). Paraffin sections were then cut at 3 μm, and frozen sections were cut at 6 μm. The following histological evaluations were performed using sections at the papillary muscle level. To confirm the degree of inflammation in the cardiac tissue, paraffin sections were stained with hematoxylin-eosin (HE), and the slides were observed under a light microscope (BX53; Olympus, Tokyo, Japan). To evaluate the differentiation potential of the transplanted cells, fluorescent immunohistochemistry was performed at weeks 2 and 8. The primary antibodies used were rabbit anti-GFP (1:500; Medical & Biological Laboratories Co., Ltd., Nagoya, Japan), goat anti-ANP (1:100; Santa Cruz Biotechnology, Dallas, TX, USA), goat anti-CD31 (Santa Cruz Biotechnology), mouse anti-sarcomeric α-actinin (1:100; Sigma-Aldrich), mouse anti-troponin-I (1:50; Merck, Billerica, MA, USA), mouse anti-connexin 43 (1:50; Abcam, Cambridge, UK), and mouse anti-α-SMA (1:500; Thermo Fisher Scientific, Waltham, MA, USA). The secondary antibodies used were Alexa 488-conjugated donkey anti-rabbit (1:500; Jackson ImmunoResearch), Alexa 594-conjugated donkey anti-goat (1:500; Jackson ImmunoResearch), and Alexa The cells were visualized with 594-conjugated donkey anti-mouse antibody (1:500; Jackson ImmunoResearch) under a Nikon A1 confocal laser scanning microscope (Nikon, Tokyo, Japan). The ratios of ANP·GFP double positive cells / GFP positive cells and troponin-I·GFP double positive cells / GFP positive cells were calculated (n=3). To evaluate apoptosis in injured tissues, TdT-mediated dUTP nick end labeling (TUNEL) staining was performed on day 3 (In Situ Cell Death Detection Kit, TMR Red; Roche Diagnostics Deutschland GmbH, Mannheim, Germany). 2Twenty regions of interest (ROIs) were observed, and the number of TUNEL-positive cells per unit area was calculated (n=3 per group). To evaluate fibrosis, Masson-Trichrome staining was performed at week 8. Slides were observed and photographed under a light microscope (Olympus). Cardiac fiber area / cardiac cross-sectional area was calculated using Adobe Photoshop (Adobe Inc., San Jose, CA, USA) (n=6 per group).

[0050] <Evaluation of cell migration factors for injured tissues in rats with cell transplants> We measured sphingosine-1-phosphate (S1P), a chemotactic factor that induces Muse cells to the site of injury (Yamada Y, Wakao S, Kushida Y, et al. S1P-S1PR2 Axis Mediates Homing of Muse Cells Into Damaged Heart for Long-Lasting Tissue Repair and Functional Recovery After Acute Myocardial Infarction. Circulation research. 2018;122:1069-1083.). On day 0, rats were sacrificed by isoflurane overdose, the hearts were removed, and the left ventricles were homogenized using a biomasher. The homogenate buffer (500 mM Tris-HCl / 150 mM NaCl / 1 mM EDTA / 1% Triton X-100, 0.5% sodium deocyl sulfate / 0.5% sodium deoxycholate) was supplemented with a protease inhibitor (cOmplete, Merck) and a phosphatase inhibitor (PhosSTOP™, Sigma-Aldrich) according to the manufacturer's protocol. After centrifugation at 4°C and 15,000 rpm for 15 min, the S1P concentration in the supernatant was measured by liquid chromatography-mass spectrometry (API 4000, AB / MDS SCIEX, Framingham, MA, USA) at Toray Research Center (Kamakura, Japan). The S1P concentration was expressed as the concentration per gram of heart tissue. The myocarditis-free group (Control group; n=6) and myocarditis-induced group (Myocarditis group; n=6) were used.

[0051] <Evaluation of biodistribution of transplanted cells> Nano-lantern-transfected cells were used to evaluate the biodistribution of transplanted cells (Saito K, Chang YF, Horikawa K, et al. Luminescent proteins for high-speed single-cell and whole-body imaging. Nature communications. 2012;3:1262.). Nano-lantern-Muse cell-treated, nano-lantern-MSC-treated, and untreated rats (n=3 per group) were administered coelenterazine 200 μg / 1 ml NS via the tail vein under sedation with isoflurane inhalation on week 2. The rats were sacrificed by isoflurane overdose, and the heart, lungs, brain, liver, pancreas, spleen, kidneys, stomach, small intestine, large intestine, femur, tibia, fibula, and quadriceps were removed. The heart was sliced ​​into 3 mm thickness, and the other organs were sliced ​​into 1 cm thickness or less, and immersed in coelenterazine 50 μg / ml solution. Images were captured using an IVIS Lumina LT (Perkin Elmer, Waltham, MA, USA), and light intensity was quantified using Living Image Software (Perkin Elmer).

[0052] <Evaluation of angiogenesis> On week 2, 2 mg of dextran, Texas Red™ (Thermo-Fisher Scientific) was administered via the tail vein under sedation with isoflurane inhalation. The rats were euthanized by isoflurane overdose, and the hearts were removed. The tissue was cleared according to the tissue clearing technique CUBIC (clear, unobstructed brain / body imaging cocktails and computational analysis) (Susaki EA, Tainaka K, Perrin D, et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell. 2014;157:726-739.). After sufficient clearing was obtained, the tissue was observed with a Multiphoton laser microscope (Nikon). 118 axial still images of 0.78 × 0.78 μm were taken at 0.85 μm intervals from the epicardial side to the endocardial side. For each sample, images were taken at nine locations in the anterior, lateral, and posterior walls of the left ventricle, one randomly selected from the base, apex, and the middle, and the vascular lumen volume was calculated using Image J software (National Institutes of Health, Bethesda, MD, USA).

[0053] <Statistical analysis> The data obtained were expressed as mean ± standard deviation. JMP Pro 14 software (SAS Institute, Cary, NC, USA) was used for statistical analysis. Comparison of continuous variables between the two groups for tissue S1P levels and signal intensity of IVIS data was performed using the Shapiro-Wilk test to prove that both were normally distributed, and Student's t-test was performed. One-way ANOVA and Tukey-Kramer's post hoc test were used for other multigroup comparisons. The significance level was set at p<0.05.

[0054] <<Experimental Results>> <Positive rate of SSEA-3 in the cell population after Muse cell enrichment> The gate of flow cytometry was set so that SSEA-3 positive cells were not detected in MSCs reacted with the isotype control. The positive rate of SSEA-3 in MSCs used in this study was 5.1 ± 1.3%, and the positive rate of SSEA-3 after cell separation by MACS was 75.0 ± 4.8% (Figure 1).

[0055] <Examination of the optimal cell transplantation time and the number of transplanted cells> To examine the optimal cell administration time, the degree of inflammation at 2 weeks, 3 weeks, 4 weeks, 6 weeks, and 8 weeks after induction of myocarditis was histologically evaluated, and the change in EF was compared by MRI. In HE staining, mononuclear cell infiltration was observed at 2 weeks, but no other inflammatory findings were observed. At 3 weeks, marked granulocyte and mononuclear cell infiltration was observed, accompanied by myocardial cell degeneration and interstitial edema (A in Figure 2). At 6 weeks, granulocyte and mononuclear cell infiltration decreased, and the appearance of fibrosis was observed by MT staining. All lesions were observed almost circumferentially on the epicardial side. EF showed a decreasing trend over time until 3 weeks and reached a plateau after 3 weeks (B in Figure 2). In this study, in order to aim for clinical verification, cell transplantation was performed 3 weeks after induction of myocarditis when cardiac function and histological severity reached their peaks. Subsequently, the optimal number of transplanted cells was examined. Three weeks after induction of myocarditis, a group administered 100,000 Muse cells, a group administered 200,000 Muse cells, and a group administered 400,000 Muse cells were established, and EF was measured 2 weeks after cell transplantation (Figure 3). EF was 74.0 ± 2.5% in the group administered 200,000 Muse cells, showing a statistically significantly higher value compared to the group administered 100,000 Muse cells (69.5 ± 2.4%, p < 0.05), but no statistically significant difference was observed compared to the group of 400,000 Muse cells (72.7 ± 1.3%). From the above, the number of transplanted cells was set to 200,000 cells.

[0056] <Migration factors and in vivo distribution of Muse cells> The concentration of S1P, a chemotactic factor for Muse cells, was measured using cardiac tissue homogenate samples on day 0 (Fig. 4). The S1P concentration was 24.9±2.9 ng / g in the control group and 32.5±4.9 ng / g in the myocarditis group, which was statistically significantly higher in the myocarditis group (p<0.05). The biodistribution of the administered cells at Week 2 was evaluated using IVIS (Figure 5A). The light intensity of each organ obtained by IVIS is shown (Figure 5B). More cell accumulation was observed in the Muse group than in the MSC group (p<0.05). Furthermore, the cells mainly accumulated around the epicardium, which coincided with the site of inflammation caused by this disease model. Although slight accumulation was observed in the lungs in the MSC group, no accumulation in the lungs was observed in the Muse group. Furthermore, no accumulation in other organs was observed in either group.

[0057] <Improvement of cardiac function in cell transplanted rats> Cardiac function at week 2 and week 8 is shown (Fig. 6A-F). At week 2, EF in the Muse group was 73.1±1.9%, which was statistically significantly higher than the Vehicle group (60.8±2.5%, p<0.001), MSC group (66.7±2.4%, p<0.001), and MSC' group (68.3±2.3%, p<0.01). No significant difference was observed between the Muse group and the Muse' group (73.2±2.0%). ESV in the Muse group was 0.11±0.01 ml, which was statistically significantly lower than the Vehicle group (0.15±0.01 ml, p<0.001). Although there was no significant difference between the Muse group and the MSC group (0.12±0.02 ml) or MSC' group (0.12±0.01 ml), the Muse group tended to have a lower EDV. There was no significant difference between the cell transplantation groups. At week 8, EF in the Muse group was 68.8±4.5%, which was statistically significantly higher than that in the Vehicle group (59.5±3.9%, p<0.001). No significant difference was observed between the Muse group and the Muse' group (70.1±3.4%). No significant differences were observed in EDV and ESV between the cell transplantation groups. Plasma BNP levels at Week 2 are shown (Figure 6G). The BNP level in the Muse group was 347.2±188.6 pg / ml, which was statistically significantly lower than the Vehicle group (980±241.1 pg / ml, p<0.001), MSC group (802.2±171.1 pg / ml, p<0.01), and MSC' group (770±316.3 pg / ml, p<0.01). No statistically significant difference was observed between the Muse group and the Muse' group (395.2±283.3 pg / ml).

[0058] <Histological evaluation of cell transplanted rats> The differentiation ability of transplanted GFP-Muse cells into cardiomyocytes and vascular cells at week 2 was histologically observed. Among the GFP-positive cells, cells expressing the immature myocardial marker ANP, mature myocardial markers sarcomeric α-actinin, troponin-I, and connexin 43, vascular cell markers αSMA, and CD31 were observed (Figure 7, A-F). The proportion of ANP among GFP-positive cells was + GFP + The percentage of double positive cells was 41.3±1.8%, and troponin-I + GFP + The percentage of double positive cells was 16.2±3.1%. To evaluate the anti-fibrotic / fibrolytic effects of transplanted cells, cardiac tissue sections at week 8 were observed by Masson-Trichrome staining. Representative images of each group are shown in Figure 8A. The percent fibrotic area of ​​each group was calculated by cardiac fiber area / cardiac cross-sectional area × 100 (Figure 8B). The Muse group was 6.0±2.0%, and the Muse' group was 4.8±3.4%, both of which were statistically significantly lower than the Vehicle group (12.1±4.5%). Although no significant differences were observed between the Muse group and the MSC group (8.6±2.6%) or MSC' group (8.5±4.5%), a tendency for the Muse group to be lower was observed.

[0059] <Comparison of apoptotic cell numbers in cell transplanted rat tissues> For the purpose of evaluating the anti-apoptotic effect by cell transplantation, the number of TUNEL-positive cells among each group was compared using heart tissue specimens on day 3. Representative images of each group are shown in Fig. 9A. The number of TUNEL-positive cells in the Muse group was 1.8 ± 0.4 cells / mm 2 , which showed a statistically significantly lower value compared to the Vehicle group (7 ± 1.6 cells / mm 2 ) (p = 0.001) (Fig. 9B).

[0060] <Evaluation of angiogenesis by Muse cells> For the purpose of evaluating angiogenesis by cell transplantation, the vascular volume in the cleared heart tissue was measured. Tomographic images of each group taken with a multiphoton microscope are shown in Fig. 10A. The percentage of the vascular volume in the heart tissue was 7.4 ± 1.2% in the Muse group, which showed a statistically significantly higher value compared to the Vehicle group (3.2 ± 0.5%) (p = 0.047) (Fig. 10B).

[0061] As described above, the effectiveness of Muse cells in the treatment of myocarditis was shown.

Industrial applicability

[0062] By administering the cell preparation of the present invention to a patient suffering from myocarditis, it can accumulate at the damaged site of myocarditis, repair the damaged site (proliferation of cardiomyocytes, angiogenesis, tissue repair, etc.), improve or restore cardiac function, and can be applied to the treatment of myocarditis.

Claims

1. The present invention comprises, as an active ingredient, a cell fraction in which SSEA-3-positive and CD105-positive pluripotent stem cells derived from a mesenchymal tissue of a living body or cultured mesenchymal cells are selectively enriched, The pluripotent stem cells have all of the following properties: Cellular preparations for treating myocarditis: (i) low or absent telomerase activity; (ii) have the ability to differentiate into cells of any of the three germ layers; (iii) does not exhibit neoplastic growth; and (iv) having self-renewal ability;

2. The cell preparation of claim 1, wherein the pluripotent stem cells are CD117-negative and CD146-negative.

3. The cell preparation of claim 1 or 2, wherein the pluripotent stem cells are CD117-negative, CD146-negative, NG2-negative, CD34-negative, vWF-negative and CD271-negative.

4. The cell preparation according to any one of claims 1 to 3, wherein the pluripotent stem cells are CD34-negative, CD117-negative, CD146-negative, CD271-negative, NG2-negative, vWF-negative, Sox10-negative, Snail-negative, Slug-negative, Tyrp1-negative and Dct-negative.

5. The cell preparation according to any one of claims 1 to 4, wherein the myocarditis is one or more selected from lymphocytic myocarditis, giant cell myocarditis, eosinophilic myocarditis and granulomatous myocarditis.

Images