WO2021045190A1 - 心筋炎の治療剤 - Google Patents
心筋炎の治療剤 Download PDFInfo
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- WO2021045190A1 WO2021045190A1 PCT/JP2020/033577 JP2020033577W WO2021045190A1 WO 2021045190 A1 WO2021045190 A1 WO 2021045190A1 JP 2020033577 W JP2020033577 W JP 2020033577W WO 2021045190 A1 WO2021045190 A1 WO 2021045190A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
- A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
Definitions
- the present invention relates to a cell preparation for regenerative medicine. More specifically, it relates to a cell preparation containing pluripotent stem cells effective for treating myocarditis.
- Myocarditis is an inflammatory disease with the myocardium as the main component. When inflammation extends to the pericardium, it is called pericarditis. There are various types of myocarditis, including acute myocarditis, chronic myocarditis, fulminant myocarditis, dilated cardiomyopathy-like type, etc., and the onset period is from several hours to one or two weeks, or even longer. The prognosis also varies from completely normal to death.
- myocarditis may develop without a clear cause being identified.
- the frequency of myocarditis is not clear, but there is a survey result of 115 people per 100,000 population.
- Myocarditis often develops as a result of a viral infection, so the symptoms that appear as precursors include cold-like symptoms (chills, fever, headache, myalgia, general malaise, etc.), loss of appetite, nausea, vomiting, diarrhea, etc. Gastrointestinal symptoms precede. As it progresses, (1) signs of heart failure, (2) chest pain due to pericardial stimulation, (3) heart symptoms associated with heart block and arrhythmia may appear, and when fatal arrhythmia develops, light-headedness and fainting appear and sudden death occurs. It may reach.
- myocarditis In the treatment of myocarditis, steroids and immunosuppressants are used in the case of eosinophilia myocarditis and giant cell myocarditis, but myocarditis is often triggered by viral infection and is the root cause. Often cannot be treated. Therefore, short-term high-dose steroid therapy, high-dose immunoglobulin therapy, plasma exchange therapy, etc. are being studied in order to relieve the suppression of myocardial function by inflammatory substances. In addition, during the course of myocarditis, if heart failure or fatal arrhythmia needs to be dealt with, diuretics and pressor agents, artificial respiration management, assisted circulatory device, and pacemaker treatment are performed to help the heart work. Intra-aortic balloon pumping, percutaneous cardiopulmonary support devices, artificial hearts, and the like are also used for assisted circulation.
- 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 not yet sufficient, and the clinical effect is unknown.
- Muse cells can be obtained from bone marrow fluid, adipose tissue (Non-Patent Document 5), dermal connective tissue of skin, and the like, and are widely known to be present in connective tissue of tissues and organs. However, the effect of Muse cells on myocarditis is unknown.
- An object of the present invention is to provide a cell preparation for the treatment of myocarditis.
- the present inventors accumulated Muse cells in damaged myocardial tissue due to myocarditis, differentiated into cardiomyocytes within the damaged myocardial tissue, repaired the myocardial tissue, and damaged myocardium. They have found that they bring about tissue shrinkage and improvement or recovery of cardiac function, and have completed the present invention.
- the present invention is as follows.
- a cell preparation for treating myocarditis which comprises SSEA-3 positive pluripotent stem cells derived from living mesenchymal tissues or cultured mesenchymal cells.
- the cell preparation according to [1] which comprises a cell fraction enriched with the pluripotent stem cells.
- pluripotent stem cell is a pluripotent stem cell having all of the following properties: (I) Low or no telomerase activity; (Ii) Has the ability to differentiate into cells of any of the three germ layers; (Iii) show no neoplastic growth; and (iv) have self-renewal ability.
- the pluripotent stem cell is a pluripotent stem cell having all of the following properties: (I) SSEA-3 positive; (Ii) CD105 positive; (Iii) Low or no telomerase activity; (Iv) Has the ability to differentiate into one of the three germ layers; (V) does not show neoplastic growth; and (vi) has self-renewal ability.
- the myocarditis is one or more selected from lymphocytic myocarditis, giant cell myocarditis, eosinophil myocarditis and granulomatous myocarditis, [1] to [8]. ]
- the cell preparation according to any one of.
- [10] Use of SSEA-3 positive pluripotent stem cells derived from living mesenchymal tissues or cultured mesenchymal cells in the production of cell preparations for treating or preventing myocarditis.
- a cell preparation for treating myocarditis which comprises Muse cells
- Muse cells can efficiently migrate to the site of myocardial tissue damage due to myocarditis and engraft, and are thought to spontaneously differentiate at the engrafted site. Therefore, differentiation induction into cells to be treated is induced prior to transplantation. Not needed. In addition, it is non-tumorogenic and has excellent safety. Furthermore, since Muse cells are not immune rejected, they can be treated with allogeneic preparations produced from donors. Therefore, Muse cells with the excellent performance described above can provide an easily viable means for the treatment of myocarditis.
- FIG. 1 shows the results of cell analysis by FACS.
- the results of the unstained cell population, the isotype control-using cell population, and the SSEA-3 antibody-using cell population are shown from the top left.
- the bottom row shows the results of the SSEA-3 positive cell population enriched with MACS.
- FIG. 2 shows the results of cardiac function measurement in myocarditis rats.
- FIG. 2A is an optical microscope observation image (drawing substitute photograph) of a paraffin section (HE stain) of heart tissue of a myocarditis rat.
- the figure on the right is an enlarged image of the figure on the left.
- FIG. 2B shows the ejection fraction (EF) obtained from the MRI imaging data.
- FIG. 3 shows the results of cardiac function measurement (EF) of cell-transplanted rats with different cell numbers.
- FIG. 4 shows the results of sphingosine-1-phosphate (S1P) concentration measurement 0 days after transplantation of cell-transplanted rats.
- FIG. 5 shows the in vivo distribution of administered cells in cell-transplanted rats.
- FIG. 5A is an analysis image (drawing substitute photograph) obtained by an in vivo imaging system.
- B in FIG. 5 shows the amount of light of each organ obtained by the in vivo imaging system.
- FIG. 6 shows the results of cardiac function measurement in cell-transplanted rats.
- a to F of FIG. 6 show the measurement results 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. .. G in FIG.
- FIG. 6 shows the measurement results of cardiac function (brain natriuretic peptide (BNP)) 2 weeks after transplantation.
- FIG. 7 shows the results of histological evaluation of cell-transplanted rats (drawing substitute photographs).
- a to F of FIG. 7 show the detection results of ANP, which is an immature myocardial marker, sarcomeric ⁇ -actinin, troponin-I, connexin 43, which are mature myocardial markers, and CD31 and ⁇ SMA, which are vascular constituent cells, respectively.
- a to F the left figure shows the merged image, the center figure shows GFP, and the right figure shows the detection results of each myocardial marker.
- FIG. 8 shows the results of histological evaluation of cell-transplanted rats.
- FIG. 8 shows the results of histological evaluation of cell-transplanted rats.
- FIG. 8A is an optical microscope observation image (drawing substitute photograph) of Masson-Trichrome staining of paraffin sections of heart tissue 8 weeks after transplantation of each cell administration group.
- FIG. 8B shows the cardiac fiber area / cardiac cross-sectional area calculated from the optical microscope observation image.
- FIG. 9 shows the measurement results of the number of apoptotic cells in the cell transplanted rat tissue.
- FIG. 9A is an optical microscope observation image (drawing substitute photograph) of TdT-mediated dUTP tick end labeling (TUNEL) staining of frozen sections of heart tissue 3 days after transplantation of each cell administration group.
- B in FIG. 9 shows the number of TUNEL-positive cells.
- FIG. 10 shows the evaluation results of angiogenesis by cell transplantation.
- FIG. 10A is a tomographic image of each group of blood vessels in the transparent heart tissue taken with a multiphoton microscope. Further, B in FIG. 10 shows the ratio of the blood vessel volume to the heart tissue.
- the present invention is for treating myocarditis containing Muse cells.
- cell preparation of the present invention includes treatment and improvement of myocarditis such as repair of damaged myocardial tissue due to myocarditis, reduction of damaged myocardial tissue and improvement or recovery of cardiac function, as well as treatment of myocarditis. Includes healing, alleviation, prevention of recurrence, etc. of symptoms.
- the cell preparation containing Muse cells of the present invention is used for the treatment of myocarditis.
- Myocarditis is classified into lymphocytic myocarditis, giant cell myocarditis, eosinophil myocarditis, and granulomatous myocarditis according to histological characteristics.
- lymphocytic myocarditis is often caused by viral infection
- giant cell myocarditis, eosinophilic myocarditis, and granulomatous myocarditis are cardiotoxic substances, drug allergies, autoimmune diseases, and systemic diseases. It is often regarded as a complication such as.
- myocarditis is divided into acute myocarditis and chronic myocarditis according to the onset pattern.
- Acute myocarditis that falls into a cardiopulmonary crisis in the early stages of onset is called fulminant myocarditis.
- chronic myocarditis refers to myocarditis that lasts for a relatively long period of time, for example, several months or more, often causes heart failure or arrhythmia, and presents a pathological condition similar to dilated cardiomyopathy.
- chronic cardiomyopathy includes those that develop subclinically and have a chronic course (subclinical) and those in which acute myocarditis persists (prolonged).
- pericarditis As an inflammatory disease of the heart, and this pericarditis and myocarditis may be combined. In such cases, it is called pericardial / myocarditis.
- acute myocarditis can lead to dilated cardiomyopathy.
- myocarditis includes all of the above. Autoimmune myocarditis is preferably exemplified, and giant cell myocarditis is more preferably exemplified, as the myocarditis targeted by the cell preparation of the present invention.
- SSEA-3 positive pluripotent stem cells derived from living mesenchymal tissues or cultured mesenchymal cells
- the pluripotent stem cell used in the cell preparation of the present invention is a cell that the inventor of the present invention, Dezawa et al., Found its existence in a human body and named it "Muse (Multipleage-differentiation Stress Enduring) cell".
- 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)), skin connective tissue, and the like. It is known that it is widely present in connective tissues of tissues and organs.
- this cell is a cell having both pluripotent stem cell and mesenchymal stem cell properties, and is, for example, a cell surface marker "SSEA-3 (Stage-specific embryonic antigen-3)" positive cell, preferably. Is identified as SSEA-3 positive and CD-105 positive double positive cells. Therefore, a Muse cell or a cell population containing a Muse cell can be separated from a living tissue using, for example, SSEA-3 alone or the expression of SSEA-3 and CD-105 as an index.
- SSEA-3 Voltage-specific embryonic antigen-3
- Muse cells can be selectively concentrated by culturing under various external stress conditions such as the presence of a substance, the presence of active oxygen, mechanical stimulation, and pressure treatment. Details of the Muse cell separation method, identification method, characteristics, concentration method and the like are disclosed in International Publication No. WO2011 / 007900 and can be referred to.
- pluripotent stem cells as a cell preparation for treating myocarditis, pluripotent stem cells (Muse) prepared from living mesenchymal tissues or cultured mesenchymal cells using SSEA-3 as an index. Cells) or cell populations containing Muse cells may simply be referred to as "SSEA-3 positive cells”.
- Muse cells or cell populations containing Muse cells can be prepared from living tissues (eg, mesenchymal tissues) using the cell surface markers SSEA-3 or SSEA-3 and CD-105 as indicators.
- the "living body” refers to a living body of a mammal. In the present invention, the living body does not include a fertilized egg or an embryo at a developmental stage before the blastogenic stage, but includes an embryo at a developmental stage after the blastogenic stage including a foetation or a blastoblast.
- 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, ferrets and the like. Be done. 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 separated from living tissues with markers. To.
- ES cells embryonic stem cells
- iPS induced pluripotent stem
- mesenchymal tissue refers to tissues including mesenchymal cells such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermatitis, ligament, tendon, dental pulp, umbilical cord, cord blood, and sheep membrane, and various types. Tissue that exists in an organ.
- Muse cells can be obtained from bone marrow, skin, adipose tissue, blood, pulp, umbilical cord, umbilical cord blood, amniotic membrane and the like.
- Muse cells may be prepared from cultured mesenchymal cells such as fibroblasts and bone marrow mesenchymal stem cells using the above-mentioned preparation means.
- the cell population containing Muse cells used in the cell preparation of the present invention can produce cells resistant to the external stress by giving an external stress stimulus to the mesenchymal tissue of the living body or the cultured mesenchymal cells. It can also be prepared by a method that involves collecting cells that have been selectively grown to increase their abundance.
- the external stress is protease treatment, culture at low oxygen concentration, culture under low phosphoric acid condition, culture at low serum concentration, culture under low nutrition condition, culture under heat shock exposure, low temperature.
- Culturing in, freezing, culturing in the presence of harmful substances, culturing in the presence of active oxygen, culturing under mechanical stimulation, culturing under shaking treatment, culturing under pressure treatment or physical impact It may be any or a combination of two or more.
- the total treatment time with the protease is preferably 0.5 to 36 hours in order to apply external stress to the cells.
- the protease concentration may be any concentration used when peeling the cells adhered to the culture vessel, breaking up the cell mass into a single cell, or recovering a single cell from the tissue.
- the protease is preferably a serine protease, an aspartic protease, a cysteine protease, a metal protease, a glutamate protease or an N-terminal threonine protease. Further, it is preferable that the protease is trypsin, collagenase or dyspase.
- the Muse cells used may be autologous or allogeneic to the recipient undergoing cell transplantation.
- Muse cells or cell populations containing Muse cells can be prepared from living tissues using, for example, SSEA-3 positive or double positive of SSEA-3 and CD-105 as an index, but human adults.
- the skin is known to contain various types of stem and progenitor cells.
- Muse cells are not the same as these cells.
- Such stem cells and progenitor cells include skin-derived progenitor cells (SKP), neural ridge stem cells (NCSC), melanoblasts (MB), perivascular cells (PC), endothelial progenitor cells (EP), and adipose-derived stem cells (ADSC).
- SSEA-3 positive or double positive of SSEA-3 and CD-105 as an index
- the skin is known to contain various types of stem and progenitor cells.
- Such stem cells and progenitor cells include skin-derived progenitor cells (SKP), neural ridge stem cells (NCSC), melanoblasts (MB), perivascular cells (PC), endothelial progenitor cells (EP), and
- Muse cells include CD34 (markers for EP and ADSC), CD117 (c-kit) (markers for MB), CD146 (markers for PC and ADSC), CD271 (NGFR) (markers for NCSC), NG2 (PC marker), vWF factor (Fonville brand factor) (EP marker), Sox10 (NCSC marker), Snai1 (SKP marker), Slug (SKP marker), Tyrp1 (MB marker), and At least one of 11 markers selected from the group consisting of Dct (MB marker), for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 The non-expression of 11 or 11 markers can be separated as an index.
- the non-expression of CD117 and CD146 can be used as an index
- the non-expression of CD117, CD146, NG2, CD34, vWF and CD271 can be used as an index
- the non-expression of 11 markers can be used as an index for preparation.
- Muse cells having the above characteristics used in the cell preparation of the present invention include: (I) Low or no telomerase activity; (Ii) Has the ability to differentiate into cells of any of the three germ layers; It may have at least one property selected from the group consisting of (iii) no neoplastic growth; and (iv) capable of self-renewal.
- the Muse cells used in the cell preparation of the present invention have all of the above properties.
- telomerase activity means that the telomerase activity is low or low when the telomerase activity is detected using a known telomerase activity detection method, for example, TRAPEZE XL telomerase detection kit (Millipore). It means that it cannot be detected.
- Low telomerase activity means, for example, telomerase having the same level of telomerase activity as somatic human fibroblasts, or 1/5 or less, preferably 1/10 or less of that of Hela cells. It means having activity.
- Muse cells have the ability to differentiate into three germ layers (endoderm lineage, mesodermal lineage, and ectodermal lineage) in vitro and in vivo, and are, for example, induced and cultured in vitro.
- hepatocytes including cells expressing hepatoblasts or hepatocyte markers
- nerve cells including cells expressing hepatoblasts or hepatocyte markers
- skeletal muscle cells including cells expressing hepatoblasts or hepatocyte markers
- smooth muscle cells smooth muscle cells
- bone cells fat cells and the like.
- fat cells and the like.
- transplanted into the testis in vivo it may show the ability to differentiate into three germ layers.
- Muse cells proliferate at a proliferation rate of about 1.3 days, but in suspension culture, they proliferate from one cell, and when they form an embryo-like cell mass and reach a certain size, they proliferate in about 14 days.
- these embryo-like cell clusters are transferred to adhesive culture, cell proliferation is started again, and the cells proliferated from the cell cluster spread at a proliferation rate of about 1.3 days. ..
- transplanted into the testis it has the property of not becoming cancerous for at least half a year. "Does not show neoplastic growth" means such non-neoplastic growth potential.
- Muse cells have a self-renewal (self-renewal) ability.
- self-renewal ability means that the differentiation of cells contained in the embryonic body-like cell mass obtained by culturing one Muse cell in suspension culture into three germ layer cells can be confirmed.
- the next generation embryo-like cell mass is formed, and from there, the three germ layer differentiation and suspension culture are performed again. It means that the germ layer-like cell mass can be confirmed in.
- Self-renewal may be repeated one or more cycles.
- the cell preparation containing Muse cells of the present invention is It is obtained by suspending the Muse cells defined in (1) above or a cell population containing Muse cells in a physiological saline solution or an appropriate buffer (for example, phosphate buffered physiological saline solution). In this case, if the number of Muse cells separated from autologous or allogeneic tissues is small, the cells may be cultured before cell transplantation and proliferated until a predetermined number of cells is obtained. As already reported (Pamphlet of International Publication No.
- Muse cells do not become tumors, so even if cells recovered from living tissues are contained in an undifferentiated state, there is a possibility of canceration. Is low and safe.
- the culture of the collected Muse cells is not particularly limited, but can be carried out in a normal growth medium (for example, ⁇ -minimum essential medium ( ⁇ -MEM) containing 10% calf serum). More specifically, with reference to the above-mentioned International Publication No. WO2011 / 007900 pamphlet, in the culture and proliferation of Muse cells, a medium, additives (for example, antibiotics, serum) and the like are appropriately selected, and a predetermined concentration of Muse cells is obtained. A solution containing the above can be prepared.
- a medium, additives for example, antibiotics, serum
- a cell preparation containing Muse cells of the present invention When a cell preparation containing Muse cells of the present invention is administered to a human subject, it is effective to collect bone marrow fluid from human intestinal bone and, for example, cultivate bone marrow mesenchymal stem cells as adherent cells from the bone marrow fluid. After increasing the therapeutic amount of Muse cells until the amount of cells obtained is reached, the Muse cells can be separated using the antigen marker of SSEA-3 as an index, and the autologous or allogeneic Muse cells can be prepared as a cell preparation.
- bone marrow mesenchymal stem cells obtained from bone marrow fluid are cultured under external stress conditions to proliferate and concentrate Muse cells until an effective therapeutic amount is reached, and then autologous or allogeneic Muse cells are cultured. It can be prepared as a preparation.
- Muse cells when using Muse cells in cell preparations, dimethyl sulfoxide (DMSO), serum albumin, etc. are contained in the cell preparations to protect the cells, and antibiotics, etc. are contained in the cell preparations to prevent bacterial contamination and proliferation. You may.
- other formulationally acceptable ingredients eg, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, saline, etc.
- Muse cells can also be used as a pharmaceutical composition containing various additives (preferably as a pharmaceutical composition for regenerative medicine).
- the number of Muse cells contained in the cell preparation prepared above takes into consideration the sex, age, body weight, condition of the affected area, condition of cells used, etc. of the subject so that the desired effect can be obtained in the treatment of myocarditis. Then, it can be adjusted as appropriate.
- the target individual is not limited to an individual who has developed myocarditis, is suspected of developing myocarditis, or is an individual after the onset of myocarditis, and includes, but is not limited to, mammals such as humans.
- the cell preparation containing Muse cells of the present invention may be used once or multiple times at appropriate intervals (for example, twice a day, once a day, once a week) until a desired therapeutic effect is obtained.
- the therapeutically effective dose is, for example, 1 ⁇ 10 3 cells to 1 ⁇ 10 10 cells per individual, and a dose of 1 to 10 times in one year. preferable.
- the total dose to be administered in one individual is not limited, but is limited to 1 ⁇ 10 3 cells to 1 ⁇ 10 11 cells, preferably 1 ⁇ 10 4 cells to 1 ⁇ 10 10 cells, and more preferably 1 ⁇ 10 5 cells to 1 ⁇ 10. 9 cells and the like can be mentioned.
- the myocarditis model can be prepared based on a known method and is not limited, for example, a rat autoimmune myocarditis model (Kodama M, Matsumoto Y, Fujiwara M, Masani), which is a human giant cell myocarditis model.
- F Izumi T, Shibata A.
- Clin Immunol Immunopathol. 1990; 57 (2): 250-262. (Silver MA, Kowalczyk D. Coronary microvascular narrowing in acute murine cox sackie B3 myocarditis. Am Heart J. 1989; 118: 173-174) and the like.
- the Muse cells used in the cell preparation of the present invention have the property of migrating to the damaged site and engrafting. Therefore, in the administration of the cell preparation, the administration site and the administration method of the cell preparation are not limited, and intravascular administration (intravenous, intraarterial), local administration and the like are exemplified.
- the cell preparation containing Muse cells of the present invention can realize repair and regeneration of a damaged site in a patient with myocarditis.
- administration of the cell preparation of the present invention can reduce the size of tissue damaged by myocarditis in a subject with myocarditis.
- the damaged tissue is not limited to, but may be a tissue in which one or more types of infiltration of inflammatory cells, degeneration of cardiomyocytes, edema of interstitium, and fibrosis are observed.
- the "disordered tissue size" is defined as the ratio (%) of the damaged tissue to the normal heart tissue.
- the size of the damaged tissue can be measured by conventional examination, analysis, and measurement techniques.
- the reduction rate with respect to the damaged tissue size of the control that is, (control damaged tissue size-damaged tissue size after cell transplantation) / control damaged tissue size ⁇ It is useful to use 100).
- the size of the damaged tissue is reduced by 100% as compared with the non-administered group (control) of the cell preparation. It is more preferably 10 to 90% reduction, even more preferably 20 to 70% reduction, and even more preferably 30 to 50% reduction.
- the damaged tissue size was 12.1 ⁇ 4.5% in the control group, whereas it was 12.1 ⁇ 4.5% in the Muse cell transplantation group.
- the damaged tissue size was 6.0 ⁇ 2.0% or 4.8 ⁇ 3.4%. From these figures, it can be seen that Muse cell transplantation was able to reduce the size of damaged tissue by about 50-60%.
- the cell preparation of the present invention can improve or restore normal (or normal value) cardiac function after myocarditis.
- improved of cardiac function means improvement of cardiac function reduced by myocarditis, and it is preferable that cardiac function is improved to the extent that it does not interfere with daily life.
- recovery normal means that the cardiac function decreased by myocarditis returns to the state before myocarditis.
- the cell preparation of the present invention can be used for the prevention and / or treatment of (chronic) heart failure after myocarditis.
- the index for evaluating cardiac function is not limited, but generally includes left ventricular end-diastolic volume (EDV), left ventricular end-systolic volume (ESV), and so on.
- Ejection fraction (EF) and brain natriuremic peptide (BNP) can be mentioned. These indicators can be measured by conventional inspection, analysis and measurement techniques.
- the improvement or recovery of cardiac function by the cell preparation of the present invention can be determined by using, for example, at least one of the above four indexes. For example, as described in Examples described later, 8 weeks after cell transplantation, the EF was 68.8 ⁇ 4.5% or 70.1 ⁇ 3.4% in the Muse cell transplantation group, and 59 in the target group.
- the cardiac function was significantly improved in the cell transplant group as compared with the control group.
- the plasma BNP value in the Muse cell transplantation group was 347.2 ⁇ 188.6 pg / ml or 395.2 ⁇ 283.3 pg / ml, and 980 ⁇ 241 in the target group. It was .1 pg / ml, and from the measured values of both, it can be seen that the cardiac function was significantly improved in the cell transplantation group as compared with the control group.
- Muse cells were isolated as follows using MSCs with 7 to 8 passages. Rat anti-stage-specific embryonic antigen-3 (SSEA-3) IgM antibody (1: 1000; BioLegend, San Diego, CA, USA) as primary antibody, goat anti-rat FITC ⁇ chain IgM antibody (1: 1000; Miltenyi Biotec, Bergisch Gladbach, Germany) was reacted.
- SSEA-3 Rat anti-stage-specific embryonic antigen-3
- SSEA-3 positive cells were isolated using autoMACS® Pro Separator (Miltenyi).
- BD FACS Aria BD Biosciences, Franklin Lakes, USA
- FACS fluorescence activated cell sorting
- Muse cells Those containing 70% or more of SSEA-3 positive cells were defined as Muse cells and used as transplanted cells.
- Some MSCs have past literature (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 depression. : official journal of the International Society of Cerebral Blood Flow and Metabolism.
- MSC'group A 3,000,000 MSC administration group (MSC'group) was set up for the purpose of examining the therapeutic effect of MSC containing cells. All transplanted cells were suspended in 1 ml of physiological saline and administered by tail vein. In cardiac function measurement and histological fibrosis evaluation experiments, MACS-enriched cells were administered. In the immunohistochemistry experiment, GFP-Muse cells isolated by FACS were transplanted, and in the in vivo distribution evaluation experiment, nano-lantern-Muse cells were transplanted. No immunosuppressant was administered in any of the cell-administered groups.
- MRI 1T ICON, Bruker, Billerica, MA, US
- a short axis image of the left ventricle from the apex of the heart to the outflow tract of the left ventricle was performed with the following imaging 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.
- EDV left ventricular end-diastolic volume
- ESV left ventricular end-systolic volume
- EF ejection fraction
- Week 2 plasma brain natriuremic peptide (BNP) was measured using the enzyme-linked immunosolvent assay (ELISA) method (BNP-32 Rat RIA Kit, Peninsula Laboratories, San Carlos, CA, USA). ..
- Rats were sacrificed to death by overdose of isoflurane on Day 3, week 2, and week 8, followed by 4% paraformaldehyde (PFA) perfusion fixation.
- the heart was excised, divided into 4 parts with a thickness of 3 mm in the short axis direction of the left ventricle, and then embedded in paraffin or frozen tissue embedding material (OCT Compound; Sakura Finetek, Tokyo, Japan). Then, a thin section was prepared with a paraffin section of 3 ⁇ m and a frozen section of 6 ⁇ m.
- OCT Compound paraffin or frozen tissue embedding material
- Hematoxylin-Eosin (HE; Hematoxylin-Eosin) staining was performed using paraffin sections to confirm the degree of inflammation in the heart tissue. The slides were observed with an optical microscope (BX53; Olympus, Tokyo, Japan). Fluorescent immunohistochemistry was performed on week 2 and week 8 for the purpose of evaluating the differentiation potential of transplanted cells.
- Rabbit anti-GFP antibody (1; 500; Medical & Biological Laboratories Co., Ltd., Nagoya, Japan), goat anti-ANP antibody (1: 100; Santa Cruz Biotechnology, Dallas, TX, USA), goat anti CD31 antibody (Santa Cruz Biotechnology), mouse anti-sarcomeric ⁇ -actinin antibody (1: 100; Sigma-Aldrich), mouse anti-troponin-I antibody (1:50; Merck, Billerica, MA, USA), mouse anti-connexin 43 antibody (1: 100; Sigma-Aldrich) 1:50; Abcam, Cambridge, UK), mouse anti- ⁇ -SMA antibody (1: 500; Thermo Fisher Scientific, Waltham, MA, USA) as secondary antibody, Alexa 488-labeled donkey anti-rabbit antibody (1: 500; Jackson ImmunoResearch), Alexa 594 labeled donkey anti-goat antibody (1: 500; Jackson ImmunoResearch), Alexa 594 labeled donkey anti-mouse antibody (1: 500; Jackson ImmunoResearch), Nikon A1
- S1P Sphingosine-1-phosphate
- 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
- protease inhibitors cOmplete, Merck
- PhosSTOP phosphatase inhibitors
- the S1P concentration in the supernatant was measured by liquid chromatography-mass spectrometry (API 4000, AB / MDS SCIEX, Framingham, MA, USA) at the Toray Research Center (Kamakura, Japan). ).
- the S1P concentration was expressed as the concentration in 1 g of heart tissue.
- Nano-lantern-introduced cells were used for the purpose of evaluating the in vivo distribution 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.).
- coelenterazine 200 ⁇ g / 1 ml NS was intravenously administered on week 2 under sedation by isoflurane inhalation. It was administered.
- Rats were sacrificed to death by overdose of isoflurane, and the heart, lung, brain, liver, pancreas, spleen, kidney, stomach, small intestine, large intestine, femur, tibia, fibula, and quadriceps femoris were removed.
- the heart was sliced to a thickness of 3 mm and the other organs were sliced to a thickness of 1 cm or less and immersed in a 50 ⁇ g / ml solution of coelenterazine.
- Photographed with IVIS Lumina LT Perkin Elmer, Waltham, MA, USA
- Quantified with Living Image Software Perkin Elmer).
- EF showed a downward trend over time until 3 weeks, and became a plateau after 3 weeks (B in Fig. 2).
- EF was 74.0 ⁇ 2.5% in the 200,000 Muse cell group, which was statistically significantly higher than that in the 100,000 Muse cell group (69.5 ⁇ 2.4%, p ⁇ 0.05), but was significantly higher in the 400,000 Muse cell group (72.7). No statistically significant difference was observed compared to ⁇ 1.3%). Based on the above, the number of transplanted cells was set to 200,000 cells.
- the concentration of S1P which is a migration factor of Muse cells, was measured using a day 0 cardiac tissue homogenate sample (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 were statistically significantly higher in the myocarditis group (p ⁇ 0.05).
- the in vivo distribution of administered cells during Week 2 was evaluated by IVIS (A in Fig. 5). The amount of light of each organ obtained by IVIS is shown (B in Fig. 5). More cell accumulation was observed in the Muse group than in the MSC group (p ⁇ 0.05).
- the cells were mainly accumulated around the epicardial side, which coincided with the site of inflammation caused by this disease model. Slight lung accumulation was observed in the MSC group, but no lung accumulation was observed in the Muse group. In addition, no accumulation in other organs was observed in any of the groups.
- the BNP value of the Muse group was 347.2 ⁇ 188.6 pg / ml, and the Statistics group (980 ⁇ 241.1 pg / ml, p ⁇ 0.001), the MSC group (802.2 ⁇ 171.1 pg / ml, p ⁇ 0.01), and the MSC'group (770). It was statistically significantly lower than ⁇ 316.3 pg / ml, p ⁇ 0.01). There was no statistically significant difference between the Muse group and the Muse'group (395.2 ⁇ 283.3 pg / ml).
- ⁇ Histological evaluation of cell transplanted rats The ability of transplanted GFP-Muse cells to differentiate into cardiomyocytes and vascular constituent cells during week 2 was histologically observed.
- GFP-positive cells cells expressing ANP, which is an immature myocardial marker, sarcomeric ⁇ -actinin, troponin-I, connexin 43, which are mature myocardial markers, and ⁇ SMA and CD31, which are vascular constituent cells, were observed (AF in Fig. 7). ).
- the proportion of ANP + GFP + double-positive cells among GFP-positive cells was 41.3 ⁇ 1.8%, and the proportion of troponin-I + GFP + double-positive cells was 16.2 ⁇ 3.1%.
- FIG. 8 shows a representative image of each group.
- the percentage fibrotic area of each group was calculated by cardiac fiber area / cardiac cross-sectional area x 100 (B in FIG. 8).
- Both the Muse group 6.0 ⁇ 2.0% and the Muse'group 4.8 ⁇ 3.4% were statistically significantly lower than the Vehicle group (12.1 ⁇ 4.5%).
- FIG. 9 shows a representative image of each group.
- FIG. 10A shows tomographic images of each group taken with a multiphoton microscope.
- the cell preparation of the present invention When the cell preparation of the present invention is administered to a patient who develops myocarditis, it accumulates at the damaged site of myocarditis, repairs the damaged site (proliferation of cardiomyocytes, angiogenesis, tissue repair, etc.), and cardiac function. Can be improved or recovered, and can be applied to the treatment of myocarditis.
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Abstract
Description
そのため、炎症性物質による心筋機能抑制からの解放のため、ステロイド短期大量療法、大量免疫グロブリン療法、血漿交換療法等が検討されている。
また、心筋炎の経過中、心不全や致死的不整脈への対応が必要とされた場合、利尿剤や昇圧剤の使用、人工呼吸管理、補助循環装置、ペースメーカー治療が行われ、心臓の働きを助ける補助循環のために、大動脈内バルーンパンピング、経皮的心肺補助装置、人工心臓等を使用することも行われる。
例えば、非特許文献1には、骨髄由来間葉系幹細胞が急性心筋炎ラットモデルにおいて心筋損傷や機能障害を軽減することが開示されている。
しかしながら、その効果はまだ十分なものとは言えず、臨床的な効果も不明である。
しかしながら、Muse細胞の心筋炎に対する効果は知られていない。
[1] 生体の間葉系組織または培養間葉系細胞に由来するSSEA-3陽性の多能性幹細胞を含む、心筋炎を治療するための細胞製剤。
[2] 前記多能性幹細胞が濃縮された細胞画分を含む、[1]に記載の細胞製剤。
[3] 前記多能性幹細胞が、CD105陽性である、請求項1または2に記載の細胞製剤。
[5] 前記多能性幹細胞が、CD117陰性、CD146陰性、NG2陰性、CD34陰性、vWF陰性およびCD271陰性である、[1]~[4]のいずれかに記載の細胞製剤。
[6] 前記多能性幹細胞が、CD34陰性、CD117陰性、CD146陰性、CD271陰性、NG2陰性、vWF陰性、Sox10陰性、Snai1陰性、Slug陰性、Tyrp1陰性およびDct陰性である、[1]~[5]のいずれかに記載の細胞製剤。
(i)テロメラーゼ活性が低いかまたは無い;
(ii)三胚葉のいずれの胚葉の細胞に分化する能力を持つ;
(iii)腫瘍性増殖を示さない;および
(iv)セルフリニューアル能を持つ。
[8] 前記多能性幹細胞が、以下の性質の全てを有する多能性幹細胞である、[7]に記載の細胞製剤:
(i)SSEA-3陽性;
(ii)CD105陽性;
(iii)テロメラーゼ活性が低いかまたは無い;
(iv)三胚葉のいずれかの胚葉に分化する能力を持つ;
(v)腫瘍性増殖を示さない;および
(vi)セルフリニューアル能を持つ。
[9] 前記心筋炎が、リンパ球性心筋炎、巨細胞性心筋炎、好酸球性心筋炎および肉芽腫性心筋炎から選ばれる1種または2種以上である、[1]~[8]のいずれかに記載の細胞製剤。
[10]生体の間葉系組織又は培養間葉系細胞に由来するSSEA-3陽性の多能性幹細胞の、心筋炎を治療又は予防するための細胞製剤の製造における使用。
[11]生体の間葉系組織又は培養間葉系細胞に由来するSSEA-3陽性の多能性幹細胞を含む細胞製剤の有効量を、治療又は予防を必要とする心筋炎患者に投与する工程を含む、心筋炎の治療方法。
Muse細胞は、心筋炎による心筋組織障害部位に効率的に遊走して生着することができ、生着した部位で自発的に分化すると考えられるので移植に先立って治療対象細胞への分化誘導が不要である。また、非腫瘍形成性であり安全性にも優れる。さらに、Muse細胞は免疫拒絶を受けないことから、ドナーから製造された他家製剤による治療も可能である。したがって、上記に示す優れた性能を有するMuse細胞によって、心筋炎の治療に対する容易に実行可能な手段を提供することができる。
<1>生体の間葉系組織または培養間葉系細胞に由来するSSEA-3陽性の多能性幹細胞(Muse細胞)を含む細胞製剤
本発明は、Muse細胞を含む、心筋炎を治療するための細胞製剤(以下、「本発明の細胞製剤」ということがある)に関する。
なお、本発明において、「心筋炎の治療」には、心筋炎による障害心筋組織の修復、障害心筋組織の縮小および心機能の改善または回復等の心筋炎の治療、改善の他、心筋炎の症状の治癒、緩和、再発防止等が含まれる。
本発明のMuse細胞を含む細胞製剤は、心筋炎の治療に使用される。
心筋炎は、組織学的特徴から、リンパ球性心筋炎、巨細胞性心筋炎、好酸球性心筋炎、肉芽腫性心筋炎に分類される。病因的には、リンパ球性心筋炎はウィルス感染によるものが多く、巨細胞性心筋炎、好酸球性心筋炎、肉芽腫性心筋炎は心毒性物質・薬物アレルギー・自己免疫・全身性疾患等の合併症としてみなされることが多い。
さらに、急性心筋炎が原因となり拡張型心筋症に至る場合もある。
本発明において、「心筋炎」には、上記全てのものが含まれる。限定されないが、本発明の細胞製剤が対象とする心筋炎として、自己免疫性心筋炎が好ましく例示され、巨細胞性心筋炎がより好ましく例示される。
(1)生体の間葉系組織または培養間葉系細胞に由来するSSEA-3陽性の多能性幹細胞(Muse細胞)
本発明の細胞製剤に使用される多能性幹細胞は、本発明者である出澤らが、ヒト生体内にその存在を見出し、「Muse(Multilineage-differentiating Stress Enduring)細胞」と命名した細胞である。Muse細胞は、骨髄液、脂肪組織(Ogura,F.,et al.,Stem Cells Dev.,Nov 20,2013(Epub)(published on Jan 17,2014))や皮膚の真皮結合組織等から得ることができるほか、広く組織や臓器の結合組織に存在することが知られている。また、この細胞は、多能性幹細胞と間葉系幹細胞の両方の性質を有する細胞であり、例えば、細胞表面マーカーである「SSEA-3(Stage-specific embryonic antigen-3)」陽性細胞、好ましくはSSEA-3陽性かつCD-105陽性の二重陽性細胞として同定される。したがって、Muse細胞またはMuse細胞を含む細胞集団は、例えば、SSEA-3単独またはSSEA-3およびCD-105の発現を指標として生体組織から分離することができる。また、Muse細胞が様々な外的ストレスに対する耐性が高いことを利用して、蛋白質分解酵素処理や、低酸素条件、低リン酸条件、低血清濃度、低栄養条件、熱ショックへの暴露、有害物質存在下、活性酸素存在下、機械的刺激下、圧力処理下等各種外的ストレス条件下での培養によりMuse細胞を選択的に濃縮することができる。Muse細胞の分離法、同定法、特徴、濃縮法等の詳細は、国際公開第WO2011/007900号に開示されており、参照することができる。
なお、本明細書においては、心筋炎を治療するための細胞製剤として、SSEA-3を指標として用いて、生体の間葉系組織または培養間葉系細胞から調製された多能性幹細胞(Muse細胞)またはMuse細胞を含む細胞集団を単に「SSEA-3陽性細胞」と記載することがある。
ここで、「生体」とは、哺乳動物の生体をいう。本発明において、生体には、受精卵や胞胚期より発生段階が前の胚は含まれないが、胎児や胞胚を含む胞胚期以降の発生段階の胚は含まれる。哺乳動物としては、限定されないが、ヒト、サル等の霊長類;マウス、ラット、ウサギ、モルモット等のげっ歯類;ネコ、イヌ、ヒツジ、ブタ、ウシ、ウマ、ロバ、ヤギ、フェレット等が挙げられる。
本発明の細胞製剤に使用されるMuse細胞は、生体の組織から直接マーカーを持って分離される点で、胚性幹細胞(ES細胞)や人工多能性幹(iPS)細胞と明確に区別される。
また、「間葉系組織」とは、骨、滑膜、脂肪、血液、骨髄、骨格筋、真皮、靭帯、腱、歯髄、臍帯、臍帯血、羊膜等の間葉系細胞を含む組織および各種臓器に存在する組織をいう。例えば、Muse細胞は、骨髄や皮膚、脂肪組織、血液、歯髄、臍帯、臍帯血、羊膜等から得ることができる。例えば、生体の間葉系組織を採取し、この組織からMuse細胞を調製し、利用することが好ましい。また、上記調製手段を用いて、線維芽細胞や骨髄間葉系幹細胞等の培養された間葉系細胞からMuse細胞を調製してもよい。
前記外的ストレスは、プロテアーゼ処理、低酸素濃度での培養、低リン酸条件下での培養、低血清濃度での培養、低栄養条件での培養、熱ショックへの暴露下での培養、低温での培養、凍結処理、有害物質存在下での培養、活性酸素存在下での培養、機械的刺激下での培養、振とう処理下での培養、圧力処理下での培養または物理的衝撃のいずれかまたは複数の組み合わせであってもよい。
前記プロテアーゼによる処理時間は、細胞に外的ストレスを与えるために合計0.5~36時間行うことが好ましい。また、プロテアーゼ濃度は、培養容器に接着した細胞を剥がすとき、細胞塊を単一細胞にばらばらにするとき、または組織から単一細胞を回収するときに用いられる濃度であればよい。
前記プロテアーゼは、セリンプロテアーゼ、アスパラギン酸プロテアーゼ、システインプロテアーゼ、金属プロテアーゼ、グルタミン酸プロテアーゼまたはN末端スレオニンプロテアーゼであることが好ましい。更に、前記プロテアーゼがトリプシン、コラゲナーゼまたはジスパーゼであることが好ましい。
(i)テロメラーゼ活性が低いかまたは無い;
(ii)三胚葉のいずれの胚葉の細胞に分化する能力を持つ;
(iii)腫瘍性増殖を示さない;および
(iv)セルフリニューアル能を持つ
からなる群から選択される少なくとも1つの性質を有してもよい。好ましくは、本発明の細胞製剤に使用されるMuse細胞は、上記性質を全て有する。
本発明のMuse細胞を含む細胞製剤は、限定されないが、上記(1)に規定するMuse細胞またはMuse細胞を含む細胞集団を生理食塩水や適切な緩衝液(例えば、リン酸緩衝生理食塩水)に懸濁させることによって得られる。この場合、自家または他家の組織から分離したMuse細胞数が少ない場合には、細胞移植前に細胞を培養して、所定の細胞数が得られるまで増殖させてもよい。なお、すでに報告されているように(国際公開第WO2011/007900号パンフレット)、Muse細胞は、腫瘍化しないため、生体組織から回収した細胞が未分化のまま含まれていても癌化の可能性が低く安全である。また、回収したMuse細胞の培養は、特に限定されないが、通常の増殖培地(例えば、10%仔牛血清を含むα-最少必須培地(α-MEM)等)において行うことができる。より詳しくは、上記国際公開第WO2011/007900号パンフレットを参照して、Muse細胞の培養および増殖において、適宜、培地、添加物(例えば、抗生物質、血清)等を選択し、所定濃度のMuse細胞を含む溶液を調製することができる。
また、細胞移植2週間後において、Muse細胞移植群では、血漿中BNP値は347.2±188.6 pg/mlまたは395.2±283.3 pg/mlであり、対象群では980±241.1 pg/mlであり、両者の測定値から、細胞移植群では、対照群と比較して、心機能が有意に改善されていることが分かる。
<心筋炎モデルの作製>
8週齢オスLewisラット (LEW/SsNSlc, SLC Japan, Hamamatsu, Japan) を用いた。イソフルラン吸入麻酔による鎮静下に、過去の文献(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.)を参考に、ブタ心筋ミオシン (10mg/ml; Sigma-Aldrich Japan, Tokyo, Japan) および同量の11 mg/ml Myocobacterium tuberculosisを含むFreund’s complete adjuvant (Difco Laboratories, Sparks, MD, USA) を0.1 mlずつ足底に皮下注射して自己免疫性心筋炎を作成した。
ヒト骨髄由来間葉系幹細胞(Mesenchymal Stem Cells:MSC) (Lonza Japan, Tokyo, Japan) を使用した。過去の文献(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.)および国際公開第WO2011/007900号(特許第5185443号)に従い、Dulbecco’s Modified Eagle’s Medium low-glucose (DMEM; Life Technologies, Carlsbad, CA, USA)、10%ウシ胎仔血清 (FBS; fetal bovine serum) (Hyclone; Thermo-Fisher Scientific, Waltham MA, USA)、0.1 mg/mLカナマイシン (Life Technologies) を用いて10cm dishで37℃、5%CO2の条件下で培養した。継代数7回から8回のMSCを使用しMuse細胞を以下の通り分離した。1次抗体としてラット抗stage-specific embryonic antigen-3 (SSEA-3) IgM抗体 (1: 1000; BioLegend, San Diego, CA, USA)、アイソタイプコントロールとしてヤギ抗ラットFITCμ鎖IgM抗体 (1:1000; Miltenyi Biotec, Bergisch Gladbach, Germany) を反応させた。2次抗体としてfluorescein isothiocyanate (FITC) 標識抗ラットIgM抗体 (1:100; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA)、3次抗体として抗FITC micro beads (1:50; Miltenyi) を反応させ、autoMACS(登録商標) Pro Separator (Miltenyi) を用いてSSEA-3陽性細胞を分離した。細胞分離後、BD FACS Aria (BD Biosciences, Franklin Lakes, USA) (FACS; fluorescence activated cell sorting) を用いて、Magnetic cell sorting (MACS)で濃縮した細胞中のSSEA-3陽性細胞の割合を解析した。SSEA-3陽性細胞が70%以上含まれているものをMuse細胞と定義し、移植細胞として用いた。
一部のMSCには過去の文献(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.)を参考にレンチウィルスを用いてgreen fluorescent protein (GFP) またはNano-lantern/pcDNA3を導入し、1次抗体としてラット抗SSEA-3 IgM抗体 (1:1000; BioLegend)、2次抗体としてallophycocyanin (APC) 標識抗ラットIgM抗体 (1:100; Jackson ImmunoResearch) を反応させ、FACSを用いてSSEA-3陽性細胞とSSEA-3陰性細胞を分離した。MACSにより分離したMuse細胞を模して、SSEA-3陽性細胞の割合が70%となるように分離した細胞を混和した。
予備実験として細胞移植時期を検討する目的で、心筋炎モデル作成2,3,4,6,8週間後にmagnetic resonance imaging (MRI) (1T ICON, Bruker, Billerica, MA, USA) による心機能評価を行い、心機能の推移を比較検討した。
また、至適移植細胞数を検討する目的で、100,000 Muse細胞投与群、200,000 Muse細胞投与群、400,000 Muse細胞投与群 (各群n=5) を設けた。至適細胞移植時期に細胞移植を行い、MRIにより心機能を比較した。
心筋炎作成3週間後に以下の如く無作為に群分けを行い、細胞移植を行った:心筋炎モデルに生理食塩水 (NS; normal saline) を投与するvehicle群 (Vehicle群)、MSC 200,000細胞投与群 (MSC群)、Muse 200,000細胞投与群 (Muse群)、心筋炎を誘導しないsham群 (S群)。また、複数回投与による治療効果を検討する目的で、心筋炎作成3週間後と4週間後にそれぞれ200,000 Muse細胞を投与する群 (Muse'群) を、Muse細胞投与群と同等のSSEA-3陽性細胞を含むMSCの治療効果を検討する目的で、3,000,000 MSC投与群 (MSC’群)を設けた。いずれの移植細胞も1 ml生理食塩水に懸濁し、経尾静脈的に投与した。心機能測定と組織学的な線維化の評価実験ではMACSで濃縮した細胞を投与した。免疫組織化学実験ではFACSで分離したGFP-Muse細胞を、生体内分布の評価実験ではnano-lantern-Muse細胞を移植した。いずれの細胞投与群においても免疫抑制剤は投与しなかった。
Week 2とweek 8に、1.5%イソフルラン吸入麻酔下にMRI (1T ICON, Bruker, Billerica, MA, US )による撮像を行った。心尖部から左室流出路までの左室短軸像を以下の撮像パラメータで行った: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。得られた撮像データについて以下の項目の測定を行った:左室拡張末期容積(EDV; end-diastolic volume)、左室収縮末期容積 (ESV; end-systolic volume)、駆出率 (EF; ejection fraction)。Week 2は各群n=9, week 8は各群n=6で評価した。
Week 2の血漿中の脳性ナトリウム利尿ペプチド (BNP; brain natriuremic peptide) をenzyme-linked immunosolvent assay (ELISA) 法を用いて測定した (BNP-32 Rat RIA Kit, Peninsula Laboratories, San Carlos, CA, USA)。
Day 3, week 2, week 8でイソフルラン過量投与によりラットを犠牲死させたのち、4%パラホルムアルデヒド (PFA; paraformaldehyde) 灌流固定を行った。心臓を摘出し、左室短軸方向に3mm厚に4分割したのち、パラフィンまたは凍結組織包埋材 (O.C.T. Compound; Sakura Finetek, Tokyo, Japan) に包埋した。その後、パラフィン切片は3 μm、凍結切片は6 μmで薄切切片を作成した。乳頭筋レベルの切片を用いて以下の組織学的評価を行った。
心臓組織中の炎症の程度を確認する目的でパラフィン切片を用いてヘマトキシリン-エオジン(HE; Hematoxylin-Eosin) 染色を行った。スライドは光学顕微鏡(BX53; Olympus, Tokyo, Japan) で観察した。
移植細胞の分化能の評価目的に、week 2,week 8において蛍光免疫組織化学を行った。1次抗体としてウサギ抗GFP抗体 (1;500; Medical & Biological Laboratories Co., Ltd., Nagoya, Japan), ヤギ抗ANP抗体 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA), ヤギ抗CD31抗体 (Santa Cruz Biotechnology), マウス抗sarcomericα-actinin抗体 (1:100; Sigma-Aldrich), マウス抗troponin-I抗体 (1:50; Merck, Billerica, MA, USA), マウス抗connexin 43抗体 (1:50; Abcam, Cambridge, UK), マウス抗α-SMA抗体 (1:500; Thermo Fisher Scientific, Waltham, MA, USA) を、2次抗体としてAlexa 488標識ロバ抗ウサギ抗体 (1:500; Jackson ImmunoResearch), Alexa 594標識ロバ抗ヤギ抗体 (1:500; Jackson ImmunoResearch), Alexa 594標識ロバ抗マウス抗体 (1:500; Jackson ImmunoResearch) を用い、Nikon A1 共焦点レーザー顕微鏡 (Nikon, Tokyo, Japan) で観察した。ANP・GFP2重陽性細胞/GFP陽性細胞およびtroponin-I・GFP2重陽性細胞/GFP陽性細胞を算出した (n=3)。
障害組織におけるアポトーシスの評価目的に、day 3においてTdT-mediated dUTP nick end labeling (TUNEL) 染色を行った (In Situ Cell Death Detection Kit, TMR Red; Roche Diagnostics Deutschland GmbH, Mannheim, Germany )。無作為に1 mm2の対象領域 (ROI; regions of interest) を20カ所観察し、単位面積当たりのTUNEL陽性細胞数を算出した (各群n=3)。
線維化の評価としてweek 8においてMasson-Trichrome染色を行った。スライドは光学顕微鏡(Olympus) で観察、撮影した。Adobe Photoshop (Adobe Inc., San Jose, CA, USA)を用いて心臓線維面積/心臓断面積を算出した (各群n=6)。
Muse細胞を損傷部位に誘導する遊走因子であるスフィンゴシン-1-リン酸 (S1P: Sphingosine-1-phosphate)(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.) の測定を行った。Day 0においてイソフルラン過量投与によりラットを犠牲死して心臓を摘出し、左室をバイオマッシャーによりホモジナイズした。ホモジネートバッファー (500 mM Tris-HCl/ 150 mM NaCl/ 1 mM EDTA/ 1% Triton X-100, 0.5% sodium deocyl sulfate/ 0.5% sodium deoxycholate) にプロテアーゼ阻害剤 (cOmplete, Merck) およびホスファターゼ阻害剤 (PhosSTOP(商標), Sigma-Aldrich) を製品プロトコールに従って添加したものを用いた。4℃, 15,000 rpm, 15分間の遠心分離後、上清中のS1P濃度を東レリサーチセンター (Kamakura, Japan) にて液体クロマトグラフィー質量分析法 (API 4000, AB/MDS SCIEX, Framingham, MA, USA) により測定した。S1P濃度は心臓組織1 g中の濃度として表記した。心筋炎非誘導群 (Control群; n=6)、心筋炎誘導群 (Myocarditis群; n=6) で行った。
移植細胞の生体内分布を評価する目的で、Nano-lanternを導入した細胞を用いた(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細胞投与、nano-lantern-MSC投与、非治療ラット(各群n=3)に対し、week 2にイソフルラン吸入による鎮静下でセレンテラジン 200 μg/ 1 ml NSを径尾静脈的に投与した。イソフルラン過量投与によりラットを犠牲死して心臓、肺、脳、肝臓、膵臓、脾臓、腎臓、胃、小腸、大腸、大腿骨、脛骨、腓骨、大腿四頭筋を摘出した。心臓を3 mm厚に、その他の臓器を1 cm厚以下にスライスしてセレンテラジン50 μg/ml溶液に浸した。IVIS Lumina LT (Perkin Elmer, Waltham, MA, USA) で撮影し、Living Image Software (Perkin Elmer) で光量の数値化を行った。
Week 2にイソフルラン吸入による鎮静下でdextran, Texas Red(商標) (Thermo-Fisher Scientific) 2 mgを径尾静脈的に投与した。ラットをイソフルラン過量投与により安楽死させ、心臓を摘出した。組織透明化技術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.) に従って組織の透明化を行った。十分な透明化が得られたのち、Multiphotonレーザー顕微鏡 (Nikon) にて観察した。心外膜側から内膜側方向へ0.85μm毎に0.78×0.78μmの軸位断静止画像を118カ所撮影した。各サンプルの左室の前壁、側壁、後壁において、心基部、心尖部およびその中間から無作為に1カ所ずつ、合計9カ所撮影し、Image J software (National Institutes of Health, Bethesda, MD, USA) により血管内腔容積を算出した。各群n=3で評価した。
得られたデータは平均±標準偏差で表示した。統計学的解析にはJMP Pro 14 software (SAS Institute, Cary, NC, USA) を用いた。組織中S1P値およびIVISデータのシグナル強度における2群間の連続変数の比較は,Shapiro-Wilk検定によりいずれも正規分布であることが証明され,Student’s t-testを行った。その他の多群間比較にはOne-way ANOVAおよびTukey-Kramer’s post hoc testを行った。有意水準はp<0.05とした。
<Muse細胞濃縮後の細胞集団中のSSEA-3陽性率>
アイソタイプコントロールを反応させたMSCsでSSEA-3陽性細胞が検出されないようフローサイトメトリーのゲートを設定した。本研究で用いたMSCのSSEA-3陽性率は5.1±1.3%で、MACSによる細胞分離後のSSEA-3陽性率は75.0±4.8%であった(図1)。
至適細胞投与時期を検討するために、心筋炎誘導後2週、3週、4週、6週、8週における炎症の程度を組織学的に評価し、EFの推移をMRIにて比較した。HE染色では2週目には単核球浸潤は認めるものの、その他の炎症所見は認めなかった。3週目には著明な顆粒球および単核球浸潤を認め、心筋細胞の変性や間質の浮腫を伴っていた(図2のA)。6週目には顆粒球および単核球浸潤は減少し、MT染色で線維化の出現を認めた。いずれの病変も心外膜側にほぼ全周性に認めた。EFは3週まで経時的に低下傾向を示し、3週以降はプラトーとなった(図2のB)。本研究では臨床に即した検証を目指すため、心機能および組織学的な重症度がピークを迎える心筋炎誘導3週後に細胞移植を行うこととした。
続いて、至適移植細胞数の検討を行った。心筋炎誘導3週後に100,000 Muse細胞投与群、200,000 Muse細胞投与群、400,000 Muse細胞投与群を設け、細胞移植2週後にEFを測定した(図3)。EFは200,000 Muse細胞投与群で74.0±2.5%で、100,000 Muse細胞投与群 (69.5±2.4%, p<0.05) に比べて統計学的に有意に高値を示したが、400,000 Muse細胞群 (72.7±1.3%) に比べて統計学的有意差は認めなかった。以上より、移植細胞数を200,000細胞に設定した。
Muse細胞の遊走因子であるS1P濃度をDay 0の心臓組織ホモジネートサンプルを用いて測定した (図4)。S1P濃度はcontrol群24.9±2.9 ng/g、myocarditis群32.5±4.9 ng/gでありmyocarditis群で統計学的に有意に高値であった (p<0.05)。
Week 2における投与された細胞の生体内分布をIVISにて評価した(図5のA)。IVISにより得られた各臓器の光量を示す (図5のB)。Muse群ではMSC群に比べより多くの細胞集積を認めた(p<0.05)。また、細胞は主に心外膜側周辺に集積しており、本疾患モデルで引き起こされる炎症部位に一致していた。MSC群ではわずかに肺の集積を認めたが、Muse群では肺への集積は認めなかった。また、いずれの群においてもその他の臓器への集積は認めなかった。
Week 2およびweek 8における心機能を示す(図6のA-F)。Week 2においてMuse群でEFは73.1±1.9%で、Vehicle群 (60.8±2.5%, p<0.001), MSC群 (66.7±2.4%, p<0.001), MSC’群 (68.3±2.3%, p<0.01) に比べて統計学的に有意に高値を示した。Muse群とMuse'群 (73.2±2.0%) との間に有意差は認めなかった。Muse群でESVは0.11±0.01 mlでVehicle群(0.15±0.01 ml, p<0.001) に比べて統計学的に有意に低値を示した。Muse群とMSC群 (0.12±0.02 ml) およびMSC’群 (0.12±0.01 ml) との間に有意差はなかったものの、Muse群で低い傾向を認めた。EDVは各細胞移植群間で有意差は認めなかった。
Week 8においてMuse群でEFは68.8±4.5%で、Vehicle群 (59.5±3.9%, p<0.001) に比べて統計学的に有意に高値を示した。Muse群とMuse'群 (70.1±3.4%) の間に有意差は認めなかった。EDVおよびESVは各細胞移植群間で有意差は認めなかった。
Week 2における血漿中BNP値を示す(図6のG)。Muse群のBNP値は347.2±188.6 pg/mlであり、Vehicle群 (980±241.1 pg/ml, p<0.001)、MSC群 (802.2±171.1 pg/ml, p<0.01)、MSC’群 (770±316.3 pg/ml, p<0.01) に比べて統計学的に有意に低値を示した。Muse群とMuse'群(395.2±283.3 pg/ml) の間に統計学的有意差は認めなかった。
移植したGFP-Muse細胞のweek 2における心筋細胞および血管構成細胞への分化能を組織学的に観察した。GFP陽性細胞の中で未熟心筋マーカーであるANP、成熟心筋マーカーであるsarcomeric α-actinin,troponin-I,connexin 43,血管構成細胞であるαSMA,CD31を発現する細胞を認めた(図7のA-F)。GFP陽性細胞に占めるANP+GFP+の2重陽性細胞の割合は41.3±1.8%であり、troponin-I+GFP+の2重陽性細胞の割合は16.2±3.1%であった。
移植細胞の抗線維化/線維分解作用を評価する目的でweek 8の心臓組織切片をMasson-Trichrome染色にて観察した。図8のAに各群の代表的画像を示す。各群のpercent fibrotic areaを心臓線維面積/心臓断面積×100で算出した(図8のB)。Muse群 6.0±2.0%, Muse'群 4.8±3.4%でいずれもVehicle群 (12.1±4.5%) と比べて統計学的に有意に低値であった。Muse群とMSC群(8.6±2.6%) およびMSC’群 (8.5±4.5%) の間には有意差は認めないものの、Muse群で低い傾向を認めた。
細胞移植による抗アポトーシス効果を評価する目的で、day 3の心臓組織標本を用いて各群間のTUNEL陽性細胞数を比較した。図9のAに各群の代表的画像を示す。Muse群でTUNEL陽性細胞数は1.8±0.4細胞/mm2で、Vehicle群 (7±1.6細胞/mm2) に比べて統計学的に有意に少ない値を示した (p=0.001)(図9のB)。
細胞移植による血管新生を評価する目的で、透明化した心臓組織中の血管容積を計測した。図10のAに多光子顕微鏡で撮影した各群の断層画像を示す。心臓組織に占める血管容積の割合はMuse群で7.4±1.2%で、Vehicle群 (3.2±0.5%) に比べて統計学的に有意に高い値を示した (p=0.047) (図10のB)。
Claims (9)
- 生体の間葉系組織または培養間葉系細胞に由来するSSEA-3陽性の多能性幹細胞を含む、心筋炎を治療するための細胞製剤。
- 前記多能性幹細胞が濃縮された細胞画分を含む、請求項1に記載の細胞製剤。
- 前記多能性幹細胞が、CD105陽性である、請求項1または2に記載の細胞製剤。
- 前記多能性幹細胞が、CD117陰性およびCD146陰性である、請求項1~3のいずれか1項に記載の細胞製剤。
- 前記多能性幹細胞が、CD117陰性、CD146陰性、NG2陰性、CD34陰性、vWF陰性およびCD271陰性である、請求項1~4のいずれか1項に記載の細胞製剤。
- 前記多能性幹細胞が、CD34陰性、CD117陰性、CD146陰性、CD271陰性、NG2陰性、vWF陰性、Sox10陰性、Snai1陰性、Slug陰性、Tyrp1陰性およびDct陰性である、請求項1~5のいずれか1項に記載の細胞製剤。
- 前記多能性幹細胞が、以下の性質の全てを有する多能性幹細胞である、請求項1~6のいずれか1項に記載の細胞製剤:
(i)テロメラーゼ活性が低いかまたは無い;
(ii)三胚葉のいずれの胚葉の細胞に分化する能力を持つ;
(iii)腫瘍性増殖を示さない;および
(iv)セルフリニューアル能を持つ。 - 前記多能性幹細胞が、以下の性質の全てを有する多能性幹細胞である、請求項7に記載の細胞製剤:
(i)SSEA-3陽性;
(ii)CD105陽性;
(iii)テロメラーゼ活性が低いかまたは無い;
(iv)三胚葉のいずれかの胚葉に分化する能力を持つ;
(v)腫瘍性増殖を示さない;および
(vi)セルフリニューアル能を持つ。 - 前記心筋炎が、リンパ球性心筋炎、巨細胞性心筋炎、好酸球性心筋炎および肉芽腫性心筋炎から選ばれる1種または2種以上である、請求項1~8のいずれか1項に記載の細胞製剤。
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- 2020-09-04 WO PCT/JP2020/033577 patent/WO2021045190A1/ja unknown
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US20220323509A1 (en) | 2022-10-13 |
JPWO2021045190A1 (ja) | 2021-03-11 |
CA3153237A1 (en) | 2021-03-11 |
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