Composition for treating and / or preventing mitochondrial disease including high-purity mesenchymal stem cells

Abstract

In one embodiment, the present invention relates to a composition for treating and / or preventing mitochondrial disease. In one embodiment, the present invention relates to an agent for ameliorating mitochondrial dysfunction.

Description

Composition for treating and / or preventing mitochondrial disease, comprising highly purified mesenchymal stem cells

[0001] The present invention relates to a composition for treating and / or preventing mitochondrial diseases, which comprises highly purified mesenchymal stem cells.

[0002] Mitochondrial dysfunction, which results in clinical symptoms, is collectively known as mitochondrial disease. Because mitochondria are present in cells throughout the body and have the function of producing energy, impaired mitochondrial function can lead to pathological conditions such as energy metabolism disorders in various organs.

[0003] The causes of mitochondrial diseases include mutations in genes on nuclear DNA and abnormalities in mitochondrial DNA (mtDNA). Nearly 200 gene mutations have been identified in nuclear DNA, and more than 100 pathogenic point mutations have been identified in mtDNA.

[0004] Regarding mitochondrial diseases, for example, Patent Document 1 describes a pharmaceutical composition for treating or preventing mitochondrial diseases containing a specific compound, and Patent Document 2 describes a pharmaceutical composition for use in mitochondrial diseases, etc., containing human stem cells enriched with functional mitochondria. However, at present, there are no therapeutic drugs other than taurine that have been rigorously confirmed to be effective in clinical trials in Japan. Furthermore, the approved use of taurine is limited to "suppression of stroke-like episodes in mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome," and there are currently no drugs effective for mitochondrial diseases.

[0005] Patent Application No. 2022-125211 Patent Application No. 2021-502844

[0006] Elad Jacoby et al., npj Regenerative Medicine volume 6, Article number: 58 (2021)

[0007] In one embodiment, an objective of the present invention is to provide a composition for treating and / or preventing mitochondrial disease. In one embodiment, an objective of the present invention is to provide an agent for improving mitochondrial dysfunction.

[0008] The present inventors have found that cell populations of fast-proliferating mesenchymal stem cell clones that are LNGFR (CD271) positive or LNGFR (CD271) and Thy-1 (CD90) co-positive, or their progeny, may be effective in treating and / or preventing mitochondrial diseases.

[0009] The present invention encompasses the following embodiments: (1) A composition for treating and / or preventing mitochondrial disease, comprising a cell population of a fast-proliferating mesenchymal stem cell clone that is positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), or its progeny. (2) The composition according to (1), wherein the mitochondrial disease is MELAS (Mitochondrial myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like episodes). (3) The composition according to (1) or (2), wherein the coefficient of variation of forward scattered light in flow cytometry of the cell population or its progeny is 40% or less. (4) The composition according to any of (1) to (3), wherein the cell population is derived from human bone marrow. (5) The composition according to any one of (1) to (4), wherein the cell population is obtained by a method comprising the steps of: (a) obtaining a population of mesenchymal stem cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), (b) single-cell culturing of clones of the mesenchymal stem cells, and (c) recovering and expanding clones that proliferate rapidly. (6) The composition according to (5), wherein the method further comprises the steps of: (d) measuring the coefficient of variation of forward scattered light by flow cytometry, and (e) recovering a population having a coefficient of variation of forward scattered light by flow cytometry of 40% or less. (7) The composition according to any one of (1) to (6), wherein the composition is administered to a subject by intravenous administration. (8) An agent for improving mitochondrial dysfunction, comprising a cell population of a fast-proliferating mesenchymal stem cell clone that is positive for LNGFR (CD271) or that is co-positive for LNGFR (CD271) and Thy-1 (CD90), or their progeny. (9) A method for treating and / or preventing mitochondrial disease, comprising administering to a subject a cell population of a fast-proliferating mesenchymal stem cell clone that is positive for LNGFR (CD271) or that is co-positive for LNGFR (CD271) and Thy-1 (CD90), or their progeny.(10) The method according to (9), wherein the mitochondrial disease is MELAS (Mitochondrial myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like episodes). (11) The method according to (9) or (10), wherein the coefficient of variation of forward scattered light in flow cytometry of the cell population or its progeny is 40% or less. (12) The method according to any of (9) to (11), wherein the cell population is derived from human bone marrow. (13) The method according to any of (9) to (12), wherein the cell population is obtained by a method comprising the steps of: (a) obtaining a population of mesenchymal stem cells that are positive for LNGFR (CD271) or that are co-positive for LNGFR (CD271) and Thy-1 (CD90); (b) culturing clones of the mesenchymal stem cells as single cells; and (c) recovering and expanding clones that proliferate rapidly. (14) The method according to (13), wherein the method for obtaining the cell population further comprises the steps of (d) measuring the coefficient of variation of forward scattered light by flow cytometry, and (e) recovering a population having a coefficient of variation of forward scattered light by flow cytometry of 40% or less. (15) A method for improving mitochondrial dysfunction, comprising administering to a subject a cell population of a fast-proliferating mesenchymal stem cell clone or a progeny thereof that is positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90).

[0010] The present invention can provide a composition for treating and / or preventing mitochondrial diseases, and can also provide an agent for improving mitochondrial dysfunction.

[0011] Figure 1 shows an overview of the method for establishing iPS cells from patients with mitochondrial disease and differentiating iPS cells into neurons. Figure 2 shows the results of measuring gene expression of OCT-4, TRA1-50, and NANOG in iPS cells derived from MELAS patients. Figure 3 shows the results of measuring gene expression and gene mutations of NeuO in neurons derived from MELAS patients. Figure 4 shows the mitochondrial transfer ability of REC and MEC to MELAS-N cells. Figure 5 shows the effect of REC and MEC on reducing mitochondrial reactive oxygen species in MELAS-N cells. Figure 6 shows the effect of REC and MEC on restoring respiratory function in MELAS-N cells. Figure 7 shows the effect of REC and MEC on reducing GDF-15 in MELAS-N cells. Figure 8 shows the mitochondrial content and properties in REC and MSCs. A shows mitochondrial quantification, B shows swollen mitochondria quantification, and C shows cellular respiratory activity. FIG. 9 shows the results of measuring the amount of REC-derived mitochondria transferred to various mouse tissues one week after intravenous administration of REC to mitochondrial pathology model mice.

[0012] In one embodiment, the present invention relates to a composition for treating and / or preventing a mitochondrial disease, comprising a cell population of a fast-proliferating mesenchymal stem cell clone that is LNGFR (CD271) positive or co-positive for LNGFR (CD271) and Thy-1 (CD90), or its progeny.

[0013] Method for Obtaining Mesenchymal Stem Cells In the present invention, mesenchymal stem cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90) can be obtained, for example, according to the methods described in WO2009 / 31678 or WO2021 / 144995. The method for obtaining such mesenchymal stem cells is outlined below.

[0014] First, mesenchymal stem cells are highly enriched by selecting a cell fraction that is LNGFR (CD271) positive (CD271+) or CD271 and CD90 (Thy-1) co-positive (CD271+CD90+) from a cell population containing human mesenchymal stem cells. If the cell population containing human mesenchymal stem cells also contains hematopoietic cells, a step of selecting CD45 and CD235a co-negative (CD45-CD235a-) cells may be added to select non-hematopoietic cells.

[0015] Materials for obtaining a cell population containing human mesenchymal stem cells are not particularly limited, and examples include bone marrow and peripheral blood (including peripheral blood after administration of G-CSF). Examples of bone marrow that can be used include bone marrow from the spine, sternum, ilium, and the like. For example, mononuclear cells prepared from human bone marrow or adipose tissue / placental chorion can be used. Cell populations can be pre-selected from these materials by flow cytometry or affinity chromatography using known mesenchymal stem cell markers (e.g., CD105 (SH2), CD73 (SH3 / 4), CD71, CD44, etc.).

[0016] In one embodiment, the human mesenchymal stem cells are obtained or derived from a patient for whom a mitochondrial disease is to be treated and / or prevented, hi another embodiment, the human mesenchymal stem cells are obtained or derived from a donor different from the patient for whom a mitochondrial disease is to be treated and / or prevented.

[0017] During cell preparation, if the material is in the form of a cell mass containing mesenchymal stem cells, the material can be subjected to physical treatment such as pipetting or enzymatic treatment with trypsin, collagenase, etc., as necessary. Furthermore, if the material contains red blood cells, it is preferable to hemolyze the red blood cells beforehand. Using the cell population prepared as described above, CD271+ cells or CD271+CD90+ cells are selected.

[0018] Methods for selecting CD271+ cells or CD271+CD90+ cells include, for example, methods using antibodies. The antibodies are anti-CD271 antibodies and / or anti-CD90 antibodies that can select CD271+ cells or CD271+CD90+ cells. When flow cytometry is used for selection, live cells can be selected in a short period of time by using anti-CD271 antibodies labeled with different fluorescent dyes such as FITC, PE, or APC, or by using an appropriate combination of anti-CD271 and anti-CD90 antibodies. In addition to flow cytometry, CD271+CD90+ cells can also be selected by methods using magnetic beads or affinity chromatography.

[0019] Before using these methods, dead cells may be removed by reacting the cell population with a fluorescent dye (e.g., PI) that stains dead cells and then removing the fluorescently stained cells.

[0020] REC cell enrichment Next, the selected LNGFR-positive cells or LNGFR and Thy1 co-positive cells are cultured as single cells (clones), and by selecting the lot with the fastest proliferation rate, highly purified human mesenchymal stem cells (REC: Rapidly Expanding Clone) with excellent proliferation, differentiation, and migration capabilities are obtained.

[0021] First, LNGFR-positive cells or LNGFR-positive and Thy1-positive cells are clonally sorted into a 96-well culture plate using flow cytometry (cell sorter). That is, one cell is seeded per well. After two weeks of single-cell culture, the culture plate is photographed under a microscope, and wells that have reached confluence or semi-confluence are selected. The cells contained in these wells are designated as RECs.

[0022] Here, "fast proliferation" and "high-speed proliferation" mean that when cells are seeded one cell per well of a 96-well culture plate and cultured, the culture plate becomes confluent or semi-confluent two weeks or earlier after the start of culture (for example, a doubling time of 26±1 hours).

[0023] Confluence refers to a state in which cultured cells cover, for example, 90% or more of the surface of the culture vessel (culture surface). Semi-confluence refers to a state in which cultured cells cover, for example, 70-90% of the surface of the culture vessel (culture surface). The size and type of culture equipment used can be adjusted appropriately depending on the cell growth rate. Moderately / slowly expanding cells, i.e., cells that do not reach semi-confluence or confluence after two weeks of single cell culture, are discarded. RECs collected from each well selected as RECs are transferred to culture flasks individually and further cultured until confluence is reached (expansion culture). The expanded cells are then collected separately. RECs from one well constitute one lot, and these may be used for the selection described below.

[0024] Here, the cells of the present invention are obtained by clonal sorting in which one cell is seeded per well, and therefore the genetic traits of the expanded cells are all the same. Therefore, in the present invention, the entire cell population may be referred to as a "clone," or each individual cell constituting the cell population may be referred to as a "clone."

[0025] In the present invention, RECs to be used for selection can be evaluated in advance using a REC marker (anti-Ror2). For example, after the expansion culture, adherent and proliferated cells are collected from all the lots, and a portion (1 to 3 × 10) of each lot is collected. 5 A small number of cells (approximately 1000 cells) are selected and single-stained with an anti-Ror2 monoclonal antibody. A technique for single-staining with an anti-Ror2 monoclonal antibody is known (WO2016 / 17795). In summary, the percentage of REC marker-positive cells among the collected cells is determined by flow cytometry analysis using the REC marker. The percentage can be determined by quantitative PCR to quantify Ror2 mRNA expression, or by manually determining the percentage using a microscope. Lots (cell populations) with a positive percentage above a certain value (e.g., 65%) are considered acceptable and can be used for the selection process described below.

[0026] Progeny of the cell populations described herein can also be used in the present invention. Progeny can be generated by subculturing the cell populations. Progeny can be LNGFR (CD271) positive (CD271+) or CD271 and CD90 (Thy-1) co-positive (CD271+CD90+), but can lose expression of these markers during the culture process. Cells lacking expression of these markers can be selected for differentiation potential, proliferation potential, cell size, and / or uniformity thereof.

[0027] Selection of stem cell populations of the present invention The cell populations described herein or the cell populations themselves may be further selected using the coefficient of variation (CV value) of forward scattered light in flow cytometry and / or the average cell size as indicators.

[0028] Forward scatter is light scattered at a small angle in the forward direction relative to the axis of the laser beam. Forward scatter consists of scattered, diffracted, and refracted light from the cell surface, and provides information about the size of the sample.

[0029] The coefficient of variation (CV) is the standard deviation divided by the mean, and is a value used to relatively evaluate the variation in data with different units, or the relationship between data and variation in relation to the mean.

[0030] In the present invention, a cell population having a CV value of 40% or less may be selected. A cell population having a CV value of 40% or less is a cell population composed of cells of uniform size. Preferably, the CV value is 35% or less, 30% or less, 25% or less, or 20% or less.

[0031] The average size of the cells in the cell population selected by the present invention may be 20 μm or less, for example, 18 μm or less, in the range of 14 μm to 18 μm.

[0032] The number of cell clones constituting the cell population selected as described above is not limited. For example, 0.8×10 7~1.2X10 7 The number of cells may be as small as 100.

[0033] A cell population with a small FSC CV value may have excellent proliferation and differentiation capabilities, and the use of such a cell population may be more effective in improving mitochondrial diseases.

[0034] The cell populations described herein or their progeny can be used as therapeutic mesenchymal stem cell populations for treating and / or preventing mitochondrial diseases. When administered to a subject, the cell populations described herein or their progeny can have at least one, e.g., two, three, or four of the following effects, compared to a control (not administered with the cell population or progeny): increasing the amount of mitochondria in the subject, reducing mitochondrial swelling, reducing reactive oxygen species in mitochondria, improving mitochondrial ATP production function, and improving mitochondrial respiratory function.

[0035] In one embodiment, the present invention relates to a pharmaceutical composition comprising the cell population described herein or its progeny. In the pharmaceutical compositions described herein, the concentration of the cell population or its progeny is not limited, but for example, if the pharmaceutical composition is a liquid, it can be 1x10 3 cell / ml or more, 1x10 4 cell / ml or more, or 1x10 7 It may be more than 1x10 cells / ml 9 cell / ml or less, 1x10 8 cell / ml or less, 1x10 7 It may be less than 1x10 cells / ml, for example 3 cell / ml to 1x10 9 cell / ml, 1x10 4 cell / ml to 1x10 8 cell / ml, 1x10 5 cell / ml to 1x10 7 cell / ml, or approximately 1 x 10 ６The concentration of the cell population may be 100 cells / ml. The pharmaceutical compositions described herein may contain one or more pharmaceutically acceptable carriers in addition to the cell population or its progeny. Examples of carriers include bulking agents, diluents, buffers, isotonicity agents, chelating agents, thickening agents, coloring agents, stabilizers, emulsifiers, dispersing agents, suspending agents, preservatives, etc. The dosage form of the pharmaceutical compositions described herein is not limited, and can be prepared, for example, into injections such as sterile solutions and suspensions, according to commonly used procedures.

[0036] In one embodiment, the present invention relates to a pharmaceutical composition for treating and / or preventing a mitochondrial disease, comprising a cell population described herein or a progeny thereof. In another embodiment, the present invention relates to a method for treating and / or preventing a mitochondrial disease, comprising administering to a subject a cell population described herein or a progeny thereof.

[0037] A "subject" to which the pharmaceutical compositions, cell populations, or progeny thereof described herein are administered means a human or non-human organism. Examples of non-human organisms include birds and non-human mammals (e.g., cows, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep, and horses).

[0038] As used herein, "treatment" includes all treatments related to mitochondrial disease, such as improvement of symptoms associated with mitochondrial disease, continuation of improved symptoms, suppression of recurrence, reduction of other treatments, etc. As used herein, "prevention" includes preventing the onset of symptoms associated with mitochondrial disease or reducing the risk thereof.

[0039] As used herein, mitochondrial disease refers to a pathological condition in which clinical symptoms appear due to impaired mitochondrial function. Examples of mitochondrial diseases include MELAS (Mitochondrial myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like episodes), Kearns-Sayre syndrome (KSS), CPEO (Chronic Progressive External Ophthalmoplegia Syndrome), MERRF (Myoclonus epilepsy associated with ragged-red fibers), Sensorineural hearing loss, Fatal, infantile encephalopathy; Leigh / Leigh-like syndrome, Leber disease (Leber's hereditary optic neuropathy [LHON]), Barth syndrome, Pearson's disease, and Friedreich's ataxia (FRDA), and may be, for example, MELAS.

[0040] In one embodiment, the present invention relates to an agent for improving mitochondrial dysfunction, comprising the cell population described herein or progeny thereof. In another embodiment, the present invention relates to a pharmaceutical composition for improving mitochondrial dysfunction, comprising the cell population described herein or progeny thereof. In yet another embodiment, the present invention relates to a method for improving mitochondrial dysfunction, comprising administering to a subject the cell population described herein or progeny thereof.

[0041] As used herein, mitochondrial dysfunction means at least one, for example, two, three, or four of the following: a decrease in mitochondrial mass, mitochondrial swelling, an increase in reactive oxygen species in mitochondria, a decrease in mitochondrial ATP production function, and a decrease in mitochondrial respiratory function.

[0042] The dosage, frequency, number of administrations, and route of administration of the cell population or progeny thereof, pharmaceutical composition, or agent for improving mitochondrial dysfunction described herein can be selected according to the age, sex, weight, symptoms, route of administration, frequency of administration, dosage form, and the like of the subject. An effective dose is an amount necessary for treating and / or preventing mitochondrial disease, or improving mitochondrial dysfunction. The dose of the cell population or progeny thereof, pharmaceutical composition, or agent for improving mitochondrial dysfunction described herein is not limited to, but may be, for example, 10 4 cells / kg or more, 10 5 cells / kg or more, or 10 6 cells / kg or more, 10 cells / kg or less, 10 9 cells / kg or less, or 10 8 cells / kg or less, for example, 10 4 cells / kg~10 10 cells / kg, 10 5 cells / kg~10 9 cells / kg, 10 6 cells / kg~10 8 cells / kg, or 10 7 The dosage may be, for example, once or more per day, once or more per 2 days, once or more per 3 days, or once or less per 2 months, once or less per month, or once or less per 2 weeks, for example, once a week. The number of administrations may be once or more, twice or more, three or more, or seven or less, six or less, five or less, for example, four times. The route of administration may be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous.

[0043] In one embodiment, the present invention relates to a method for producing a composition for treating and / or preventing a mitochondrial disease, comprising obtaining a cell population described herein or progeny thereof, and preparing a pharmaceutical composition described herein comprising said cell population or progeny thereof. In another embodiment, the present invention relates to a method for treating and / or preventing a mitochondrial disease, comprising administering to a subject a cell population described herein or progeny thereof.

[0044] Example 1 Cells Neurons differentiated from iPS cells established from three MELAS patients and one normal healthy individual (MELAS-N, Control-N) were used. iPS cells established from normal healthy individuals (201B7, HSP-0063) and iPS cells established from MELAS patients (HSP-2864, HSP-0458, and HSP-0461) were obtained from RIKEN CELL BANK (Japan).

[0045] An overview of the method for generating iPS cells from a patient with mitochondrial disease and differentiating them into neurons is shown in Figure 1. Specifically, iPS cells were generated by transfecting peripheral blood mononuclear cells (PBMCs) from a patient with mitochondrial disease with OCT3 / 4, SOX2, KLF4, L-MYC, LIN28, mp53DD, and EBNA1 using episomal vectors (K. Fujimori et al., Stem Cell Reports, 9 (2017) 1675-1691). Neuronal differentiation from iPS cells was performed according to the method described above. Specifically, cells were cultured in StemFit AK02N medium containing three inhibitors: 3 μM CHIR-99021 (REPROCELL, Japan), 3 μM SB-431542 (Fujifilm, Japan), and 3 μM dorsomorphin (Sigma, USA). After 5 days, the cells were dissociated using TrypLE Select and cultured in MHM medium (KBM Neural Stem Cell Kit, KOHJIN BIO, Japan) containing 2% B27-Minus vitamin A (Thermo Fisher Scientific, USA) and 20 ng / ml bFGF (Peprotech, Japan). Other details of the method were as described in K. Fujimori et al. (supra).

[0046] Frozen GMP-equivalent RECs were purchased from PuREC Co., Ltd. RECs were obtained from bone marrow mononuclear cells (BMMCs) by seeding LNGFR and Thy-1 co-positive cells as single cells in 96-well plates and then growing rapidly to confluence after 2 weeks. MSCs were purchased from Lonza.

[0047] Neuronal culture: Neurons differentiated from iPSCs established from three MELAS patients and one healthy individual (MELAS-N, Control-N) were cultured in an undifferentiated state in StemFit AK02N medium (Ajinomoto, Japan) without MEF feeder cells. Subsequently, iPSCs were dissociated into single cells using TrypLE select (Thermo Fisher Scientific, USA) and plated at 2 × 10 cells per well in a 6-well plate treated with iMatrix511 (Nippi, Japan). 4 The cells were replated at a density of 1000 μg / ml and cultured in StemFit AK02N containing Y27632 (Fujifilm, Japan) at 5% CO2 and 37°C. The day after subculture, the medium was changed to remove Y27632, and thereafter the medium was changed every 2–3 days.

[0048] Gene expression in iPS cells derived from MELAS patients and properties of neurons derived from MELAS patients. Gene expression of stem cell markers OCT-4, TRA1-50, and NANOG was examined in iPS cells derived from MELAS patients. The results are shown in Figure 2. Furthermore, gene expression and gene mutation of the neuron-specific marker NeuO were examined in neurons derived from MELAS patients. The results are shown in Figure 3.

[0049] Mitochondrial Transfer into MELAS-N Cells MELAS-N cells were incubated with 5 μL / mL of Vybrant TM MSCs and RECs were labeled with DiD Cell-Labeling Solution (Thermo Fisher Scientific, USA) for 20 minutes at 37°C. TM Labeling was performed using Green FM (Thermo Fisher Scientific, USA) for 20 minutes at 37°C. The labeled MELAS-N cells were co-cultured with MSCs or RECs in RPMI1640 growth medium (Wako, Japan) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS; HyClone) for 48 hours. The cells were then washed with phosphate-buffered saline (PBS) and incubated with TrypLE. TMCells were detached by incubating them in Express Enzyme (Thermo Fisher Scientific, USA) at 37°C for 5 minutes. Cell viability was assessed using 4% trypan blue exclusion and light microscopy and was consistently greater than 95%. Cells were harvested using CytoFLEX (BECKMAN COULTER, USA) and analyzed by FlowJoint. TM Data were analyzed using software version 10 (BD Biosciences, USA). Mitochondria-specific dye transfer from MSCs or RECs was measured by gating DiD events, excluding doublets (forward scatter width vs. height), and measuring fluorescence in the fluorescein channel.

[0050] The results are shown in Figure 4. As shown in Figure 4, RECs were significantly more effective than MSCs in transferring mitochondria to MELAS-N cells.

[0051] Reduction of reactive oxygen species MitoSOX TM A red fluorescent probe (Thermo Fisher Scientific, USA) was used to visualize mitochondrial superoxide production according to the manufacturer's protocol. Specifically, cells (1 × 10) grown on 24-well plates were cultured in 10% CO2-free medium. 5 ) were washed twice with PBS to remove the medium and 5 μM MitoSOX TM The cells were incubated with the working solution at 37°C for 10 minutes, then gently washed three times with warm buffer (Thermo Fisher Scientific, USA), and immediately imaged under a fluorescence microscope (BZ-X710, Keyence, Japan). TM To confirm the mitochondrial localization of SOX-Red, cells were labeled with 0.1 μmol / L MitoBright LT-Green (Dojindo, Japan) for 15 min at 37°C. The mean fluorescence intensities of MitoSOX-Red and MitoBright LT-Green were detected using a GloMax® Discover Microplate Reader (Promega, USA).

[0052] The results are shown in Figure 5. As shown in Figure 5, the level of reactive oxygen species was high in MELAS-N, but the level of reactive oxygen species was reduced by REC and MSC, and the degree of reduction was significantly greater with REC than with MSC.

[0053] Respiratory function recovery. Key parameters of mitochondrial function were measured by directly measuring the oxygen consumption rate (OCR) using a Seahorse XF HS mini (Agilent Technologies, USA). Specifically, cells from each group were seeded at a density of 15,000 cells per well in XF cell culture microplates (Agilent Technologies, USA) according to the manufacturer's protocol. After overnight culture, the medium was replaced with Seahorse XF RPMI medium (Agilent Technologies, USA) supplemented with 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate (pH 7.4) and transferred to a non-CO2 incubator for 60 min. Mitochondrial stress assays were performed by sequentially adding 1.5 μM / well oligomycin (Olig), 1 μM / well FCCP, and 0.5 μM / well rotenone / antimycin A (AA / Rot). Each plotted value of real-time assessment of mitochondrial respiration was expressed as a percentage of the basal OCR. Results were analyzed using XFe Wave software (Agilent Technologies, USA).

[0054] The results are shown in Figure 6. As shown in Figure 6, MELAS-N had a low oxygen consumption rate (OCR), but the OCR level was increased by REC and MSC, and the degree of increase was significantly greater with REC than with MSC.

[0055] Reduction of GDF-15 Cell culture supernatants were prepared according to the protocol, and GDF-15, a biomarker for mitochondrial disease, was measured using an enzyme-linked immunosorbent assay (ELISA) kit (Thermo Fisher Scientific, USA) according to the manufacturer's protocol. The optical density of the samples was measured at 450 nm using a GloMax® Discover Microplate Reader (Promega, USA). The results are shown in Figure 7. As shown in Figure 7, GDF-15 expression levels were high in MELAS-N, but the level of GDF-15 expression was reduced by co-culture with RECs and MSCs.

[0056] Comparison of RECs and normal MSCs. Mitochondrial quantification. Three RECs and three MSC clones were treated with 200 nM MitoTracker. TM The cells were labeled with MitoTracker Green FM (Thermo Fisher Scientific, USA) for 20 minutes at 37°C. TM The mean fluorescence intensity of Green FM was detected by flow cytometry (CytoFLEX, BECKMAN COULTER, USA), and the data were analyzed by FlowJoint. TM Quantification was performed using software version 10 (BD Biosciences, USA).

[0057] The results are shown in Figure 8. As shown in Figure 8A, the amount of mitochondria was greater in RECs than in MSCs.

[0058] Quantification of swollen mitochondria. Three REC and MSC clones each were grown in 35-mm dishes for 10–12 h. After prefixation in 2.5% glutaraldehyde solution (Wako, Japan), 2% PFA, and 0.1 M phosphate buffer (Wako, Japan) for electron microscopy at room temperature for 2 h, the cells were then washed three times with water. They were then washed with PBS and dehydrated for 8 min each in graded ethanol (50%, 70%, 90%, 95%, and 100%). Next, the cells were mounted in epoxy resin using propylene oxide to aid penetration and cured at 60°C for 24 h. Ultrathin sections were taken using a diamond knife, mounted on copper mesh grids, stained with uranyl acetate, and imaged using a Topcon EB-002B transmission electron microscope (Keyence, Japan). The resulting images were quantitatively analyzed for mitochondrial swelling using Image J software (Wayne Rasband, USA).

[0059] The results are shown in Figure 8. As shown in Figure 8B, there were many expanded cells in MELAS-N, but the expanded cells were reduced by REC and MSC, and the degree of reduction was significantly greater with REC than with MSC.

[0060] Mitochondrial respiratory function. Oxygen consumption rate (OCR) was tested using the Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, USA) and measured using a flux analyzer (Seahorse XF HS mini, Agilent Technologies, USA) according to the protocol. Specifically, cells were uniformly seeded (1.5 × 10) on an XF HSmini cell culture plate. 4 Cells were equilibrated in XF RPMI medium (Agilent Technologies, USA) supplemented with 10 mM glucose, 1 mM sodium pyruvate, and 2 mM l-glutamine and incubated in a non-CO2 atmosphere. 2 The cells were then transferred to an incubator and left for 1 hour. Coupled and uncoupled respiration were then measured using 1.5 μM oligomycin (Olig). Maximal respiration was determined using 1 μM FCCP. Finally, non-mitochondrial respiration was measured using a combination of 0.5 μM antimycin A and rotenone (AA / Rot). Each plot of real-time mitochondrial respiration was expressed as a percentage of basal OCR. Data sets were analyzed using Wave software (Agilent Technologies, USA).

[0061] The results are shown in Figure 8. As shown in Figure 8C, the respiratory function of MELAS-N was restored to the level of normal neurons by co-culture with REC and MSC, and the respiratory function recovery effect of REC was significantly superior to that of MSC.

[0062] Example 2: In vivo mitochondrial transfer test In a mitochondrial pathology model mouse (ND6 mitomycin) with reduced mitochondrial function due to a mutation in the ND6 gene, we examined whether REC-derived mitochondria could transfer to various organs in vivo and whether they could restore mitochondrial function. If efficacy is proven in this test, REC could be an effective treatment for patients with mitochondrial disease, for which no established treatment exists.

[0063] Animal treatment: Eight to nine-week-old ND6 Mito mice were divided into two groups (four mice each) and administered an injection of 2 × 10 REC into the tail vein under anesthesia (isoflurane) [pain category: B]. 6 The REC cells were suspended in 0.2 mL of a mixed solvent consisting of equal parts HSA-CP-1 and 0.5% HSA / RPMI and administered. The control group received 0.2 mL of the solvent alone. One and two weeks after administration, mice from each group were euthanized by cervical dislocation (pain category: B), and tissues were removed. The removed tissues and mice were then disposed of appropriately. Because the ND6 mutation has been confirmed to be 100% transmitted to offspring through maternal inheritance, all mice obtained in this study were used for analysis.

[0064] One week after REC administration (4 mice in the treatment group, 4 mice in the control group) and two weeks after (4 mice in the treatment group, 4 mice in the control group), tissues from the target organs (lungs, kidneys, liver, soleus muscle, heart, and brain) were excised and subjected to genomic analysis (qPCR; copy number analysis) to measure the copy number of the ND2 gene derived from mouse mitochondrial DNA (Ms-mtDNA) and human mitochondrial DNA (H-mtDNA, derived from REC) and verify the transfer efficiency of human mtDNA (mitochondria derived from REC). The results after 1 week of REC administration are shown in Figure 9.

[0065] Mitochondrial respiratory function Mitochondria were isolated from tissues one week after REC administration (four mice in the treatment group, four mice in the control group) and two weeks after REC administration (four mice in the treatment group, four mice in the control group). Mitochondrial respiratory function was measured using a flux analyzer in the same manner as in Example 1, and the enzyme activity of the respiratory chain complexes was measured.

Claims

1. A composition for treating and / or preventing mitochondrial disease, comprising a cell population of a fast-proliferating mesenchymal stem cell clone that is LNGFR (CD271) positive, or LNGFR (CD271) and Thy-1 (CD90) co-positive, or its progeny.

2. The composition described in claim 1, wherein the mitochondrial disease is MELAS (Mitochondrial myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like episodes).

3. The composition described in claim 1 or 2, wherein the coefficient of variation of forward scatter in flow cytometry of the cell population or its progeny is 40% or less.

4. The composition of claim 1, wherein the cell population is derived from human bone marrow.

5. The composition according to claim 1, wherein the cell population is obtained by a method comprising the steps of: (a) obtaining a population of mesenchymal stem cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90); (b) culturing clones of the mesenchymal stem cells as single cells; and (c) recovering and expanding clones that proliferate rapidly.

6. The composition of claim 5, wherein the method further comprises the steps of: (d) measuring the coefficient of variation of forward scatter in flow cytometry; and (e) recovering a population having a coefficient of variation of forward scatter in flow cytometry of 40% or less.

7. The composition of claim 1, which is administered to a subject by intravenous administration.

8. An agent for improving mitochondrial dysfunction, comprising a cell population of a fast-proliferating mesenchymal stem cell clone that is LNGFR (CD271) positive, or LNGFR (CD271) and Thy-1 (CD90) co-positive, or its progeny.

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