Pluripotent stem cells effective in treating motor neuron disease (MND)

Abstract

Amyotrophic lateral sclerosis (ALS) is a life-threatening neurodegenerative disease characterized by the progressive loss of motor neurons. Muse cells are endogenous reparative pluripotent-like stem cells distributed to various tissues. Once intravenously injected, such cells, upon sensing sphingosine-1-phosphate produced by damaged cells, settle selectively on damaged sites, and after homing, can exhibit multiphase effects including natural differentiation into tissue-protecting and tissue-reconstructing cells. In the present invention, with G93A-Tg mice serving as an ALS model, human Muse cells injected intravenously successfully homed into the lumbar cord, mainly at the pia mater and the lower white matter, and expressed glial-like forms and GFAP. Meanwhile, no such homing or differentiation was observed in the case of human mesenchymal stem cells (MSC), and, rather, MSCs were found distributed to the lungs. The Muse group demonstrated significant improvement in the scores of rotarod, hanging wire, and lower extremity muscle strength, recovered the motor neuron number, and significantly reduced denervation and muscle fiber atrophy of lower extremity muscles as compared to the medium group. Furthermore, the Muse cell effect on motor function of TDP-43 Tg mice serving as an ALS model was evaluated. The results shows that, compared to the medium group, the Muse group had a significantly improved hanging wire test score. These results indicate that Muse cells perform homing in a damaged-site-dependent manner, and protect the spinal cord in response to the death of motor neurons, and thus Muse cells are useful for treatment of ALS patients.

Description

[Technical Field]

[0001] The present invention relates to cell preparations for regenerative medicine. More specifically, the present invention relates to cell preparations comprising pluripotent stem cells that are effective for treating, preventing, alleviating, and / or delaying the onset of motor neuron disease (MND) in a subject. [Background technology]

[0002] Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive motor neuron loss. Approximately 10% of ALS patients have a genetic phenotype associated with mutations in Cu / Zn superoxide dismutase (SOD1) [Non-Patent Documents 1-3][1-3], TAR DNA-binding protein 43 (TDP-43) [Non-Patent Documents 4 and 5][4, 5], and a hexanucleotide repeat expansion in the C9orf72 gene [Non-Patent Documents 6 and 7][6, 7]. In addition to the oral drug riluzole, the free radical scavenger edaravone was recently approved as a new anti-ALS drug [Non-Patent Documents 8 and 9][8, 9]. However, the therapeutic efficacy of these treatments remains very limited, and new therapeutic strategies for ALS are needed.

[0003] Muse (multilineage-differentiating stress-enduring) cells are endogenous pluripotent-like stem cells that can be recovered as cells expressing stage-specific embryonic antigen (SSEA)-3, a surface marker for pluripotent stem cells. They are normally localized in bone marrow, peripheral blood, and connective tissues of organs and are non-tumorigenic [Non-Patent Documents 10-13][10-13]. Muse cells express the sphingosine-1-phosphate receptor (S1PR2), which recognizes damaged tissue and sphingosine-1-phosphate (S1P) produced by damaged or apoptotic cells. Therefore, upon intravenous injection, Muse cells selectively accumulate at the site of injury. After homing to the injury site, Muse cells spontaneously differentiate into damaged or apoptotic cells, replacing the damaged or apoptotic cells and contributing to tissue repair have been reported in animal models of stroke, acute myocardial infarction, epidermolysis bullosa, chronic kidney disease, and liver cirrhosis [Non-Patent Documents 14-18][14-18]. In addition to their tissue repair properties, Muse cells possess pleiotropic functions, including angiogenesis, immunomodulation, nutrition, antiapoptosis, and antifibrosis [Non-Patent Documents 18 and 19][18, 19]. Another important unique feature is that allogeneic Muse cells evade host immune rejection after intravenous administration and survive as differentiated cells within host tissues for more than six months without immunosuppressive treatment [Non-Patent Document 18]

[18] . This is partly explained by the expression of human leukocyte antigen (HLA)-G, a histocompatibility antigen that mediates immune tolerance, in the placenta [Non-Patent Document 18]

[18] . Based on these properties, intravenously administered allogeneic Muse cells have been approved by drug regulatory authorities and have been applied in clinical trials for acute myocardial infarction, stroke, spinal cord injury, epidermolysis bullosa, and neonatal cerebral palsy, although none of these have been HLA-matched or administered long-term immunosuppressively [Non-Patent Document 20]

[20] . Because Muse cells can target damaged tissue, the number of cells required for treatment is an order of magnitude lower than that of mesenchymal stem cells (MSCs)

[21] . Based on these properties, the inventors investigated the possible therapeutic potential of Muse cells in ALS animal models. [Prior art documents] [Non-patent literature]

[0004] [Non-licensed document 1] Aoki, M. et al. Nat Genet 5, 323-324, doi:10.1038 / ng1293-323 (1993). [Non-licensed document 2] Gurney, ME et al. Science 264, 1772-1775, doi:10.1126 / science.8209258 (1994). [Non-licensed document 3] Rosen, DR et al. Nature 362, 59-62, doi:10.1038 / 362059a0 (1993).

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

[0005] The present invention provides novel medical applications of pluripotent stem cells (e.g., Muse cells) in regenerative medicine. More specifically, the present invention provides cell preparations and / or pharmaceutical compositions containing Muse cells that are effective for treating, preventing, alleviating, and / or delaying the onset of motor neuron disease (MND) in a subject, as well as methods for treating subjects with the above-mentioned diseases using the same. [Means for solving the problem]

[0006] Using a mutant SOD1 (G93A) transgenic mouse model, the present inventors found that intravenously (systemically) administered Muse cells accumulated in the injured spinal cord and reduced lower limb muscle denervation and muscle fiber atrophy, making it possible to treat and prevent ALS. Furthermore, the present inventors found that injecting or administering Muse cells into an ALS mouse model overexpressing ubiquitinated proteins (e.g., TDP-43) locally accumulated Muse cells at the lesion site, differentiated into tissue-constituting cells at the lesion site, and repaired ALS. Based on these findings, the present invention was completed.

[0007] That is, the present invention is as follows. [1] A cell preparation for treating, preventing, alleviating and / or delaying the onset of motor neuron disease (MND) in a subject, comprising SSEA-3-positive pluripotent stem cells isolated from biological mesenchymal tissue or cultured mesenchymal cells. [2] The cell preparation described in [1] above, which contains a cell fraction enriched in SSEA-3-positive pluripotent stem cells due to an external stress stimulus. [3] The cell preparation according to [1] or [2] above, wherein the pluripotent stem cells are CD105 positive. [4] The cell preparation according to any one of [1] to [3] above, wherein the pluripotent stem cells are CD117-negative and CD146-negative. [5] The cell preparation according to any one of [1] to [4] above, wherein the pluripotent stem cells are CD117-negative, CD146-negative, NG2-negative, CD34-negative, vWF-negative, and CD271-negative. [6] The cell preparation according to any one of [1] to [5] above, wherein the pluripotent stem cells are CD34-negative, CD117-negative, CD146-negative, CD271-negative, NG2-negative, vWF-negative, Sox10-negative, Snail-negative, Slug-negative, Tyrp1-negative, and Dct-negative. [7] The cell preparation according to any one of [1] to [6] above, wherein the pluripotent stem cells are pluripotent stem cells having all of the following properties: (i) low or absent telomerase activity; (ii) have the ability to differentiate into cells of any of the three germ layers; (iii) does not exhibit neoplastic growth; and (iv) It has self-renewal ability. [8] The cell preparation according to any one of [1] to [7] above, wherein the MND is amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), spinal muscular atrophy (SMA), progressive muscular atrophy (PMA), or spinal-bulbar muscular atrophy (SBMA). [9] The cell preparation according to any one of [1] to [8] above, wherein the pluripotent stem cells have the ability to be engrafted in the spinal cord.

[0008] The present invention enables the repair of motor neuron disease (MND) in subjects by administering Muse cells directly to the site of injury or intravenously, etc., through a tissue regeneration mechanism in which Muse cells differentiate at the site of injury into cells that constitute normal tissue surrounding the site. [Brief explanation of the drawings]

[0009] [Figure 1] (a) Distribution of GFP-labeled Muse cells in the spinal cord 7 days after intravenous (iv) or intrathecal (it) injection. The dotted boxes in the panels indicate higher magnifications of each panel. Notably, only iv injection delivered a large number of GFP-labeled Muse cells to the pia mater and the lower white matter of both the cervical and lumbar spinal cord. (b) The number of GFP-labeled Muse cells was higher after iv injection than after it injection. (c) Distribution of nano-lantern-labeled human MSCs and Muse cells in the spinal cord, brain, muscle, lung, and leg bones 7 days after iv administration. Notably, only Muse cells were detected in the spinal cord. (d) Nano-lantern-labeled Muse cells were observed in the pulmonary vascular lumen (left panel, arrow) and bone marrow (right panel, arrow). Scale bars: (a) 100 μm, (a, box) 20 μm; 2 mm (c, spinal cord), 1 cm (c, others), and (d) 20 μm. [Figure 2](a-d) Clinical analysis of G93A Tg mice treated with vehicle (n = 10), MSCs (n = 9), and Muse cells (n = 9) for (a) body weight, (b) rotarod test, (c) hanging-wire test, and (d) lower limb muscle strength. Compared with vehicle, intravenous treatment with Muse cells showed significant improvements in the rotarod test, hanging-wire test, and lower limb muscle strength (*p < 0.05 vs. vehicle). (e) There were no GFP-positive cells in the lumbar spinal cord of vehicle-treated G93A Tg mice. (f) Some GFP-positive cells were only observed in the pia mater of the MSC group. (g) More GFP-positive cells were detected from the pia mater to the ventral horn of the Muse cell group. The boxes in (g) are enlarged in i (solid box) and j (dotted box), respectively. Treatment with Muse cells revealed clear GFP-positive (h) and GFP / GFAP double-positive cells in the pia mater (i, arrow) and ventral horn (j, arrowhead) showing morphology typical of astroglia (arrows), but no GFP + microglial markers such as Iba-1 (k), Tuj1 (l), or double positivity for NeuN (m). Scale bars: (e) 100 μm and (h) 50 μm. [Figure 3] (a-b) The number of Nissl-stained motor neurons in the lumbar spinal cord was significantly reduced in G93A Tg mice (*p<0.05, vs. wild type = WT), and significantly improved by intravenous treatment with Muse cells (#p<0.05, vs. vehicle). (c-d) Neuromuscular junction (NMJ) staining showed denervation in the tibialis anterior muscle of G93A Tg mice and recovery in the Muse cell group (VAChT-positive motor terminals; green, acetylcholine receptors stained with BTX; red, arrowheads; *p<0.05, vs. WT; #p<0.05, vs. vehicle). (e-f) HE-stained neurogenic muscle fiber atrophy in the tibialis anterior muscle of G93A Tg mice, and significantly improved by treatment with Muse cells (*p<0.05, vs. vehicle; #p<0.05, vs. MSC). Scale bars: (a) 500 μm, (c) 50 μm, and (d) 50 μm. [Figure 4]Section levels are indicated, approximately at the midsagittal level, as obtained according to the sectioning protocol. Stereotaxic coordinates are shown, taken from Paxinos & Franklin, "The Mouse Brain Atlas" (2nd ed.). [Figure 5] Section levels in the spinal cord tissue array are shown. Diagrams of spinal cord sections are taken from the Allen Brain Atlas (http: / / mousespinal.brain-map.org / mageseries / showref.html). [Figure 6] Body weight: The graph shows the evolution of body weight [g] per group (A, C, D) measured once a week throughout the treatment period. Each point represents the mean ± standard error of the mean across all animals for each group and week. Group C was compared with groups A and D (p<0.01 group D vs. group C, repeated measures ANOVA, factor: group). [Figure 7] Body weight: The graph shows the evolution of body weight [g] per group (B, C, and D) measured once a week throughout the treatment period. Each point represents the mean ± SEM of all animals for each group and week. Group C was compared with groups B and D (p<0.01 group D vs. group C, repeated measures ANOVA, factor: group). [Figure 8] Hanging wire: The graph shows the time spent hanging on the wire [seconds] per group (A, C, and D) measured after 1 week and then every 2 weeks throughout the treatment period. Each point represents the mean ± SEM for all animals in each group and week. Group C was compared with groups A and D (p<0.05 group C vs. group A, p<0.01 group D vs. group C, repeated measures ANOVA, factor: group). [Figure 9] Hanging wire: The graph shows the time course of the latency to fall off the wire [seconds] per group (B, C, and D) measured at week 1 and every two weeks throughout the treatment period. Each point represents the mean ± SEM for all animals in each group and week. Group C was compared with groups B and D ($$: p<0.01 group D vs. group C, repeated measures ANOVA, factor: group). [Figure 10]Rotarod: Graphs represent the mean latency to fall [seconds] for each group in trials 1, 2, and 3, and at baseline and week 12. Group C was compared with group D, and groups A-B were compared with group C (##: p<0.01, t-test). Data are presented as bar graphs of the mean ± standard error of the mean across all animals in each group. [Figure 11] Clasping: The graph shows the mean clasping score [n] for each group at baseline and at week 12. Group C was compared with Group D, and groups A-B were compared with Group C (##: p<0.01, t-test). Data are presented as bar graphs of the mean ± standard error for all animals in each group. [Figure 12] hTDP-43: The graph shows the mean immunofluorescence signal measured within the ROI of one spinal cord tissue array per mouse (n = 8 per group). Each value represents the mean ± SEM. *: p < 0.05 vs. group C (Dunnett's test); #: p < 0.05, ##: p < 0.01 vs. group D (t-test); $$: p < 0.01 vs. group C (Dunnett's test followed by two-way ANOVA; factor: group); %%: p < 0.01 vs. group D (two-way ANOVA; factor: group). [Figure 13] GFAP: The graph shows the average immunofluorescence signal measured within the ROI of one spinal cord tissue array per mouse (n = 8 per group). Each value represents the mean ± SEM. *: p < 0.05, **: p < 0.01 vs. group C (Dunnett's test); #: p < 0.05, ##: p < 0.01 vs. group D (t-test); $$: p < 0.01 vs. group C (Dunnett's test, two-way ANOVA; factor: group); %: p < 0.05, %%: p < 0.01 vs. group D (two-way ANOVA; factor: group). [Figure 14] MAP2: The graph shows the mean immunofluorescence signal measured within the ROI of one spinal cord tissue array per mouse (n = 8 per group). Each value represents the mean ± SEM. $: p < 0.05 vs. group C (2-way ANOVA; factor: group after Dunnett's test); %: p < 0.05, %%: p < 0.01 vs. group D (2-way ANOVA; factor: group). [Figure 15]Iba1: The graph shows the mean immunofluorescence signal measured within the ROI of one spinal cord tissue array per mouse (n = 8 per group). Each value represents the mean ± SEM. #: p < 0.05, ##: p < 0.01 vs. group D (t-test); $: p < 0.05 vs. group C (Two-way ANOVA after Dunnett's test; factor: group); %: p < 0.05, %%: p < 0.01 vs. group D (Two-way ANOVA; factor: group). [Figure 16] hTDP-43: The graph shows the mean immunofluorescence signal measured within ROIs of five brain sections per mouse (n = 8 per group). Each value represents the mean ± SEM. ##: p < 0.01 vs. group D (t-test). [Figure 17] GFAP: The graph shows the mean immunofluorescence signal measured within the ROI of five brain sections per mouse (n = 8 per group). Each value represents the mean ± SEM. ##: p < 0.01 vs. group D (t-test). [Figure 18] Iba1: The graph shows the mean immunofluorescence signal measured within ROIs of five brain sections per mouse (n = 8 per group). Each value represents the mean ± SEM. #: p < 0.05, ##: p < 0.01 vs. group D (t-test). [Figure 19] NeuN: The graph shows the mean immunofluorescence signal measured within ROIs of five brain sections per mouse (n = 8 per group). Each value represents the mean ± SEM. #: p < 0.05, ##: p < 0.01 vs. group D (t-test). [Figure 20] Western blot: The graph shows the mean TDP-43 signal (AU) or calculated ratio (TDP-43 normalized to tubulin isotype III) for all animals (hippocampus: n = 15 per group, spinal cord: n = 7 per group). Each value represents the mean ± SEM. $: p < 0.05 vs. group C (2-way ANOVA; factor: group after Dunnett's test); %: p < 0.05, %%: p < 0.01 vs. group D (2-way ANOVA; factor: group). ##: p < 0.01 vs. group D (t-test). DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention relates to a cell preparation and pharmaceutical composition containing SSEA-3-positive pluripotent stem cells (e.g., Muse cells) for treating, preventing, alleviating, and / or delaying the onset of motor neuron disease (MND) in a subject, as well as a method for treating MND using the cell preparation, etc. The present invention will be described in detail below.

[0011] Abbreviations used in this specification are detailed below. Abbreviations commonly used in the technical field will follow conventional conventions.

[0012] Abbreviations used Ang1: Angiopoietin-1 ANOVA: Analysis of variance BW: Weight BRET: Bioluminescence Resonance EGF: epidermal growth factor energy transfer; FACS: Fluorescence-activated cell sorting GFAP: glial fibrillary acidic protein GFP: green fluorescent protein HBSS: Hanks' Balanced Salt Solution HE: Hematoxylin and eosin HGF: Hepatocyte growth factor Iba1: ionized calcium-binding adaptor molecule 1 IGF-1: insulin-like growth factor 1 it: intrathecal iv: intravenous MAP2: microtubule-associated protein 2 MSC: Mesenchymal stem cell NMJ: neuromuscular junction NeuN: neuronal nucleus PGE2: Prostaglandin E2 SOD1: superoxide dismutase S1P: sphingosine-1-phosphate S1PR2: sphingosine-1-phosphate receptor 2 TDP-43:TAR DNA-binding protein 43 Tg: transgenic VAChT: vesicular acetylcholine transporter VEGF: Vascular endothelial growth factor WT: wild type

[0013] 1.Applicable diseases The present invention uses a cell preparation or pharmaceutical composition containing SSEA-3-positive pluripotent stem cells (Muse cells) that can be used to treat, prevent, alleviate, and / or delay the onset of motor neuron diseases (hereinafter sometimes simply abbreviated as "MND") such as amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), spinal muscular atrophy (SMA), progressive muscular atrophy (PMA), or spinal-bulbar muscular atrophy (SBMA).

[0014] Furthermore, the present invention can be used to treat, prevent, alleviate, and / or delay the onset of FTLD-TDP, a type of frontotemporal lobar degeneration (FTLD) in which TDP-43 accumulates in neurons, using a cell preparation or pharmaceutical composition containing SSEA-3-positive pluripotent stem cells (Muse cells).

[0015] As used herein, the term "motor neuron disease (MND)" refers to a neurological disorder that selectively destroys motor neurons. Generally, motor neuron disease (also known as motor neuron degenerative disease) is classified in [G12: spinal muscular atrophy and related syndromes], preferably [G12.2: motor neuron disease], of Chapter VI (G) "Diseases of the nervous system" based on the diagnostic criteria of the World Health Organization's (WHO) International Statistical Classification of Diseases and Related Health Problems (10th edition) ICD-10. Examples of MNDs include amyotrophic lateral sclerosis (including familial amyotrophic lateral sclerosis), primary lateral sclerosis, spinal muscular atrophy (including distal spinal muscular atrophy, familial spinal muscular atrophy, and scapuloperoneal spinal muscular atrophy), progressive muscular atrophy (such as juvenile progressive muscular atrophy, childhood progressive muscular atrophy, infantile progressive bulbar palsy, and spinal progressive muscular atrophy), spinal and bulbar muscular atrophy, Werdnig-Hoffmann disease, diffuse atrophic paralysis, motor neuron disease, pseudobulbar palsy, bulbar palsy, juvenile unilateral upper limb muscular atrophy, progressive bulbar palsy, traumatic bulbar palsy, and cervical spondylotic muscular atrophy.

[0016] MND is a neurological disorder that selectively destroys motor neurons, which can lead to their degeneration and death. Motor neurons, including primary (upper) and secondary (lower) motor neurons, act on voluntary muscles, stimulating them to contract. Primary motor neurons originate in the cerebral cortex and send fibers through the brainstem and spinal cord, responsible for controlling secondary motor neurons. Secondary motor neurons are located in the brainstem and spinal cord and send fibers to muscles. Secondary motor neuron disease is a disorder involving the degeneration of secondary motor neurons. When secondary motor neurons degenerate, the muscle fibers they normally activate are severed and no longer contract, causing muscle weakness and decreased reflexes. Loss of either type of neuron leads to weakness, and muscle atrophy (muscle wasting) and painless weakness are the clinical hallmarks of MND.

[0017] Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease characterized by the selective and progressive loss of motor neurons in the spinal cord, brainstem, and cerebral cortex. It typically leads to progressive muscle weakness and neuromuscular respiratory failure. Approximately 10% of ALS cases are associated with point mutations in the gene encoding the Cu / Zn superoxide dismutase 1 enzyme (SOD1). Herein, the therapeutic efficacy of the cell preparation of the present invention was examined using a mutant SOD1 (G93A) transgenic mouse model (see Example 1). On the other hand, ALS development is known to be associated with the abnormal accumulation of phosphorylated or ubiquitinated TDP-43 in neurons. Abnormalities in the gene encoding TDP-43 and in the TDP-43 degradation mechanism have been reported, and these abnormalities are observed in some familial ALS cases (ALS10, associated with mutations in the TARDBP gene) and approximately 90% of sporadic ALS cases. In the present specification, an ALS mouse model in which TDP-43 was overexpressed was prepared, and the therapeutic efficacy of the cell preparation of the present invention was examined (see Example 2).

[0018] Primary lateral sclerosis (PLS) is a disease of unknown cause in which only primary (upper) motor neurons are selectively and progressively damaged, sparing secondary (lower) motor neurons. PLS can be difficult to distinguish from ALS, which primarily affects primary motor neurons, and is sometimes associated with frontotemporal lobar degeneration (FTLD).

[0019] Spinal muscular atrophy (SMA) is a neurogenic muscular atrophy caused by damage to motor neurons (anterior horn cells) in the spinal cord. It progressively causes muscle weakness and atrophy in the trunk and limbs. SMA is classified into Type I (severe type) (also known as Werdnig-Hoffmann disease), which develops in childhood; Type II (intermediate type) (also known as Dubowitz disease); Type III (mild type) (also known as Kugelberg-Welander disease); and Type IV (adult-onset type).

[0020] Progressive muscular atrophy (PMA) is a disease that causes progressive weakness and atrophy of the skeletal muscles of the limbs and trunk. Examples of PMA that are caused by the muscles themselves include muscular dystrophy and myotonia. Muscular dystrophy is defined as a disease caused by a gene mutation whose main pathology is necrosis and regeneration of skeletal muscles.

[0021] Spinal and bulbar muscular atrophy (SBMA) is a neurological disorder that occurs only in adult males due to a gradual loss of nerves (secondary motor neurons) that move muscles in the brainstem and spinal cord. It is known that the cause of spinal and bulbar muscular atrophy is an abnormality in the androgen receptor gene on the X chromosome, which determines sex.

[0022] Frontotemporal lobar degeneration (FTLD) is a neurodegenerative disease characterized by the slowly progressive degeneration and loss of neurons, primarily in the frontal and temporal lobes of the brain, resulting in personality changes, behavioral abnormalities, aphasia, cognitive impairment, and movement disorders. In FTLD, accumulation of protein inclusions is observed within neurons, and the disease is classified according to the type of inclusion protein: FTLD-TDP (accumulation of TDP-43), FTLD-TAU (accumulation of TAU), FTLD-FUS (accumulation of FUS), and FTLD-others (accumulation of other proteins). FTLD-TDP accounts for approximately 45% of FTLD cases and is classified into types A to D based on histopathological characteristics.

[0023] 2. Cell Preparation (1) Pluripotent stem cells (Muse cells) The pluripotent stem cells used in the cell preparations of the present invention are cells that Idezawa, one of the present inventors, discovered in the human body and named "Muse (multilineage-differentiating stress-enduring) cells." Muse cells can be obtained from bone marrow fluid, adipose tissue (Ogura, F., et al., Stem Cells Dev., Nov 20, 2013 (Epub) (published on Jan 17, 2014)), and skin tissue such as the dermal connective tissue, and are also scattered in the connective tissue of various organs. These cells possess properties of both pluripotent stem cells and mesenchymal stem cells and are identified, for example, by double positivity for the cell surface markers "SSEA-3 (Stage-specific embryonic antigen-3)" and "CD105." Therefore, Muse cells or cell populations containing Muse cells can be isolated from biological tissues, for example, using these antigen markers as indicators. Details of isolation, identification, and characteristics of Muse cells are disclosed in International Publication No. WO 2011 / 007900. Furthermore, as reported by Wakao et al. (S. Wakao, et al., Proc. Natl. Acad. Sci. USA, Vol. 108, pp. 9875-9880 (2011)), when mesenchymal cells are cultured from bone marrow, skin, or the like and used as a population of Muse cells, all of the SSEA-3-positive cells are known to be CD105-positive cells. Therefore, when Muse cells are isolated from mesenchymal tissues of living organisms or cultured mesenchymal stem cells in the cell preparations of the present invention, the Muse cells can be purified and used simply by using SSEA-3 as an antigen marker. In this specification, pluripotent stem cells (Muse cells) isolated from biological mesenchymal tissue or cultured mesenchymal tissue using SSEA-3 as an antigen marker, which can be used in cell preparations (including pharmaceutical compositions) for treating motor neuron diseases, or cell populations containing Muse cells, may be referred to simply as "SSEA-3-positive cells." Furthermore, in this specification, "non-Muse cells" refer to cells contained in biological mesenchymal tissue or cultured mesenchymal tissue, other than "SSEA-3-positive cells."

[0024] Briefly, Muse cells or cell populations containing Muse cells can be isolated from biological tissues (e.g., mesenchymal tissues) using an antibody against the cell surface marker SSEA-3 alone or using antibodies against SSEA-3 and CD105 together. Here, "biological organism" refers to a mammalian organism. In the present invention, "biological organism" does not include fertilized eggs or embryos at developmental stages earlier than the blastula stage, but does include embryos at developmental stages after the blastula stage, including fetuses and blastulas. Mammals include, but are not limited to, humans, primates such as monkeys, rodents such as mice, rats, rabbits, and guinea pigs, cats, dogs, sheep, pigs, cows, horses, donkeys, goats, and ferrets. The Muse cells used in the cell preparations of the present invention are clearly distinguished from embryonic stem cells (ES cells) and iPS cells in that they are directly isolated from biological tissues using markers. Furthermore, "mesenchymal tissue" refers to tissues such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, umbilical cord, and umbilical cord blood, as well as tissues present in various organs. For example, Muse cells can be obtained from bone marrow, skin, or adipose tissue. For example, it is preferable to collect mesenchymal tissue from a living body and isolate and use Muse cells from this tissue. Alternatively, Muse cells may be isolated from cultured mesenchymal cells such as fibroblasts or bone marrow mesenchymal stem cells using the above-mentioned isolation methods. In the cell preparation of the present invention, the Muse cells used may be autologous or allogeneic to the recipient of the cell transplant.

[0025] As described above, Muse cells or cell populations containing Muse cells can be isolated from biological tissues using, for example, SSEA-3 positivity and SSEA-3 and CD105 double positivity as indicators. However, adult human skin is known to contain various types of stem and progenitor cells. However, Muse cells are not the same as these cells. These stem and progenitor cells include skin-derived progenitor cells (SKPs), neural crest stem cells (NCSCs), melanoblasts (MBs), pericytes (PCs), endothelial progenitor cells (EPs), and adipose-derived stem cells (ADSCs). Muse cells can be isolated by detecting the absence of markers specific to these cells. More specifically, Muse cells can be separated using non-expression of at least one, for example, two, three, four, five, six, seven, eight, nine, ten, or eleven, of eleven markers selected from the group consisting of CD34 (a marker for EPs and ADSCs), CD117 (c-kit) (a marker for MBs), CD146 (a marker for PCs and ADSCs), CD271 (NGFR) (a marker for NCSCs), NG2 (a marker for PCs), vWF factor (von Willebrand factor) (a marker for EPs), Sox10 (a marker for NCSCs), Snail (a marker for SKPs), Slug (a marker for SKPs), Tyrp1 (a marker for MBs), and Dct (a marker for MBs). For example, but not limited to, separation can be performed using the non-expression of CD117 and CD146 as an indicator, and further separation can be performed using the non-expression of CD117, CD146, NG2, CD34, vWF, and CD271 as an indicator, and further separation can be performed using the non-expression of the above 11 markers as an indicator.

[0026] Furthermore, the Muse cells having the above characteristics used in the cell preparation of the present invention are as follows: (i) low or absent telomerase activity; (ii) have the ability to differentiate into cells of any of the three germ layers; (iii) does not exhibit neoplastic growth; and (iv) Self-renewing ability In one aspect of the present invention, Muse cells used in the cell preparation of the present invention have all of the above properties. Regarding (i) above, "low or no telomerase activity" refers to low or undetectable telomerase activity when detected using, for example, a TRAPEZE XL telomerase detection kit (Millipore). "Low" telomerase activity refers to telomerase activity comparable to that of human fibroblasts, which are somatic cells, or telomerase activity that is one-fifth or less, preferably one-tenth or less, of that of HeLa cells. Regarding (ii) above, Muse cells have the ability to differentiate into three germ layers (endodermal, mesodermal, and ectodermal) in vitro and in vivo. For example, by in vitro induction culture, they can differentiate into hepatocytes, neurons, skeletal muscle cells, smooth muscle cells, osteocytes, adipocytes, etc. Furthermore, they may also exhibit the ability to differentiate into three germ layers when transplanted into testes in vivo. Furthermore, when transplanted intravenously into the body, Muse cells have the ability to migrate and engraft in damaged organs (heart, skin, spinal cord, liver, muscle, etc.) and differentiate into cells appropriate for the tissue. Regarding (iii) above, Muse cells proliferate at a rate of approximately 1.3 days in suspension culture. However, in suspension culture, they proliferate from a single cell, form embryoid-like cell clusters, and proliferation ceases after approximately 14 days. However, when these embryoid-like cell clusters are cultured in adherent culture, cell proliferation resumes, and the cells proliferate from the cell clusters and spread. Furthermore, when transplanted into the testis, they do not become cancerous for at least six months. Regarding (iv) above, Muse cells have the ability to self-renew (self-replicate). Here, "self-renewal" refers to the process by which differentiation into three germ layers can be confirmed from cells contained in an embryoid-like cell mass obtained by culturing a single Muse cell in suspension culture, and at the same time, by bringing the cells from the embryoid-like cell mass back into suspension culture as a single cell, an embryoid-like cell mass of the next generation can be formed, from which differentiation into three germ layers and the formation of an embryoid-like cell mass in suspension culture can again be confirmed. Self-renewal can be performed by repeating one or more cycles.

[0027] Furthermore, the cell fraction containing Muse cells used in the cell preparation of the present invention may be a cell fraction enriched in SSEA-3-positive and CD105-positive pluripotent stem cells that have at least one, and preferably all, of the following properties, and that is obtained by a method comprising applying an external stress stimulus to mesenchymal tissue of a living body or cultured mesenchymal cells, killing cells other than those resistant to the external stress, and recovering the surviving cells: (i) SSEA-3 positive; (ii) CD105 positive; (iii) low or absent telomerase activity; (iv) have the ability to differentiate into three germ layers; (v) does not exhibit neoplastic growth; and (vi) It has self-renewal ability.

[0028] The external stress may be any one or a combination of protease treatment, culture under low oxygen concentration, culture under low phosphate conditions, culture under low serum concentration, culture under low nutrient conditions, culture under heat shock, culture at low temperature, freezing treatment, culture in the presence of harmful substances, culture in the presence of active oxygen, culture under mechanical stimulation, culture under shaking treatment, culture under pressure treatment, or physical impact. For example, the protease treatment time is preferably 0.5 to 36 hours in total to impart external stress to the cells. Furthermore, the protease concentration may be any concentration used when detaching cells adhered to a culture vessel, disaggregating cell clumps into single cells, or recovering single cells from tissue. The protease is preferably a serine protease, an aspartic acid protease, a cysteine ​​protease, a metalloprotease, a glutamic acid protease, or an N-terminal threonine protease. Furthermore, the protease is preferably trypsin, collagenase, or dispase.

[0029] (2) Preparation and use of cell preparations The cell preparation of the present invention can be obtained by suspending the Muse cells or a cell population containing Muse cells obtained in (1) above in physiological saline or an appropriate buffer solution (e.g., phosphate-buffered saline). In this case, if the number of Muse cells isolated from autologous or allogeneic tissue is low, the cells may be cultured and expanded to a predetermined cell concentration before cell transplantation. As previously reported (International Publication No. WO 2011 / 007900), Muse cells do not become tumorigenic. Therefore, even if undifferentiated cells are contained in biological tissue, the possibility of cancer formation is low and safe. Furthermore, the culture of recovered Muse cells can be performed in a standard growth medium (e.g., α-minimal essential medium (α-MEM) containing 10% fetal bovine serum). For more details, referring to International Publication No. WO 2011 / 007900, appropriate medium and additives (e.g., antibiotics, serum) can be selected for the culture and expansion of Muse cells to prepare a solution containing Muse cells at a predetermined concentration. When the cell preparation of the present invention is administered to a human subject, several milliliters of bone marrow fluid can be collected from the human ilium, and bone marrow mesenchymal stem cells can be cultured as adherent cells from the bone marrow fluid to expand the number of cells sufficient to isolate an effective therapeutic dose of Muse cells. The Muse cells can then be isolated using the SSEA-3 antigen marker as an indicator, and autologous or allogeneic Muse cells can be prepared as a cell preparation. Alternatively, Muse cells can be isolated using the SSEA-3 antigen marker as an indicator, and the cells can be cultured and expanded to an effective therapeutic dose, and the autologous or allogeneic Muse cells can be prepared as a cell preparation.

[0030] Furthermore, when Muse cells are used in cell preparations, the cell preparation may contain dimethyl sulfoxide (DMSO) or serum albumin to protect the cells, or antibiotics to prevent bacterial contamination and proliferation. Furthermore, the cell preparation may contain other pharmaceutical acceptable ingredients (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.) as well as cells or components contained in mesenchymal stem cells other than Muse cells. Those skilled in the art can add these factors and drugs to cell preparations at appropriate concentrations. In this way, Muse cells can also be used as pharmaceutical compositions containing various additives.

[0031] The number of Muse cells contained in the cell preparation or pharmaceutical composition prepared as described above can be adjusted appropriately to achieve the desired effect on motor neuron disease (e.g., increased muscle strength, suppression of the progression of muscle atrophy, etc.) taking into consideration the subject's gender, age, weight, condition of the affected area, and the condition of the cells used. Target individuals include, but are not limited to, mammals such as humans. The cell preparation of the present invention may be administered once, or multiple times (e.g., 2 to 10 times or more) at appropriate intervals (e.g., twice a day, once a day, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, once every three months, once every six months, etc.) until the desired therapeutic effect is achieved. The administration time may be early, middle, or late in the onset of disease, as long as a therapeutic effect is achieved.

[0032] The therapeutically effective amount varies depending on the condition of the subject, but is, for example, 1 × 10 per individual per administration. 3 cells ~1×10 11 cells, preferably 1 x 10 4 cells ~1×10 10 cells, more preferably 1 x 10 5 cells ~1×10 9The cell preparation of the present invention may be administered directly to the brain (brainstem, cerebral cortex) or spinal cord, or may be administered intravenously, although this is not particularly limited. Based on the examples and the like, intravenous administration is also a particularly preferred embodiment.

[0033] The cell preparations and pharmaceutical compositions of the present invention may use human-derived Muse cells. However, if the recipient is a subject of a different species from the cells (e.g., a mouse or rat), an immunosuppressant (e.g., cyclosporine) may be administered before or simultaneously with the administration of the xenogeneic cells to suppress in vivo rejection of the xenogeneic cells. According to the present invention, when the cell preparations and pharmaceutical compositions of the present invention are used for the treatment of motor neuron diseases, such an immunosuppressant may or may not be used in combination. Furthermore, when the cell preparations and pharmaceutical compositions of the present invention are administered to patients, it is particularly preferred not to use an immunosuppressant in combination.

[0034] 3. Therapeutic effects of Muse cells In an embodiment of the present invention, the cell preparations and pharmaceutical compositions of the present invention can treat, prevent, alleviate, and / or delay the onset of motor neuron disease (MND). More specifically, the present invention can restore muscle strength in the trunk and limbs or inhibit the progression of muscle atrophy caused by motor neuron disease. Here, "treatment" refers to the suppression or complete elimination of various symptoms caused by MND. Furthermore, "alleviation" refers to the alleviation of various symptoms caused by MND and the inhibition of their progression, preferably to the extent that symptoms are alleviated to the extent that they do not interfere with daily life. The term "alleviation" can also mean "delaying symptoms."

[0035] As shown in the Examples below, the efficacy of the cell preparation or pharmaceutical composition of the present invention can be evaluated by measuring the motor ability of ALS model SOD1(G93A) Tg mice or TDP-43 Tg mice. Evaluation methods include, but are not limited to, commonly used methods such as the hanging wire test, rotarod test, clasping test, grip strength test, and beam-balance test.

[0036] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples in any way. [Example]

[0037] All animal experiments in this study were approved by the Okayama University Animal Experimentation Committee (OKU-2019289) and were conducted in accordance with the Okayama University Guidelines for Animal Experiments. [Example]

[0038] Materials and Methods Animals and experimental groups Transgenic (Tg) mice carrying the G93A human SOD1 mutation (G1H / +) were obtained from Jackson Laboratories (Bar Harbor, ME, USA) [2] and maintained as hemizygotes by mating Tg males with C57BL / 6J females. For experiments comparing intravenous (i.v.) and intrathecal (i.t.) injection, three animals were used per group. For ex vivo imaging using nano-lanterns, two animals were used, using vehicle, MSCs, and Muse cells. For evaluation of the effects of intravenous injection, 10 animals (5 males and 5 females) were used in the vehicle group, 9 animals (4 males and 5 females) in the MSC group, and 9 animals (5 males and 4 females) in the Muse cell group.

[0039] Preparation of GFP-labeled MSCs and Muse cells GFP-labeled MSCs were prepared by lentiviral GFP labeling of human bone marrow-MSCs (Lonza, Basel, Switzerland) as previously reported

[22] . GFP-labeled Muse cells were isolated from GFP-MSCs as SSEA-3-positive cells by fluorescence-activated cell sorting (FACS) as previously reported [10, 11]. The GFP-MSCs and Muse cells were frozen in Beissel freezing containers (Nihon Freezer, Tokyo, Japan) and kept in liquid nitrogen until use.

[0040] In vivo comparison cell engraftment for injection route The GFP-labeled Muse cells (2.0 × 10 ) were cultured in 250 μl of Hank's balanced salt solution (HBSS, pH 7.4). 5 (individually) were injected into the tail vein for i.v. injection or into the cisterna magna for intrathecal injection, as previously reported [23, 24]. On day 7, all animals were sacrificed.

[0041] Ex vivo dynamics of nano-lanterns Nano-lantern labels with bioluminescence resonance energy transfer (BRET) efficacy are bright luminescent proteins that allow the detection of even small numbers of transplanted cells

[25] . Human bone marrow-MSCs (Lonza) were labeled with nano-lanterns as previously reported

[25] . Nano-lantern-labeled Muse cells were isolated from nano-lantern-labeled MSCs by FACS as SSEA-3-positive cells. For ex vivo kinetics of i.v. injection, a mixture of medium (HBSS), nano-lantern-labeled MSCs, and Muse cells (1.0 × 10 5 Cells / 250 μl) were injected intravenously at 14 weeks of age. Seven days later, animals were sacrificed under deep anesthesia and analyzed as previously reported

[26] .

[0042] Evaluation of treatment effect Vehicle, GFP-MSCs, and GFP-Muse cells (both 5.0 × 10 4A total of 250 μl of cells was injected into the tail vein of each animal over a 1-minute period. Because we and other groups have confirmed that G93A Tg mice exhibit early onset of disease at approximately 56 days of age

[27] , we decided to start cell administration at 56 days of age and continue weekly administration (10 injections total) using the same number of cells until day 119 (10 injections total). The immunosuppressant cyclosporine A (10 mg / kg / day, Novartis International, Basel, Switzerland) was also administered intraperitoneally (i.p.) to all animals immediately after administration of the vehicle, MSC, or Muse cell groups, and every other day until sacrifice. Survival was checked daily, and body weight (BW) and rotarod scores were measured twice weekly from day 56 of age. Rotarod testing and wheel running activity were performed according to our previous methods [24, 28]. The hanging wire test was performed weekly to assess muscle strength and coordination, as previously reported.

[29] Lower limb muscle strength was measured weekly using a precision spring scale (Ooba Keiki Co., Tokyo, Japan).

[0043] Immunohistological analysis Mice were euthanized when they were unable to stand up within 15 seconds of falling to one side, and this was recorded as the time of death. Each animal was deeply anesthetized with an intraperitoneal injection of pentobarbital (20 mg / kg), and samples were taken as previously described

[26] . The primary antibodies used were goat anti-GFP (1:500, Abcam, Cambridge, UK); rabbit anti-GFP (1:500, MBL, Woburn, USA); rabbit anti-Iba1 (1:500, Osaka, Japan); mouse anti-βIII tubulin (Tuj1) (1:100, Santa Cruz Biotechnology); rabbit anti-glial fibrillary acidic protein (GFAP) (1:500, Dako, Glostrup, Denmark); anti-NeuN (1:100, Millipore, MA, USA); and goat anti-vesicular acetylcholine transporter (VAChT) (1:200, Thermo Scientific). The secondary antibodies used were either anti-goat, rabbit, or mouse IgG conjugated with Alexa 488 or 546 (1:500, Alexa Fluor™, Invitrogen, Carlsbad, CA, USA). α-Bungarotoxin conjugated with Alexa 594 (1:500, Millipore) was also used to detect acetylcholine receptors.

[0044] Five to six randomly selected regions from the pia mater to the ventral horn were analyzed for cell type marker / GFP double labeling. Nissl-stained motor neurons from L4 to L5 were counted using five transverse sections from each lumbar spinal cord

[30] . Cells larger than 20 μm in diameter with distinct nucleoli were counted as motor neurons in both ventral horns below the lateral line, extending from the central canal across the spinal cord

[31] . For denervation, approximately 100 neuromuscular junctions (NMJs) from each mouse were analyzed. For muscle fiber size, approximately 180 muscle fibers from three hematoxylin and eosin (HE)-stained sections of the tibialis anterior muscle per mouse were analyzed by researchers blinded to treatment conditions.

[0045] statistical analysis Investigators were blinded to the experimental groups during data collection and analysis. Data supporting the findings of this study are available from the appropriate personnel upon reasonable request. Data were analyzed using SPSS version 22.0.0.0 (IBM Corp., Armonk, New York, USA) and expressed as mean ± SD. Treatment effects were assessed by non-repeated measures analysis of variance (ANOVA) and Dunnett's t test. Immunohistochemical data were analyzed using Kruskall-Wallis followed by the Mann-Whitney U test with Bonferroni correction. For all statistical analyses, significance was considered at p < 0.05.

[0046] result To determine the administration route, we compared the homing of GFP-Muse cells between i.v. and i.t. injections by immunohistological analysis of the spinal cord of G93A mice on day 7. Preliminary studies showed that the number of GFP-Muse cells was consistently low or negligible in the cervical, thoracic, and lumbar spinal cord in the i.t. injection group, whereas it was higher in the cervical and lumbar spinal cord in the i.v. injection group compared to the i.t. injection group. GFP-Muse cells were primarily located in the pia mater and lower white matter. GFP-Muse cells were rarely detected in the thoracic spinal cord, even after i.v. injection (Table 1, Figure 1a, b). Therefore, i.v. injection was chosen as the administration route for the following experiments.

[0047] [Table 1]

[0048] Nanolantern-labeled cells were used to examine the in vivo dynamics of MSC and Muse cells after i.v. injection. In the MSC group, strong signals were detected in the lungs and trace signals in the femur at day 7, but no signals were detected in other organs, including the brain, cervical spinal cord, and lumbar spinal cord. In the Muse group, signals were detected in the cervical and lumbar spinal cord (Figure 1c, upper right panel) and lungs, but not in the brain (Figure 1c, middle panel). The femur signal was higher in the Muse group than in the MSC group. Immunohistological analysis confirmed the presence of nanolantern-Muse cells in the pulmonary vascular lumen and bone marrow (Figure 1d). This signal was consistently below the detection limit in all organs examined in the vehicle group (Figure 1c).

[0049] The mean survival time was not significantly different among the three groups (vehicle; 144.4 ± 8.0 days, MSC; 143.9 ± 6.9 days, Muse cell; 142.6 ± 6.7 days). Furthermore, there was no statistically significant difference in body weight among the three groups over the entire period (Fig. 2a). In contrast, the rotarod test showed a reduction in the Muse group on days 67 and 70, with statistical significance observed in the vehicle group (Fig. 2b). Furthermore, the hanging wire scores on days 84, 112, and 133, as well as lower limb muscle strength on days 126 and 140, were also significantly improved only in the Muse cell group compared with the vehicle group (Fig. 2c, d).

[0050] In the lumbar spinal cord, GFP-positive cells were not detectable in the vehicle group (Figure 2e), whereas a small number of GFP-positive cells were observed in the MSC group (Figure 2f). In the Muse cell group, GFP-positive cells were observed in the spinal pia mater (Figure 2g, solid box), lower white matter, and ventral horn (Figure 2g, dotted box, 2h). 85.7% of these cells (180 of 201 GFP-positive cells) co-expressed the astrocyte marker GFAP, located in both the pia mater (Figure 2i) and ventral horn (Figure 2j). The remaining GFP-positive cells (14.3%) did not show positivity for the microglial marker Iba-1 (Fig. 2k, 0 / 120 GFP-positive cells), the neuronal marker Tuj1 (Fig. 2l, 0 / 97 GFP-positive cells), or NeuN (Fig. 2m, 0 / 109 GFP-positive cells), suggesting that the majority of i.v. injected Muse cells spontaneously differentiated into cells of the astrocyte lineage after homing to the lumbar spinal cord.

[0051] Compared with the wild-type (WT) group, the number of surviving motor neurons in the ventral horn was significantly lower in the vehicle, MSC, and Muse groups (WT, Fig. 3a, b, * p<0.05). However, the Muse group had a statistically significant higher number of motor neurons compared to the vehicle group (Fig. 3a, b, #p<0.05). No statistical significance was observed between the vehicle and MSC groups (Fig. 3a, b).

[0052] In the tibialis anterior muscle, the number of innervated synapses was significantly reduced in the vehicle, MSC, and Muse cell groups compared with WT (Fig. 3c, d, * p<0.05), and statistically significant recovery in the Muse cell group compared with the vehicle group (Fig. 3c, d, #p<0.05). Myofiber size analysis showed severe neurogenic myofiber atrophy in the vehicle and MSC groups, which was significantly greater than in the vehicle group ( * p<0.05) and MSC group (#p<0.05) were improved in the Muse cell group with statistical significance (Fig. 3e, f).

[0053] Consideration This study is the first to demonstrate that multiple intravenous administrations of Muse cells improve muscle strength in the rotarod, hanging wire, and lower limbs in the ALS model G93A Tg mice. The intravenous injections of Muse cells homed to the lumbar spinal cord, lungs, and bone (Figure 1c, d). In the lumbar spinal cord, GFP-positive Muse cells primarily expressed the astroglial marker GFAP and exhibited a glial-like morphology at terminal stages (154 days of age, Figure 2g-j). Furthermore, the number of surviving motor neurons in the lumbar spinal cord was statistically significantly higher than in the vehicle group (Figure 3a, b). This may have resulted in the reduction of both lower limb muscle denervation and muscle fiber atrophy (Figure 3c-f).

[0054] In ALS model mice, intravenous injection was superior to intravenous injection in delivering Muse cells to the spinal cord, an important therapeutic target (Table 1, Figure 1a, b). Intravenous administration offers advantages over intravenous administration due to its ease of access, minimal invasiveness, and minimal burden on patients. This allows for repeated administration of Muse cells to ALS patients. Recent studies have demonstrated that Muse cells expressing sphingosine-1-phosphate receptor 2 (S1PR2) can specifically home to the injury site by sensing sphingosine-1-phosphate (S1P) produced by injured cells

[18] . S1P is a general injury signal common to all organs because it is produced by phosphorylation of sphingosine, a component of the cell membrane. Therefore, Muse cells can reach the spinal cord of ALS mice by intravenous injection.

[0055] Nano-lantern imaging revealed a strong signal of Muse cells in the femur, rather than MSCs (Figure 1c). Bone marrow abnormalities have been reported in ALS

[32] . Accordingly, Muse cells selectively and actively accumulated in the femoral bone marrow and spinal cord in a lesion-dependent manner, in contrast to their passive entrapment in pulmonary capillaries. Another interesting point is that intravenously injected Muse cells migrated to the spinal pia, lower white matter, and ventral horn (Figure 2g-j), suggesting that Muse cells home to the spinal cord via pial perforating arteries. In contrast, intravenously injected Muse cells were barely detected in the spinal cord (Figure 1a, b). These results suggest that the homing factors of Muse cells are primarily secreted into the circulation rather than paracrinely into the spinal subarachnoid space [32-35].

[0056] In animal disease models, intravenously injected Muse cells specifically homed to damaged tissue and spontaneously differentiated into tissue-constituting cells; i.e., differentiated into neurons and oligodendrocytes in a stroke model

[16] . In contrast to stroke, ALS is chronic and progressive, and its pathology is completely different from that of stroke. Intravenously injected Muse cells differentiated primarily into the astroglial lineage, rather than the neuronal lineage, in the spinal cord of ALS mice. Reactive astrocytes are an important therapeutic target for ALS

[36] . Muse cells produce various neurotrophic factors, such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), prostaglandin E2 (PGE2), and angiopoietin-1 (Ang1) [17-19]. Therefore, they may provide beneficial factors to motor neurons and astrocytes and prevent muscle fiber atrophy in ALS models. Furthermore, clinical trials for acute myocardial infarction, stroke, spinal cord injury, epidermolysis bullosa, and neonatal cerebral palsy are currently being conducted using donor Muse cell infusions without the need for HLA matching or long-term immunosuppressive drugs. Clinical trials for acute myocardial infarction have reported safety and significant cardiac recovery

[21] .

[0057] Overall, this study demonstrated for the first time that systemic administration of Muse cells showed significant clinical benefits in an ALS mouse model, and that intravenously injected Muse cells preferentially migrated to the spinal cord, supplied astroglia, supported motor neuron survival, and suppressed muscle fiber atrophy. Muse cells may be a promising cell resource for the treatment of ALS patients. [Example]

[0058] 1. Research purpose The purpose of this study was to evaluate the effects of Muse cells on the motor function of TDP-43 Tg mice.

[0059] 2. Test system and its validity Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by degeneration of motor neurons in the brain and spinal cord. Evidence suggests that a ubiquitinated protein called human transactivation response (TAR) DNA-binding protein 43 kDa (TDP-43) plays a central role in the pathogenesis of ALS. TDP-43 Tg mice are an appropriate model for studying TDP-43 production and intracellular deposition. This model, which mimics the clinical features of ALS, is useful for testing the effects of drugs on ALS. In the proposed study, we use mice overexpressing human wild-type TDP-43 (hTDP-43) under the control of the neuronal mouse Thy-1 promoter. In homozygous mice, human TDP-43 protein expression is 3.8-fold higher than that of endogenous protein in non-transgenic littermates.

[0060] 3. Preparation of Human Muse Cells Human Muse cells were isolated and identified according to the method described in WO2011 / 007900. More specifically, Muse cells were obtained by expanding and enriching mesenchymal stem cells under stress conditions. Animals were cultured at 2 × 10 6 5 ml / kg of viable cells / ml of working solution was administered.

[0061] 4. Medium Hanks' balanced salt solution, calcium-free, magnesium-free, phenol red-free (HBSS; Gibco 14175-046) was used as the vehicle.

[0062] 5. Animal Care 5-1. Breeding facilities Animals were housed in individually ventilated cages with standard rodent bedding supplied by Rettenmaier. A maximum of five animals per cage were housed in the same group. Room temperature was maintained at approximately 21°C, and relative humidity was maintained at 40-70%. Animals were kept under a constant light / dark cycle (12 h / 12 ​​h). Animals were fed standard rodent chow (Altromin) in the form of dry pellets and regular tap water ad libitum.

[0063] 5-2.Environmental enrichment From the start of the first treatment, all animals were housed with Mouse Igloo and FAST Trac (from PLEXX) as environmental enrichment.

[0064] 5-3.Identification Animals were numbered consecutively by classical ear punching, and each cage was identified by a color card indicating study number, sex, individual registration number (IRN), date of birth, date of genotyping, and treatment group assignment.

[0065] The genotype of each animal was determined by polymerase chain reaction specific for the transgenic construct. Prior to the start of the study, each mouse was genotyped using DNA isolated from ear tissue obtained by ear punching for animal identification.

[0066] 5-4. Group assignment Only animals in apparent good health were included. All animals were randomly assigned to cohorts consisting of animals from all treatment groups (taking into account the mean weight and sufficient sex distribution across all groups and cohorts). The number of animals in the initial cohort was limited to ensure age-matching and uniform handling.

[0067] 5-5. Health condition and cage observation Prior to study allocation, each animal's health status was assessed. During the study, notable observations from outside the cage were recorded and subsequent disposition (e.g., euthanasia) was determined.

[0068] The weight of all animals was recorded weekly. If animals showed severe motor dysfunction and could no longer reach the food / water bottles, the mice were given access to food pellets and water at the bottom of the cage. Drinking bottles with long caps were also used.

[0069] 5-6. Handling of animals that die early The animal that died prematurely (IRN 5921) was necropsied. Mortality was not assessed as no other animals were euthanized or died prematurely.

[0070] 6. Materials and Methods Forty-five homozygous TDP-43 Tg mice, divided into three groups of 15 mice each, and 15 non-transgenic littermates at 8 weeks of age were used in this study. Animals received two intravenous (iv) injections of either Muse cells or vehicle.

[0071] All animals underwent baseline testing once with a modified Irwin test, including reflex testing and the hanging wire. Motor coordination was assessed with the hanging wire test (six times in total) throughout the study period, rotarod twice (at baseline and at the end of the study), and clasping behavior was assessed twice (at baseline and at the end of the study) every other week.

[0072] At the end of the study, all animals were euthanized by intraperitoneal injection of pentobarbital. After transcardial perfusion with saline, the brains were removed and separated into left and right hemibrains. The left hemibrain was divided into the hippocampus and other parts and frozen on dry ice for protein expression analysis. The right hemibrain was post-fixed in 4% PFA and cryopreserved for immunohistological evaluation. Spinal cords were also harvested; n = 7 were frozen on dry ice, and the remaining n = 8 spinal cords were fixed and processed for immunohistological evaluation.

[0073] 6-1.Animals

[0074] [Table 2]

[0075] 6-2. Treatment This study used 45 homozygous TDP-43 Tg mice, divided into three groups of 15 mice each, and 15 non-transgenic littermates at 8 weeks of age as non-disease controls. During the 12-week study, each animal received two intravenous (iv) injections of either Muse cells and vehicle, vehicle and Muse cells, or vehicle and vehicle, at weeks 1 and 8, respectively. Muse cells were administered only once, either at week 1 (group A) or week 8 (group B). The following groups were used:

[0076] [Table 3]

[0077] 6-3. Action As a baseline test, all animals underwent a modified Irwin test, including a reflex test and a hanging wire test, once. Motor coordination was assessed using the hanging wire test (six times in total) throughout the test period, rotarod twice (at baseline and at the end of the test), and clasping behavior was assessed twice (at baseline and at the end of the test) every other week. Tests were conducted randomly.

[0078] (1) Hanging wire test The hanging wire test, which assessed neuromuscular abnormalities in motor strength, was performed biweekly during the study period (starting from week 2 of treatment) and as part of the Irwin test (for baseline behavior). This test used a wire cage lid with duct tape around the perimeter to prevent the mouse from slipping off the edge. The animal was placed on top of the cage lid. The lid was gently shaken three times to force the mouse to grasp the wire, and then the cage was inverted. The lid was held approximately 55–60 cm above a soft underlayment, high enough to prevent the mouse from jumping off but not so high that it would cause harm if it fell. The latency to fall was quantified. A cutoff time of 300 seconds was used. Normal mice can hang upside down for several minutes.

[0079] (2) Rotarod test The rotarod test was performed twice, at the beginning (baseline) and end of the test. In this test, motor coordination was assessed by placing the animal on a rotating rod (a four-lane rotarod; Ugo Basile) that moved at a constant or accelerating speed. If the mouse lost balance and fell onto the platform below, the rod was automatically stopped, and measurements of the fall speed and latency were recorded.

[0080] Prior to the first test session, mice were habituated to the testing system and allowed to remain on the rod at a constant speed of 2 rpm for approximately 1 min. During testing, each animal was exposed to the apparatus three times for 180 s. The initial speed increased from 2 rpm to 20 rpm with an acceleration time of 180 s. If the mouse fell, the session ended and the Ugo Basile Program stopped the timer.

[0081] (3) Clasping test Clasping behavior was assessed twice: at the beginning of the study (baseline) and at the end of the study. A score of 4: When suspended by the tail, the mouse can fully extend its hindlimbs away from the lateral midline and maintain this position for 2 seconds. When suspended 2-3 times for several seconds, the mouse demonstrates movement of both hindlimbs at least twice during the suspension. Score 3: When the tail is suspended, there is a deflection or partial deflection of the leg extension towards the lateral midline, or trembling of the hind limbs. Score 2: Toes curled downward or any part of the foot dragged along the cage bottom / table at least twice during a 30 cm (12 in) walk. Score 1: Rigid paralysis or minimal joint movement, with the foot not being used for forward movement. Score 0: The mouse falls to one side and is unable to get up within 15–30 seconds.

[0082] 6-4. Tissue sampling and processing At the end of the study, all animals were euthanized with an injection of pentobarbital (600 mg / kg), and the hemibrain and spinal cord were harvested.

[0083] (1) Perfusion Mice were transcardially perfused with 0.9% saline. To this end, a 23-gauge needle connected to a 0.9% saline bottle was inserted into the left ventricle. The right ventricle was opened with scissors. A constant pressure of 100–120 mm Hg was maintained on the perfusion solution by connecting the solution bottle to a manometer-controlled air compressor. Perfusion continued until the liver turned pale and only perfusate, instead of blood, exited the right ventricle.

[0084] (2) Brain sampling After perfusion, the skull was opened, and the brain was carefully removed and cut on one side on a cooled surface. The right hemibrain was fixed by immersion in freshly prepared 4% paraformaldehyde / PB (pH = 7.4) at room temperature for 2 hours. The left hemibrain was further separated into the hippocampus and the rest of the brain. All parts were weighed, snap-frozen on dry ice, and stored at -80°C.

[0085] (3) Spinal cord sampling Immunohistological examination (n=8 per group) For immunohistological analysis, the spinal column was dissected from the animal and much of the muscle tissue was removed to facilitate subsequent immersion fixation. The entire spinal column, including the cervical, thoracic, and lumbar regions, was transferred in an upright position to a 15 ml tube containing freshly prepared 4% paraformaldehyde / PB for fixation. The spinal column was fixed at 4°C for 24 hours.

[0086] Protein expression analysis (n=7 per group) For protein expression analysis by Western blotting, the vertebral column was dissected from the animals, the spinal cord was removed, weighed, frozen on dry ice, and stored at -80°C.

[0087] 6-5.Immunohistological analysis (1) Tissue preparation Right hemibrains (n ​​= 15 per group) were fixed by immersion in freshly prepared 4% paraformaldehyde in phosphate buffer (PB; pH 7.4) for 2 hours at room temperature. The specimens were then transferred to a 15% sucrose-PBS solution overnight for cryoprotection. Tissue blocks were then trimmed as needed, transferred to cryomolds, embedded in OCT medium, snap-frozen in dry-ice-cooled liquid isopentane, and stored in an ultra-deep freezer (set at a target temperature of -80°C) until sectioning.

[0088] Whole spinal columns (n ​​= 8 per group) were fixed by immersion in freshly prepared 4% paraformaldehyde in PB (pH 7.4) for 24 hours at 4°C. The following day, spinal cords were dissected from the fixed columns and transferred to a 15% sucrose-PBS solution overnight for cryoprotection. The tissue blocks were then cut into eight pieces, placed in cryomolds as tissue arrays, and embedded in OCT medium. The samples were then snap-frozen in dry-ice-cooled liquid isopentane and stored in an ultra-deep freezer (set at a target temperature of -80°C) until sectioning.

[0089] (2) Sectioning Right hemibrain (n = 8 per group) Five frozen sections from 12 mediolateral levels (60 sections total) were cut sagittally at 10 μm thickness on a Leica cryotome. The next 23 sections per level were discarded. Sectioning levels were selected according to Paxinos and Franklin (The Mouse Brain in Stereotaxic Coordinates, 2nd ed., 2001). Sections were collected through the hemisphere, starting at a level approximately 0.2 mm lateral to the midline, to ensure systematic random sampling throughout the target region (Figure 4). Sections were stored in a deep freezer (target temperature setting -20°C).

[0090] Spinal cord tissue array (n=8 per group) Five frozen sections from the spinal cord tissue array were cut at 10 μm thickness on a Leica cryotome. The next 15 sections were discarded. This collection scheme was repeated at five levels (30 sections total). As a result, sections were collected from the cervical, thoracic, lumbar, and sacral regions, allowing systematic random sampling throughout the spinal cord (Figure 5). Sections were stored in a deep freezer (target temperature setting -20°C).

[0091] (3) Immunohistological examination Experiments with I-GFAP, NeuN, and hTDP-43 In experiment I, glial fibrillary acidic protein (GFAP), neuronal nuclei (NeuN), and hTDP-43 were assessed in a uniform systematic random set of five sections per mouse brain (one section from L2, L4, L6, L8, and L10; total n = 160 sections) and one section from level 3 of the spinal cord tissue array (total n = 32 sections).

[0092] Experiment II - MBP, Iba1, MAP-2 In experiment II, myelin basic protein (MBP), ionized calcium-binding adaptor molecule 1 (Iba1), and microtubule-associated protein 2 (MAP-2) were assessed in a uniform systematic random set of five sections per mouse brain (one section from L2, L4, L6, L8, and L10; n = 160 sections total) and one section from level 3 of the spinal cord tissue array (n = 32 sections total).

[0093] (4) Imaging Whole slide scans of immunofluorescence stained sections were recorded on a Zeiss automated microscope AxioScan Z1 with high aperture lenses, equipped with a Zeiss Axiocam 506 mono and Hitachi 3CCD HV-F202SCL camera and Zeiss ZEN 2.3 software.

[0094] Immunofluorescently labeled tissue sections from Experiments I and II were analyzed by quantitative image analysis. Image analysis was performed using Image Pro 10 (Media Cybernetics). First, a region of interest (ROI) was drawn on the image to identify the target area ( Right hemibrain :Cortex and brainstem; spinal cord The following signals were then quantitatively evaluated within the identified regions: the cervical spinal cord, the thoracic spinal cord, and the left and right ventral horns of the lumbar spinal cord. Additional ROIs were added to exclude wrinkles, air bubbles, or other artifacts that could interfere with the measurements. ●GFAP, ●Iba1, ●hTDP-43, ●MBP, ●MAP2, ●NeuN

[0095] Quantification included background correction using Edge Plus filtering, followed by additional morphological filtering (size, shape) to detect immunoreactive objects by appropriate thresholding, as in the case of GFAP, Iba1, hTDP-43, and NeuN.

[0096] Then, as a readout, the characteristics of the different objects were quantified between them. Immunoreactive area [%]: the percentage of the ROI covered by objects above threshold (e.g., cell somas); this is the most comprehensive parameter indicating whether there is an overall difference in immunoreactivity. ●Object density [number of objects / mm 2 ]: the number of objects above a threshold normalized to the size of the target area. This is particularly useful for detecting changes in the density of immunoreactive objects. Object intensity [au]: average brightness of pixels of immunoreactive objects above threshold; this indicates whether there is a difference in the cellular expression level of the target protein. ●Object size [μm 2 ]: size of immunoreactive objects above a threshold; this is particularly useful for detecting changes in size of immunoreactive objects. ROI intensity [au]: average brightness of pixels within the ROI; this indicates whether there is an overall difference in the immunofluorescence signal. Once the target object parameters have been defined in the test run, quantitative image analysis is performed automatically, and the results are operator independent and fully reproducible.

[0097] In addition to the region size of the investigated ROI, the following data were provided for each readout (brain and spinal cord region): ●GFAP: Average object size [μm 2 ], average object strength [au], object density [n / mm 2 ], immune active area [%] ●Iba1: Average object size [μm 2 ], average object strength [au], object density [n / mm 2 ], immune active area [%] ●hTDP-43: Average object size [μm 2 ], average object strength [au], object density [n / mm 2 ], immune active area [%] ●MBP: ROI average value [au], immunoreactive area [%] ●MAP2: ROI average value [au], immunoreactive area [%] ●NeuN: Average object size [μm 2 ], average object strength [au], object density [n / mm 2 ], immune active area [%]

[0098] 6-6. Protein expression analysis (1) Sample preparation Frozen, unfixed tissue samples (hippocampi from all 15 animals per group and spinal cords from 7 animals per group) were homogenized in 5 volumes of RIPA buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% NP-40 or 0.1% Triton-X, 2% SDS, 1 μM NaF, 0.2 mM sodium deoxycholate, 80 μM glycerophosphate, 1× protease inhibitor (Calbiochem, Germany), 1× phosphatase inhibitor) by weight. Protein concentration was measured by BCA assay. Homogenates were stored at −80°C until further use.

[0099] (2) Western blotting Equal amounts of protein from each sample were separated by molecular weight via SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis; e.g., Any kD™ Mini-PROTEAN® TGX™ Precast Protein Gels, BioRad). Prestained protein markers on each gel visualized the correct separation of proteins and later confirmed the correct protein band sizes on Western blot film. Proteins were then transferred onto nitrocellulose membranes, blocked with 5% nonfat dry milk in 1x TBS (Tris-buffered saline), and incubated with specific antibodies: human TDP-43 antibody (1:1000, Abnova, Germany, H00023435-M01) and β-tubulin isotype III (1:5000, Sigma-Aldrich, USA, T8660). HPR (horseradish peroxidase)-conjugated secondary antibodies raised against the species of the primary antibody, ECL (enhanced chemiluminescence) and a C-digit blot scanner (Licor) were used for visualization and semi-quantification of the desired protein bands.

[0100] 7.Results Weight Statistical analysis was performed to evaluate the effect of Muse cells on body weight by comparing groups over the entire experimental period. Repeated measures ANOVA was performed to evaluate the significance level of the between-group factors. The results are shown in Figures 6 and 7. (1) Comparison between Group D and Group C, and Group C and Group A From weeks 1 to 12, group C showed a significant difference compared to group D, while group A showed no significant difference compared to group C. (2) Comparison between Group D and Group C, and Group C and Group B Group C showed significant differences compared to Group D during weeks 1 to 7 and weeks 8 to 12, while Group B showed no significant differences compared to Group C during each period.

[0101] 7-2. Hanging wire test To evaluate the effects of Muse cells in the hanging-wire test, statistical analysis was performed by comparing groups over the entire experimental period. Repeated-measures ANOVA was performed to evaluate the significance level of the between-group factor. The results are shown in Figures 8 and 9. (1) Comparison between Group D and Group C, and Group C and Group A From 0 to 12 weeks, a significant difference was observed between Group C and Group D, and a significant difference was observed between Group A and Group C. (2) Comparison between Group D and Group C, and Group C and Group B Group C showed significant differences compared to Group D from weeks 0 to 6 and weeks 8 to 12, while Group B showed no significant differences compared to Group C at any of the periods.

[0102] 7-3.Rotarod The results are shown in Figure 10. In tests 1 to 3 and in the average values ​​at baseline and 12 weeks, Group C showed a significant decrease compared to Group D, while Groups A and B showed no significant difference compared to Group C.

[0103] 7-4. Clasping The results are shown in Figure 11. At baseline and at week 12, Group C showed a significant decrease compared to Group D, while Groups A and B showed no significant difference compared to Group C.

[0104] 7-5. Evaluation of immunofluorescence in spinal cord tissue Immunofluorescence signals were quantified in the spinal cord (cervical, thoracic and lumbar) and statistically evaluated.

[0105] 7-5-1.hTDP-43 The results are shown in Figure 12. (1) Comparison between Group D and Group C Group C showed significant increases in mean cervical and thoracic object intensity, object density, immunoreactive area, and total lumbar readout compared to Group D, and two-way ANOVA showed significant differences between the factors: groups. (2) Comparison of Group C with Groups A and B Group A showed a significant decrease in mean object strength and immune reaction area in the chest and lumbar region compared to Group C. However, two-way ANOVA revealed significant differences between the factors: groups in mean object strength, object density, and immune reaction area. A Dunnett's test was then performed, revealing significant differences between Groups A and B compared to Group C.

[0106] 7-5-2.GFAP The results are shown in Figure 13. (1) Comparison between Group D and Group C Group C showed significant increases in mean object size, object density, and immunoreactive area in the cervical region, and mean object intensity, object density, and immunoreactive area in the thoracic and lumbar regions compared with Group D. As a result of two-way ANOVA, the factor: group showed significant differences in all readouts. (2) Comparison of Group C with Groups A and B Groups A and B showed a significant decrease in mean cervical object size compared to Group C. However, two-way ANOVA showed a significant difference in mean object size between the factors: Group. Therefore, a Dunnett's test was performed, and groups A and B showed a significant difference compared to Group C.

[0107] 7-5-3.MAP2 The results are shown in Figure 14. (1) Comparison between Group D and Group C Group C showed a significant increase in the immunoreactive area of ​​the chest compared with Group D. As a result of two-way ANOVA, the factor: group showed significant differences in the mean ROI intensity and immunoreactive area. (2) Comparison of Group C with Groups A and B In all readouts, groups A and B showed no significant differences compared to group C. However, two-way ANOVA showed significant differences in the mean ROI intensity and immunoreactive area for the factor: group. Therefore, a Dunnett's test showed significant differences in the mean ROI intensity for groups A and B compared to group C, and in the immunoreactive area for group A compared to group C.

[0108] 7-5-4.Iba-1 The results are shown in Figure 15. (1) Comparison between Group D and Group C Group C showed a significant increase in mean cervical object size, thoracic object density, and lumbar object density and immunoreactive area compared with Group D. As a result of two-way ANOVA, the factor: group showed significant differences in all readouts. (2) Comparison of Group C with Groups A and B In all readouts, groups A and B showed no significant differences compared to group C. As a result of two-way ANOVA, the factor: group showed a significant difference in mean object intensity. Therefore, a Dunnett's test was performed, and group A showed a significant difference compared to group C.

[0109] 7-5-5.NeuN Group C showed a significant increase in cervical mass density compared to Group D. Two-way ANOVA showed significant differences in mass density for the factor: Group. Groups A and B did not show any significant effect on these changes (data not shown).

[0110] 7-5-6.MBP In all readouts, Group C showed no significant differences compared to Group D. As a result of two-way ANOVA, the factor: Group showed no significant differences (data not shown).

[0111] 7-6. Evaluation of immunofluorescence in brain tissue Immunofluorescence signals were quantified in the brain (cerebral cortex and brainstem) and statistically evaluated.

[0112] 7-6-1.hTDP-43 The results are shown in Figure 16. In all brainstem and cortex readings, Group C showed a significant increase compared to Group D. Groups A to B showed no significant difference compared to Group C.

[0113] 7-6-2.GFAP The results are shown in Figure 17. Group C showed a significant increase in object density and immunoreactive area in the brainstem, as well as in all readings of the cerebral cortex, compared with Group D. Groups A and B showed no significant differences compared with Group C.

[0114] 7-6-3.MAP2 In all brainstem and cortical readings, group C showed no significant differences compared with group D (data not shown).

[0115] 7-6-4.Iba-1 The results are shown in Figure 18. In all brainstem readings and in the mean cortical object intensity, Group C showed a significant increase compared to Group D. Groups A and B showed no significant differences compared to Group C.

[0116] 7-6-5.NeuN The results are shown in Figure 19. Group C showed a significant decrease in mean object size in the brainstem compared to Group D, and a significant increase in cortical object density compared to Group D. In all readings, Groups A to B showed no significant differences compared to Group C.

[0117] 7-6-6.MBP In all brainstem and cortical readings, group C showed no significant differences compared with group D (data not shown).

[0118] Western blot analysis TDP-43 levels determined by Western blot were quantified in the spinal cord and hippocampus and statistically evaluated. In the spinal cord and hippocampus, TDP-43 / β-tubulin isotype III levels in Group C were significantly increased compared to Group D. Groups A and B showed no significant difference compared to Group C (Figure 20).

[0119] Consideration This study is the first to demonstrate that a single intravenous injection of Muse cells at 8 weeks of age improves the performance of the hanging-wire test in hTDP-43 Tg mice, an ALS model. Immunohistological evaluation revealed that the increased hTDP-43 positive reaction in the spinal cord of hTDP-43 Tg mice was reduced, suggesting that the administration of Muse cells may have improved motor function by suppressing the aggregation of hTDP-43 in the cytoplasm of spinal neurons.

[0120] Immunohistological analysis From the seven right hemibrains prepared in 6-5(1) that were not used in the immunohistological analysis described above, frozen sections were prepared in the same manner as in (2). In addition, frozen sections were prepared in the same manner using the remaining samples from the spinal cord tissue array (n = 8 per group). Using the prepared sections, ubiquitin (an indicator of TDP-43 protein accumulation in the cytoplasm), translocator protein (an indicator of neuroinflammation in neurodegenerative diseases), and choline acetyltransferase (a marker of primary and secondary motor neurons) were evaluated by fluorescent immunostaining as described above.

[0121] By evaluating the expression state of these proteins in the brain and spinal cord by immunohistochemistry, we will be able to further clarify the characteristics of the ameliorative effects of Muse cells on the pathology of the ALS model hTDP-43 Tg mouse (e.g., inhibitory effects on TDP-43 protein accumulation in the cytoplasm, inhibitory effects on neuroinflammation, and protective effects on motor neurons).

[0122] Protein expression analysis Using the left hemibrain sections (except the hippocampus, 15 cases) not used in the Western blot analysis, nuclear and cytoplasmic fractions were separated and Western blot analysis was performed on each fraction using a human TDP-43 antibody. Because TDP-43-induced neurodegeneration is induced by the translocation of TDP-43 from the nuclear to the cytoplasmic fraction, examining the effect of Muse cell administration on the distribution of TDP-43 in each fraction will enable further characterization of the ameliorative effect of Muse cells on the pathology of the ALS model hTDP-43 Tg mice.

[0123] The entire disclosures of all references cited above and corresponding applications are incorporated herein by reference in their entirety.

[0124] References [1] Aoki, M. et al. Mild ALS in Japan associated with novel SOD mutation. Nat Genet 5, 323-324, doi:10.1038 / ng1293-323 (1993). [2] Gurney, ME et al. Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science 264, 1772-1775, doi:10.1126 / science.8209258 (1994). [3] Rosen, DR et al. Mutations in Cu / Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362, 59-62, doi:10.1038 / 362059a0 (1993). [4] Arai, T. et al. TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem Biophys Res Commun 351, 602-611, doi:10.1016 / j.bbrc.2006.10.093 (2006). [5] Kabashi, E. et al. TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet 40, 572-574, doi:10.1038 / ng.132 (2008). [6] DeJesus-Hernandez, M. et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245-256, doi:10.1016 / j.neuron.2011.09.011 (2011). [7] Renton, A. E. et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257-268, doi:10.1016 / j.neuron.2011.09.010 (2011). [8] Abe, K. et al. Exploratory double-blind, parallel-group, placebo-controlled study of edaravone(MCI-186) in amyotrophic lateral sclerosis (Japan ALS severity classification: Grade 3, requiring assistance for eating, excretion or ambulation). Amyotroph Lat Scl Fr 18, 40-48, doi:10.1080 / 21678421.2017.1361441 (2017). [9] Writing, G. & Edaravone, A. L. S. S. G. Safety and efficacy of edaravone in well defined patients with amyotrophic lateral sclerosis: a randomised, double-blind, placebo-controlled trial. Lancet Neurol 16, 505-512, doi:10.1016 / S1474-4422(17)30115-1 (2017).

[10] Kuroda, Y. et al. Unique multipotent cells in adult human mesenchymal cell populations. Proc Natl Acad Sci USA 107, 8639-8643, doi:10.1073 / pnas.0911647107 (2010).

[11] Kuroda, Y. et al. Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells. Nat Protoc 8, 1391-1415, doi:10.1038 / nprot.2013.076 (2013).

[12] Tanaka, T. et al. Mobilized Muse Cells After Acute Myocardial Infarction Predict Cardiac Function and Remodeling in the Chronic Phase. Circ J 82, 561-571, doi:10.1253 / circj.CJ-17-0552 (2018).

[13] Wakao, S. et al. Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts. Proc Natl Acad Sci USA 108, 9875-9880, doi:10.1073 / pnas.1100816108 (2011).

[14] Fujita, Y. et al. Intravenous injection of Muse cells as a potential therapeutic approach for epidermolysis bullosa. J Invest Dermatol, doi:10.1016 / j.jid.2020.05.092 (2020).

[15] Iseki, M. et al. Muse Cells, Nontumorigenic Pluripotent-Like Stem Cells, Have Liver Regeneration Capacity Through Specific Homing and Cell Replacement in a Mouse Model of Liver Fibrosis. Cell Transplant 26, 821-840, doi:10.3727 / 096368916X693662 (2017).

[16] Uchida, H. et al. Human Muse Cells Reconstruct Neuronal Circuitry in Subacute Lacunar Stroke Model. Stroke 48, 428-435, doi:10.1161 / STROKEAHA.116.014950 (2017).

[17] Uchida, N. et al. Beneficial Effects of Systemically Administered Human Muse Cells in Adriamycin Nephropathy. J Am Soc Nephrol 28, 2946-2960, doi:10.1681 / ASN.2016070775 (2017).

[18] Yamada, 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. Circ Res 122, 1069-1083, doi:10.1161 / CIRCRESAHA.117.311648 (2018).

[19] Yabuki, H., Wakao, S., Kushida, Y., Dezawa, M. & Okada, Y. Human Multilineage-differentiating Stress-Enduring Cells Exert Pleiotropic Effects to Ameliorate Acute Lung Ischemia-Reperfusion Injury in a Rat Model. Cell Transplant 27, 979-993, doi:10.1177 / 0963689718761657 (2018).

[20] Dezawa, M. Clinical trials of Muse cells. Adv. Exp. Med. Biol. 1103, 305-307, doi:10.1007 / 978-4-431-56847-6 17 (2018).

[21] Noda, T., Nishigaki, K. & Minatoguchi, S. Safety and Efficacy of Human Muse Cell-Based Product for Acute Myocardial Infarction in a First-in-Human Trial. Circ J, doi:10.1253 / circj.CJ-20-0307 (2020).

[22] Hayase, M. et al. Committed neural progenitor cells derived from genetically modified bone marrow stromal cells ameliorate deficits in a rat model of stroke. J Cerebr Blood F Met 29, 1409-1420, doi:10.1038 / jcbfm.2009.62 (2009).

[23] Chen, Y., Imai, H., Ito, A. & Saito, N. Novel modified method for injection into the cerebrospinal fluid via the cerebellomedullary cistern in mice. Acta Neurobiol Exp (Wars) 73, 304-311 (2013).

[24] Ohta, Y. et al. Therapeutic benefits of intrathecal protein therapy in a mouse model of amyotrophic lateral sclerosis. J Neurosci Res 86, 3028-3037, doi:10.1002 / jnr.21747 (2008).

[25] Saito, K. et al. Luminescent proteins for high-speed single-cell and whole-body imaging. Nat Commun 3, 1262, doi:10.1038 / ncomms2248 (2012).

[26] Abe, T. et al. Intravenously Transplanted Human Multilineage-Differentiating Stress-Enduring Cells Afford Brain Repair in a Mouse Lacunar Stroke Model. Stroke 51, 601-611, doi:10.1161 / STROKEAHA.119.026589 (2020).

[27] Vinsant, S. et al. Characterization of early pathogenesis in the SOD1(G93A) mouse model of ALS: part I, background and methods. Brain Behav 3, 335-350, doi:10.1002 / brb3.143 (2013).

[28] Abe, K., Morita, S., Kikuchi, T. & Itoyama, Y. Protective effect of a novel free radical scavenger, OPC-14117, on wobbler mouse motor neuron disease. J Neurosci Res 48, 63-70 (1997).

[29] Hosaka, Y. et al. Alpha1-syntrophin-deficient skeletal muscle exhibits hypertrophy and aberrant formation of neuromuscular junctions during regeneration. J Cell Biol 158, 1097-1107, doi:10.1083 / jcb.200204076 (2002).

[30] Nagano, I. et al. Therapeutic benefit of intrathecal injection of insulin-like growth factor-1 in a mouse model of Amyotrophic Lateral Sclerosis. J Neurol Sci 235, 61-68, doi:10.1016 / j.jns.2005.04.011 (2005).

[31] Manabe, Y. et al. Glial cell line-derived neurotrophic factor protein prevents motor neuron loss of transgenic model mice for amyotrophic lateral sclerosis. Neurol Res 25, 195-200, doi:10.1179 / 016164103101201193 (2003).

[32] Ohta, Y. et al. Neuroprotective and Angiogenic Effects of Bone Marrow Transplantation Combined With Granulocyte Colony-Stimulating Factor in a Mouse Model of Amyotrophic Lateral Sclerosis. Cell Med 2, 69-83, doi:10.3727 / 215517910X582779 (2011).

[33] Bossolasco, P. et al. Metalloproteinase alterations in the bone marrow of ALS patients. J Mol Med (Berl) 88, 553-564, doi:10.1007 / s00109-009-0584-7 (2010).

[34] Kondo, T. et al. Focal transplantation of human iPSC-derived glial-rich neural progenitors improves lifespan of ALS mice. Stem Cell Reports 3, 242-249, doi:10.1016 / j.stemcr.2014.05.017 (2014).

[35] Miyazaki, K. et al. Disruption of neurovascular unit prior to motor neuron degeneration in amyotrophic lateral sclerosis. J Neurosci Res 89, 718-728, doi:10.1002 / jnr.22594 (2011).

[36] Yamanaka, K. et al. Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis. Nat Neurosci 11, 251-253, doi:10.1038 / nn2047 (2008).

Claims

1. A cell preparation for treating, preventing, alleviating, and / or delaying the onset of motor neuron disease (MND) in a subject, comprising SSEA-3-positive and CD105-positive pluripotent stem cells isolated from mesenchymal tissue of a living body or cultured mesenchymal cells, wherein the pluripotent stem cells have all of the following properties: (i) low or absent telomerase activity; (ii) have the ability to differentiate into cells of any of the three germ layers; (iii) does not exhibit neoplastic growth; and (iv) It has self-renewal ability.

2. The cell preparation according to claim 1, comprising a cell fraction enriched in SSEA-3-positive pluripotent stem cells due to an external stress stimulus.

3. The cell preparation according to claim 1 or 2, wherein the pluripotent stem cells are CD117-negative and CD146-negative.

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

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

6. The cell preparation according to any one of claims 1 to 5, wherein the MND is amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), spinal muscular atrophy (SMA), progressive muscular atrophy (PMA), or spinal-bulbar muscular atrophy (SBMA).

7. The cell preparation according to any one of claims 1 to 6, wherein the pluripotent stem cells have the ability to engraft in the spinal cord.

Images