Pluripotent stem cells for treatment of spinal cord infarction

Abstract

The purpose of the present invention is to provide a new medical use of pluripotent stem cells (Muse cells) in regenerative therapy. The present invention provides a cell preparation and a pharmaceutical composition for treating, preventing, alleviating and / or delaying onset of spinal cord infarction, comprising SSEA-3-positive pluripotent stem cells isolated from biologically derived mesenchymal tissues or cultured mesenchymal cells. The present invention is based on a mechanism in which Muse cells are administered to a subject having spinal cord infarction, and the cells are grafted to the tissue of the spinal cord infarction site, thereby treating spinal cord infarction.

Description

Pluripotent stem cells for the treatment of spinal cord infarction

[0001] The present invention relates to a cell preparation for regenerative medicine. More specifically, the present invention relates to a cell preparation comprising pluripotent stem cells that is effective for treating, preventing, alleviating, and / or delaying the onset of spinal cord infarction in a subject.

[0002] Paraplegia, a perioperative complication of aortic surgery, is caused by spinal cord ischemic injury or spinal cord ischemia-reperfusion injury, with spinal cord infarction being the primary pathology. In addition to decreased lower limb motor function, it can also cause sensory impairment and bladder-rectum dysfunction, significantly impacting quality of life and prognosis. The incidence of paraplegia following thoracic-abdominal aortic surgery has been reported to be 8.3% for thoracic-abdominal aortic artificial vascular replacement (Non-Patent Document 1) and 11% for thoracic-abdominal aortic stent grafting (Non-Patent Document 2), even in facilities with extensive surgical experience. Despite dramatic advances in preoperative imaging diagnostic techniques and intraoperative monitoring techniques, the incidence of spinal cord injury has not significantly improved over the past decade. Cerebrospinal fluid drainage (Non-Patent Document 3), permissive hypertension, and drug administration such as steroids and naloxone are currently used as treatments, but their effectiveness is limited, and no established treatment method exists.

[0003] Numerous experimental studies using mesenchymal stem cells (MSCs) have demonstrated therapeutic effects for spinal cord ischemic injury, but these studies have only been conducted over a short period of time, and the therapeutic mechanism is believed to be due to paracrine or anti-inflammatory effects (Non-Patent Documents 4-10). Multilineage-differentiating stress-enduring (Muse) cells were reported in 2010 as a new pluripotent stem cell present in the human body (Non-Patent Document 11). Muse cells are present in all connective tissues, blood, and bone marrow in adults and can be identified by the pluripotency marker SSEA-3 (stage-specific embryonic antigen-3) (Non-Patent Document 12). Unlike embryonic stem (ES) cells and induced-pluripotent stem (iPS) cells, Muse cells are present in vivo and therefore do not exhibit tumorigenicity. Furthermore, Muse cells migrate to damaged tissues by simply administering them intravascularly, recognizing sphingosine-1-phosphate (S1P) as a neurotransmitter (Non-Patent Document 13). They then phagocytose apoptotic cells, spontaneously differentiating into tissue-specific cells and leading to tissue repair (Non-Patent Document 14). Previous studies on myocardial infarction models (Non-Patent Document 13), cerebral infarction models (Non-Patent Document 15), aortic aneurysm models (Non-Patent Document 16), renal failure models (Non-Patent Document 17), and acute liver injury models (Non-Patent Document 18) have shown that intravenous or local administration of Muse cells results in tissue-specific differentiation and structural and functional improvement (Patent Documents 1 to 4). Furthermore, because Muse cells express human leukocyte antigen (HLA)-G, which is expressed in the placenta, allogeneic donor Muse cells administered intravenously are less susceptible to immune rejection, selectively home to the recipient's damaged tissue, and survive as functional, differentiated cells for a long period of time, more than six months. In a clinical trial (JapicCTI-184103) in which Muse cell administration treatment was performed on patients with cerebral infarction, it was observed that the Muse cell administration group maintained a statistically significant improvement in upper limb motor function compared to the placebo group (Non-Patent Document 19).The bystander effect of Muse cells has also been reported to have anti-apoptotic effects (Non-Patent Document 20), anti-fibrotic and fibrolytic effects (Non-Patent Document 17), and angiogenic effects (Non-Patent Document 13). Furthermore, Muse cells possess stress resistance and anti-inflammatory properties (Non-Patent Documents 21 and 22), and are expected to persist even under stressful inflammatory conditions. Because Muse cells are endogenous stem cells, functional improvement and tissue repair of damaged tissues may occur as a biological response. Furthermore, administration of Muse cells into the body from an exogenous source is expected to enhance the therapeutic effects. These unique properties may enable the establishment of spinal cord infarction treatment with Muse cells as a new proactive treatment that goes beyond conventional conservative treatments and other cell therapies.

[0004] Patent No. 5968442 Patent No. 6604492 Patent No. 7029729 Patent No. 7072777

[0005] Moulakakis KG, Karaolanis G, Antonopoulos CN, et al. Open repair of thoracoabdominal aortic aneurysms in experienced centers. J Vasc Surg. Aug 2018;68(2):634-645 e12. doi:10.1016 / j.jvs.2018.03.410Pini R, Faggioli G, Paraskevas KI, et al. A systematic review and meta-analysis of the occurrence of spinal cord ischemia after endovascular repair of thoracoabdominal aortic aneurysms. J Vasc Surg. Apr 2022;75(4):1466-1477 e8. doi:10.1016 / j.jvs.2021.10.015Coselli JS, LeMaire SA, Koksoy C, Schmittling ZC, Curling PE. Cerebrospinal fluid drainage reduces paraplegia after thoracoabdominal aortic aneurysm repair: results of a randomized clinical trial. J Vasc Surg. Apr 2002;35(4):631-9. doi:10.1067 / mva.2002.122024Yin F, Guo L, Meng CY, et al. Transplantation of mesenchymal stem cells exerts anti-apoptotic effects in adult rats after spinal cord ischemia-reperfusion injury. Brain Res. May 2 2014;1561:1-10. doi:10.1016 / j.brainres.2014.02.047Wang Z, Fang B, Tan Z, Zhang D, Ma H.Hypoxic preconditioning increases the protective effect of bone marrow mesenchymal stem cells on spinal cord ischemia / reperfusion injury. Mol Med Rep. Mar 2016;13(3):1953-60. doi:10.3892 / mmr.2016.4753Fang B, Wang H, Sun XJ, et al. Intrathecal transplantation of bone marrow stromal cells attenuates blood-spinal cord barrier disruption induced by spinal cord ischemia-reperfusion injury in rabbits. J Vasc Surg. Oct 2013;58(4):1043-52. doi:10.1016 / j.jvs.2012.11.087Yasuda N, Kuroda Y, Ito T, et al. Postoperative spinal cord ischaemia: magnetic resonance imaging and clinical features. Eur J Cardiothorac Surg. Jul 14 2021;60(1):164-174. doi:10.1093 / ejcts / ezaa476Nakai H, Fujita Y, Masuda S, et al. Intravenous injection of adult human bone marrow mesenchymal stromal cells attenuates spinal cord ischemia / reperfusion injury in a murine aortic arch crossclamping model. JTCVS Open. Sep 2021;7:23-40. doi:10.1016 / j.xjon.2021.06.008Takahashi S, Nakagawa K, Tomiyasu M, et al.Mesenchymal Stem Cell-Based Therapy Improves Lower Limb Movement After Spinal Cord Ischemia in Rats. Ann Thorac Surg. May 2018;105(5):1523-1530. doi:10.1016 / j.athoracsur.2017.12.014Kurose T, Takahashi S, Otsuka T, et al. Simulated microgravity-cultured mesenchymal stem cells improve recovery following spinal cord ischemia in rats. Stem Cell Res. Dec 2019;41:101601. doi:10.1016 / j.scr.2019.101601Kuroda Y, Kitada M, Wakao S, et al. Unique multipotent cells in adult human mesenchymal cell populations. Proceedings of the National Academy of Sciences of the United States of America. May 11 2010;107(19):8639-43. doi:10.1073 / pnas.0911647107Dezawa M. Muse Cells Provide the Pluripotency of Mesenchymal Stem Cells: Direct Contribution of Muse Cells to Tissue Regeneration. Cell Transplant. 2016;25(5):849-61. doi:10.3727 / 096368916X690881Yamada Y, Wakao S, Kushida Y, et al.S1P-S1PR2 Axis Mediates Homing of Muse Cells Into Damaged Heart for Long-Lasting Tissue Repair and Functional Recovery After Acute Myocardial Infarction. Circ Res. Apr 13 2018;122(8):1069-1083. doi:10.1161 / CIRCRESAHA.117.311648Wakao S, Oguma Y, Kushida Y, Kuroda Y, Tatsumi K, Dezawa M. Phagocytosing differentiated cell-fragments is a novel mechanism for controlling somatic stem cell differentiation within a short time frame. Cell Mol Life Sci. Oct 6 2022;79(11):542. doi:10.1007 / s00018-022-04555-0Uchida H, Niizuma K, Kushida Y, et al. Human Muse Cells Reconstruct Neuronal Circuitry in Subacute Lacunar Stroke Model. Stroke. Feb 2017;48(2):428-435. doi:10.1161 / STROKEAHA.116.014950Hosoyama K, Wakao S, Kushida Y, et al. Intravenously injected human multilineage-differentiating stress-enduring cells selectively engraft into mouse aortic aneurysms and attenuate dilatation by differentiating into multiple cell types. J Thorac Cardiovasc Surg. Jun 2018;155(6):2301-2313.e4. doi:10.1016 / j.jtcvs.2018.01.098Uchida N, Kushida Y, Kitada M, et al. Beneficial Effects of Systemically Administered Human Muse Cells in Adriamycin Nephropathy. J Am Soc Nephrol. Oct 2017;28(10):2946-2960. doi:10.1681 / ASN.2016070775Iseki M, Kushida Y, Wakao S, 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. May 9 2017;26(5):821-840. doi:10.3727 / 096368916X693662Niizuma K, Osawa SI, Endo H, et al. Randomized placebo-controlled trial of CL2020, an allogenic muse cell-based product, in subacute ischemic stroke. J Cereb Blood Flow Metab. Sep 27 2023:271678X231202594. doi:10.1177 / 0271678X231202594Yabuki 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. Jun 2018;27(6):979-993. doi:10.1177 / 0963689718761657Alessio N, Ozcan S, Tatsumi K, et al. The secretome of MUSE cells contains factors that may play a role in regulation of stemness, apoptosis and immunomodulation. Cell Cycle. Jan 2 2017;16(1):33-44. doi:10.1080 / 15384101.2016.1211215Gimeno ML, Fuertes F, Barcala Tabarrozzi AE, et al. Pluripotent Nontumorigenic Adipose Tissue-Derived Muse Cells have Immunomodulatory Capacity Mediated by Transforming Growth Factor-beta1. Stem Cells Transl Med. Jan 2017;6(1):161-173. doi:10.5966 / sctm.2016-0014.

[0006] 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 spinal cord infarction in a subject, as well as methods for treating subjects with the above-mentioned diseases using the same.

[0007] The present inventors prepared a rat spinal cord infarction model and used the rat model to examine the therapeutic effect of spinal cord infarction by administering Muse cells. As a result, they found that a significant improvement in motor function was achieved compared to the control group, leading to the completion of the present invention.

[0008] That is, the present invention is as follows: [1] A cell preparation for treating, preventing, alleviating, and / or delaying the onset of spinal cord infarction in a subject, comprising SSEA-3-positive pluripotent stem cells isolated from mesenchymal tissue of a living body or cultured mesenchymal cells. [2] The cell preparation according to [1], comprising a cell fraction enriched in SSEA-3-positive pluripotent stem cells by external stress stimulation. [3] The cell preparation according to [1] or [2], comprising a cell fraction enriched in SSEA-3-positive pluripotent stem cells by MACS and / or FACS. [4] The cell preparation according to any of [1] to [3], wherein the pluripotent stem cells are CD105-positive. [5] The cell preparation according to any of [1] to [4], wherein the pluripotent stem cells are CD117-negative and CD146-negative. [6] The cell preparation according to any one of [1] to [5], wherein the pluripotent stem cells are CD117-negative, CD146-negative, NG2-negative, CD34-negative, vWF-negative, and CD271-negative. [7] The cell preparation according to any one of [1] to [6], 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. [8] The cell preparation according to any one of [1] to [7], wherein the pluripotent stem cells have all of the following properties: (i) low or no telomerase activity; (ii) the ability to differentiate into cells of any of the three germ layers; (iii) the lack of neoplastic growth; and (iv) the ability to self-renew. [9] The cell preparation according to any one of [1] to [8], wherein the spinal cord infarction is motor dysfunction, sensory dysfunction, or bladder-rectum dysfunction.

[10] The cell preparation according to any one of [1] to [9], wherein the pluripotent stem cells have the ability to engraft into tissue at the site of spinal cord infarction.

[11] A method for producing a cell preparation for treating, preventing, alleviating, and / or delaying the onset of spinal cord infarction in a subject, comprising SSEA-3-positive pluripotent stem cells, the method comprising a step of isolating the SSEA-3-positive pluripotent stem cells from mesenchymal tissue of a living organism or cultured mesenchymal cells.

[12] The method according to

[11] , comprising a step of enriching SSEA-3-positive pluripotent stem cells by external stress stimulation and / or MACS.

[0009] The present invention can improve spinal cord infarction by administering Muse cells intravenously or otherwise to a subject suffering from spinal cord infarction, thereby facilitating differentiation of Muse cells at the site of spinal cord infarction into cells that constitute normal tissue surrounding the site through a tissue regeneration mechanism.

[0010] The experimental system schema is shown. The day the spinal cord infarction model was created was defined as Day 0 (Day 0). Behavioral evaluation was performed using the BBB Locomotor Scale on Day 1, and animals with a BBB Locomotor Score of 0 were used in the experiment. Mice were randomly assigned to the MACS-Muse group, MSC group, or vehicle group, and behavioral evaluation and weight measurement were performed on Days 2, 3, 5, and 7, and every other week thereafter until Day 56. Mice were then sacrificed. Mice to be used in the in vivo imaging system (IVIS) were sacrificed on Day 7 and photographed. SSEA-3-positive cells were collected from MSCs using MACS. The SSEA-3 positivity rate in the final cell group is shown by FACS analysis. MSCs passaged 7 to 9 were used. SSEA-3-positive cells were enriched by MACS, and a cell population with an SSEA-3 positivity rate of >70% was defined as MACS-Muse cells. The time course of the BBB motor function score in each group is shown. The time course of the BBB motor function score for the MACS-Muse group (n = 13), MSC group (n = 12), and vehicle group (n = 12) is shown. The BBB motor function score at day 56 was 9.5 ± 1.7 for the MACS-Muse group, 4.7 ± 1.7 for the MSC group, and 4.5 ± 1.3 for the vehicle group (MACS-Muse group vs. MSC group; p < 0.01, MACS-Muse vs. vehicle group; p < 0.01, MSC group vs. vehicle group; no significant difference). The time course of body weight in each group up to day 56 is shown. MACS-Muse group (n=13), MSC group (n=12), vehicle group (n=12). The average preoperative body weight was defined as 1.0. On Day 1, the weights were 0.93 for the MACS-Muse group, 0.93 for the MSC group, and 0.94 for the vehicle group. On Day 14, the weights were 1.00 for the MACS-Muse group, 0.96 for the MSC group, and 0.96 for the vehicle group. On Day 56, the weights were 1.25 for the MACS-Muse group, 1.22 for the MSC group, and 1.20 for the vehicle group. There were no statistically significant differences in the preoperative weight ratios between groups at each observation time point. The in vivo localization and distribution of administered cells by IVIS is shown. On Day 7, (A) accumulation of Muse cells and MSCs in the spinal cord and (B) in the lungs. (C) IVIS images of the brain, heart, stomach, small intestine, large intestine, pancreas, spleen, liver, kidney, bladder, femur, tibia, fibula, and thigh muscle. (D) Total luminosity of each organ.Akaluc-Muse group (n=3), Akaluc-MSC group (n=3), ＊ p<0.05. The localization of intravenously administered Muse cells in spinal cord cross sections is shown. (A) Spinal cord tissue from the Akaluc-Muse group (Day 7). Akaluc-GFP-positive cells were observed mainly in the ventral horn of the spinal cord. The white dotted line indicates the outline of the gray matter. (B) Spinal cord tissue from the MACS-Muse group (Day 56). Human mitochondrial-positive cells were observed mainly in the ventral horn of the spinal cord. The white dotted line indicates the outline of the gray matter. Positive and negative controls are shown for each staining. Human umbilical cord was used in a and b. The cerebrum of a GFP mouse was used in c and d. The localization of intravenously administered Muse cells in spinal cord cross sections is shown. (A) Seven cross sections, 3 mm rostral, 6 mm caudal, and 9 mm from the spinal cord infarction (puncture site), were randomly selected from the left and right sides, with 800 μm square fields centered on the ventral horn of the spinal cord. (B) Number of human mitochondrial-positive cells in each cross section (n = 3). The number of human mitochondrial-positive cells in the infarct center was statistically significantly lower than those 6 mm rostral and 6 mm caudal (p < 0.05). This shows differentiation of Muse cells into neurons and vascular cells. Immunohistological staining of the spinal cord in the MACS-Muse group on Day 56. Double positivity was observed for human mitochondria and neuronal markers NeuN, MAP-2, and TUJ-1, oligodendrocyte marker GST3 / GST-pi, and vascular endothelial marker CD31. No double-positive cells were identified for the astrocyte marker GFAP or the microglia marker Iba-1. White arrows indicate double-positive cells. Bar = 50 μm. Positive and negative controls for each staining are shown. a-f: Rat spinal cord gray matter was used. g and h: Rat spinal cord white matter was used. i and j: Rat anterior spinal artery was used. The differentiation efficiency into neural cells is shown. The percentage of cells double-positive with human mitochondria was NeuN 70.6±5.4%, MAP-2 68.0±11.4%, and GST3 / GST-pi 10.6±0.3%. No cells double-positive with GFAP or Iba-1 were confirmed.

[0011] 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 spinal cord infarction in a subject, as well as a method for treating spinal cord infarction using the cell preparation, etc. The present invention will be described in detail below.

[0012] Abbreviations used in this specification are detailed below. Abbreviations commonly used in the technical field will follow conventional conventions.

[0013] Abbreviations used BBB: Basso, Beattie, Bresnahan CED: Convection-enhanced delivery DMEM: Dulbecco's modified Eagle's medium ET-1: Endothelin-1 FACS: Fluorescence-activated cell sorting FBS: Fetal bovine serum FITC: Fluorescein isothiocyanate GFAP: Glial fibrillary acidic protein GFP: Green fluorescent protein GST-pi: Glutathione S-transferase-pi hMit: Human mitochondrial HLA: Human leukocyte antigen Iba1: Ionized calcium-binding adaptor molecule 1 iPS: Induced pluripotent stem cells IVIS: In vivo imaging system MACS: Magnetic-activated cell sorting MAP-2: Microtubule-associated protein-2 MSC: Mesenchymal stem cells Muse: Multilineage-differentiating stress-ending NeuN: neuronal nucleus NO: nitric oxide PBS: phosphate-buffered saline PFA: paraformaldehyde S1P: sphingosine-1-phosphate S1PR2: sphingosine-1-phosphate receptor 2 SSEA-3: stage-specific embryonic antigen-3 TTC: triphenyltetrazolium chloride TUJ-1: anti-β-tubulin 3 TUNEL: TdT-mediated dUTP nick end labeling

[0014] 1. Applicable Diseases The present invention can be used to treat, prevent, alleviate, and / or delay the onset of spinal cord infarction (ischemic myelopathy) using a cell preparation or pharmaceutical composition containing SSEA-3-positive pluripotent stem cells (Muse cells). Spinal cord infarction is usually caused by ischemia originating from an artery outside the spinal canal and is characterized by sudden severe back pain, followed immediately by rapidly progressive, bilateral flaccid muscle weakness and sensory loss (particularly thermal pain sensation) in the limbs.

[0015] According to the present invention, the cell preparation of the present invention can be used to treat disorders caused by spinal cord infarction, preferably motor dysfunction and sensory (functional) dysfunction. As used herein, "motor dysfunction" refers to a condition in which voluntary movement is difficult, impossible, or cannot be performed smoothly, and includes motor paralysis and ataxia. Specific examples include disorders of dexterity, Babinski's sign, spasticity, spasm (chronic phase), increased deep tendon reflexes (chronic phase), muscle rigidity, bradykinesia, involuntary movement (tremor, chorea, athetosis, dystonia, etc.), ataxia (limb / trunk), and gait dysfunction. "Sensory impairment" is used interchangeably with "sensory dysfunction" and refers to a condition in which sensations such as superficial sensations like temperature, pressure, and touch, deep sensations like position sense and vibration, and complex sensations like two-point discrimination and cutaneous writing sense are not recognized normally due to damage to the spinal cord. Depending on the severity, symptoms include sensory loss (anesthesias), hypoesthesia (decreased sensation), hyperesthesia, and abnormal sensations (paresthesia).

[0016] 2. Cell Preparations and Pharmaceutical Compositions (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. Furthermore, these cells have the properties of both pluripotent stem cells and mesenchymal stem cells, and are identified, for example, as being double-positive for the respective 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 using, for example, these antigen markers as indicators. Details of the isolation, identification, and characteristics of Muse cells are disclosed in International Publication No. WO2011 / 007900. Furthermore, as reported by Wakao et al. (S. Wakao, et al., Proc. Natl. Acad. Sci. USA, Vol. 108, pp. 9875-9880 (2011)), it has been found that 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 CD105-positive cells. Therefore, in the cell preparation of the present invention, when Muse cells are isolated from biological mesenchymal tissue or cultured mesenchymal stem cells, the Muse cells can be purified and used simply using SSEA-3 as an antigen marker. Note that, herein, pluripotent stem cells (Muse cells) or cell populations containing 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 for treating motor neuron diseases, may be simply referred to as "SSEA-3-positive cells."In addition, as used herein, "non-Muse cells" refer to cells contained in biological mesenchymal tissue or cultured mesenchymal tissue, other than "SSEA-3-positive cells." Note that, as used herein, the term "pharmaceutical composition" is used as a broader concept than "cell preparation," or may be used synonymously with "cell preparation."

[0017] 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 both antibodies against SSEA-3 and CD105. Here, "living organism" refers to a living mammalian organism. In the present invention, living organisms do not include fertilized eggs or embryos at developmental stages earlier than the blastula stage, but do include embryos at developmental stages after the blastula stage, including fetuses and blastulas. Mammals include, but are not limited to, primates such as humans and monkeys, rodents such as mice, rats, rabbits, and guinea pigs, cats, dogs, sheep, pigs, cows, horses, donkeys, goats, and ferrets. The Muse cells used in the cell 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. Furthermore, Muse cells may be isolated from cultured mesenchymal cells such as fibroblasts or bone marrow mesenchymal stem cells using the above-mentioned isolation method. In the cell preparation of the present invention, the Muse cells used may be autologous or allogeneic to the recipient of the cell transplant.

[0018] As described above, Muse cells or cell populations containing Muse cells can be isolated from biological tissues using, for example, SSEA-3 positivity and double positivity for SSEA-3 and CD105 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. Such stem and progenitor cells include skin-derived progenitor cells (SKPs), neural crest stem cells (NCSCs), melanoblasts (MBs), perivascular cells (PCs), endothelial progenitor cells (EPs), and adipose-derived stem cells (ADSCs). Muse cells can be isolated using the "non-expression" of markers specific to these cells as an indicator. More specifically, Muse cells can be separated using as an indicator 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 EP and ADSC), CD117 (c-kit) (a marker for MB), CD146 (a marker for PC and ADSC), CD271 (NGFR) (a marker for NCSC), NG2 (a marker for PC), vWF factor (von Willebrand factor) (a marker for EP), Sox10 (a marker for NCSC), Snail (a marker for SKP), Slug (a marker for SKP), Tyrp1 (a marker for MB), and Dct (a marker for MB). 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.

[0019] Furthermore, the Muse cells having the above characteristics used in the cell preparation of the present invention may have at least one property selected from the group consisting of: (i) low or no telomerase activity; (ii) the ability to differentiate into cells of any of the three germ layers; (iii) the lack of neoplastic growth; and (iv) the ability to self-renew. In one aspect of the present invention, the Muse cells used in the cell preparation of the present invention have all of the above properties. Here, with regard to (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, for example, to telomerase activity at the same level as 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, nerve cells, skeletal muscle cells, smooth muscle cells, osteocytes, adipocytes, etc. In addition, when transplanted into the testis in vivo, they may also exhibit the ability to differentiate into three germ layers. Furthermore, when transplanted into a living body by intravenous injection, they have the ability to migrate and engraft in damaged organs (heart, skin, spinal cord, liver, muscle, etc.) and differentiate into cells appropriate for the tissue. Regarding (iii) above, Muse cells proliferate at a rate of approximately 1.3 days in suspension culture, but in suspension culture they proliferate from a single cell, form embryoid-like cell masses, and then stop proliferating after about 14 days. However, when these embryoid-like cell masses are cultured in adherent culture, cell proliferation resumes, and the cells that proliferate from the cell masses spread. Furthermore, when transplanted into the testis, they do not become cancerous for at least six months. Furthermore, regarding (iv) above, Muse cells have the ability to self-renew (self-replicate).Here, "self-renewal" refers to the fact that differentiation into three germ layer cells can be confirmed from cells contained in an embryoid-like cell mass obtained by culturing a single Muse cell in suspension culture, and at the same time, by bringing the cells of 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 an embryoid-like cell mass in suspension culture can again be confirmed. Self-renewal can be performed by repeating one or more cycles.

[0020] 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 having at least one, preferably all, of the following properties, which are 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 no telomerase activity; (iv) the ability to differentiate into three germ layers; (v) not showing neoplastic growth; and (vi) the ability to self-renew.

[0021] 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.

[0022] Furthermore, the cell fraction containing Muse cells used in the cell preparation of the present invention may be enriched using magnetically activated cell sorting (MACS) (e.g., autoMACS (registered trademark) Pro Separator; Miltenyi Biotec Inc.) in addition to or instead of the above-mentioned means using external stress stimulation.

[0023] Furthermore, the Muse cells having the above-described characteristics used in the cell preparation of the present invention migrate to and engraft in the tissue at the site of spinal cord infarction after being administered to the body by intravenous administration or the like. Thereafter, the Muse cells are thought to differentiate into cells that constitute the tissue and treat spinal cord infarction or related symptoms and diseases.

[0024] (2) Preparation and Use of Cell Preparations and Pharmaceutical Compositions The cell preparations of the present invention can be obtained by suspending the Muse cells or cell populations containing Muse cells obtained in (1) above in physiological saline or an appropriate buffer (e.g., phosphate-buffered saline). In this case, if the number of Muse cells isolated from autologous or allogeneic tissue is small, 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 the preparation is safe. Furthermore, the culture of the recovered Muse cells can be carried out in a standard growth medium (e.g., α-minimal essential medium (α-MEM) containing 10% fetal bovine serum). More specifically, with reference to the above-mentioned International Publication No. WO 2011 / 007900, a solution containing a predetermined concentration of Muse cells can be prepared by appropriately selecting a medium, additives (e.g., antibiotics, serum), etc., for the culture and proliferation of Muse cells. When the cell preparation of the present invention is administered to a human subject, approximately several milliliters of bone marrow fluid is collected from the human ilium, and bone marrow mesenchymal stem cells are cultured as adhesive cells from the bone marrow fluid and expanded to a cell quantity that allows for the isolation of an effective therapeutic amount of Muse cells. The Muse cells are then isolated using the SSEA-3 antigen marker as an indicator, and autologous or allogeneic Muse cells can be prepared as a cell preparation. Alternatively, for example, Muse cells can be isolated using the SSEA-3 antigen marker as an indicator, and then cultured and expanded to a cell quantity that allows for the isolation of an effective therapeutic amount of Muse cells, and the autologous or allogeneic Muse cells can be prepared as a cell preparation.

[0025] 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.) or cells or components other than Muse cells contained in mesenchymal stem cells. Those skilled in the art can add these factors and drugs to cell preparations at appropriate concentrations. Thus, Muse cells can also be used as pharmaceutical compositions containing various additives.

[0026] The number of Muse cells contained in the cell preparation or pharmaceutical composition prepared as described above can be appropriately adjusted taking into consideration the subject's gender, age, weight, condition of the affected area, the condition of the cells used, and other factors so as to achieve a therapeutic effect against spinal cord infarction, such as improvement of motor dysfunction and sensory impairment. Target individuals include, but are not limited to, mammals such as humans. As used herein, "amelioration" refers to the treatment, prevention, improvement, prevention of deterioration, or delay of spinal cord infarction and symptoms or conditions caused by spinal cord infarction, or the reversal, prevention, or delay of the progression of the disease. The cell preparation of the present invention may be administered in a single dose, 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 timing of administration may be early, middle or late in the onset of the disease, as long as a therapeutic effect is obtained.

[0027] The therapeutically effective amount varies depending on the condition of the subject, but is, for example, 1 x 10 per individual per administration. ３ Cell ~1×10 １１ cells, preferably 1 x 10 ４ Cell ~1×10 １０ cells, more preferably 1 x 10 ５ Cell ~1×10 ９Furthermore, the cell preparation of the present invention may be administered directly to the spinal cord or intravenously, although this is not particularly limited.

[0028] The cell preparations (or pharmaceutical compositions) of the present invention may use human-derived Muse cells, but if the recipient is a subject that is heterologous to the cells (e.g., mouse, rat), an immunosuppressant (cyclosporine, etc.) 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 applied to the treatment of motor neuron diseases, such immunosuppressants 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.

[0029] Thus, the cell preparation of the present invention is intended to treat, prevent, alleviate, and / or delay the onset of spinal cord infarction in a subject by appropriate use. As used herein, "treatment" refers to suppressing or completely eliminating disorders (e.g., motor dysfunction and sensory impairment) caused by spinal cord infarction. "Prevention" refers to preventing or delaying the onset of disorders caused by spinal cord infarction, or reducing the risk of such disorders.

[0030] 3. Preparation of a Rat Spinal Cord Infarction Model In this specification, an artificially prepared rat spinal cord infarction model can be used to examine the amelioration and therapeutic effects of the cell preparations of the present invention on brain disorders associated with spinal cord infarction (e.g., abnormalities in motor quality, abnormalities in neurological development). As described in Example 1 below, this model can be prepared by injecting, for example, 0.7 μL of enodocerin-1 (ET-1) (2.5 mg / ml) into the left and right spinal cord at an angle of 40° to a depth of 1.5 mm (the fused silica tube is adjusted to a length of 1.5 mm) into the 13th thoracic spinal cord.

[0031] 4. Improvement and Therapeutic Effects of Muse Cells in a Rat Spinal Cord Infarction Model The cell preparation and pharmaceutical composition of the present invention can improve and / or treat spinal cord ischemic disorders (e.g., motor dysfunction, sensory impairment) caused by spinal cord infarction in mammals, including humans. According to the present invention, the spinal cord infarction rat model prepared above can be used to experimentally examine the improvement of symptoms caused by spinal cord ischemic disorders caused by spinal cord infarction in rats by administering Muse cells, thereby evaluating the effects of the Muse cells. Specific evaluation methods can be performed using standard experimental systems for evaluating motor dysfunction (e.g., motor paralysis), sensory impairment, and bladder-rectum disorders in rats. Examples of motor function evaluation include, but are not limited to, the open field test, catwalk test, footfault test, and treadmill test. Examples of sensory function evaluation include, but are not limited to, the von Frey test (pain sensation) and the hot plate test (temperature).

[0032] The "open field test" is based on placing a test animal in a novel, fixed space (e.g., a box measuring 60 (W) x 60 (D) x 40 (H) cm) and observing the emotional behavior of the test animal, such as exploratory behavior. The observer actually records the behavior of the test animal and also records it with a video camera, and data can be extracted to evaluate activity / emotionality indicators (e.g., distance traveled, time spent stationary, rate of staying in the center).

[0033] In the "catwalk test," the test animal is made to walk across a transparent glass plate illuminated with an LED light from inside. The footprints are then videotaped from below, using the principle of total internal reflection to illuminate only the surface of contact. Images are then analyzed using computer software, allowing the test animal's limb movement and walking condition to be evaluated.

[0034] The "foot fault test" is a method for assessing motor deficits in limb function (generally the hind limbs) and placement disorders during locomotion. The test animal is placed on a high, flat grid with openings, and each time the foot slips off the open grid, it is recorded as a "foot fault," allowing the degree of impairment to be easily measured.

[0035] The "treadmill test" is a test in which a subject animal is made to walk on a treadmill at a constant speed and gradually increases the load to evaluate motor function.

[0036] The "von Frey test" is a physiological method for assessing pain sensitivity, in which a test animal's touch or pain threshold is measured by mechanical stimulation.

[0037] The "hot plate test" is a method for assessing the pain sensitivity of test animals. The test involves having the test animal touch a heated surface, observing its pain response, and assessing the presence and severity of pain.

[0038] 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.

[0039] Materials and Methods 1. Preparation of Rat Spinal Cord Infarction Model The animal experiments in this study were conducted under the review and approval of the Animal Experimentation Committee of the Tohoku University Graduate School of Medicine (Approval Numbers: 2020-146, 2022-073). Eight-week-old male Wistar rats (Japan SLC, Inc., Hamamatsu, Japan) were used. Rats were housed in cages in a room maintained at 22-25°C with a 12-hour light-dark cycle (lights on from 8:00 AM to 8:00 PM) and allowed free access to food and water. For details of the rat spinal cord infarction model and its preparation method used in this example, please also refer to the present inventors' publication in Stroke Vasc Neurol. 2024 Jun 21:svn-2023-002962. doi: 10.1136 / svn-2023-002962.

[0040] (1) Convection-enhanced delivery (CED) technology. To create a rat spinal cord infarction model, we used the convection-enhanced delivery (CED) technology based on previous literature (Endo T, Fujii Y, Sugiyama SI, et al. Properties of convective delivery in spinal cord gray matter: laboratory investigation and computational simulations. J Neurosurg Spine. Feb 2016;24(2):359-366. doi:10.3171 / 2015.5.SPINE141148; Ogita S, Endo T, Sugiyama S, et al. Convection-enhanced delivery of a hydrophilic nitrosourea ameliorates deficits and suppresses tumor growth in experimental spinal cord glioma models. Acta Neurochir (Wien). May 2017;159(5):939-946). doi:10.1007 / s00701-017-3123-2). A puncture system was established by connecting a 10 μL Hamilton syringe to a polyethylene tube (6010-35606; inner diameter: 250 μm, GL Sciences, Tokyo, Japan), and attaching a 27G needle and fused silica tubing (TSP100170; inner diameter / outer diameter: 100 / 170 μm, Molex, Lisle, IL) to the tip. Anesthesia was induced with 5.0% isoflurane and maintained at 2.0%. The rats were fixed in a prone position in a stereotaxic apparatus (NARISHIGE, Tokyo, Japan), and the rectal temperature was maintained at 36-37°C during surgery using a heating pad (BWT-100A, Bio Research Center Co., Ltd., Nagoya, Japan). The back was shaved, and a dorsal midline incision approximately 3 cm long was made from the 12th thoracic vertebra to the 1st lumbar vertebra. The 13th thoracic vertebral arch was resected, and the dorsal spinal cord was widely exposed.The puncture site was selected as the lateral funiculus, slightly ventral to the level where the dorsal root originates. The dura and arachnoid mater were punctured with a 27G needle, and a fused silica tube adjusted to a 1.5 mm diameter was inserted. Next, 4.0% Evans Blue (056-04061, FUJIFILM, Tokyo, Japan) dissolved in phosphate-buffered saline (PBS) was injected to optimize the puncture angle. The injection rate was 0.2 μL / min. Rats were sacrificed by isoflurane overdose, and the spinal cord was removed and immersion-fixed in 4% paraformaldehyde (PFA). Spinal cord cross sections were observed under a microscope (Stemi 305, ZEISS, Oberkochen, Germany).

[0041] (2) Stereotactic Local Injection of Endothelin-1 (ET-1) Endothelin-1 (ET-1; E7764, Sigma-Aldrich, St. Louis, MO) (Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. Mar 31 1988;332(6163):411-5. doi:10.1038 / 332411a0) was dissolved in saline to a concentration of 2.5 mg / ml, and 0.7 μL was injected at a rate of 0.2 μL / min. The needle tip was left stationary for 5 minutes and then removed over 1 minute. The muscle outside the puncture site was marked with 8-0 monofilament suture. After confirming hemostasis, the erector spinae muscle and skin were sutured closed. The animals received daily manual pressure to assist voiding until voiding ability was restored.

[0042] (3) Behavioral Evaluation All animals were evaluated for hindlimb motor function before surgery, 2 hours after surgery, and on postoperative days 1, 3, 5, and 7, and then once a week until postoperative day 56, until death. Hindlimb movements of each rat were videotaped and recorded for 5 minutes and assessed using the Basso, Beattie, and Bresnahan (BBB) ​​locomotor scale (Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma. Feb 1995;12(1):1-21. doi:10.1089 / neu.1995.12.1; Metz GA, Merkler D, Dietz V, Schwab ME, Fouad K. Efficient testing of motor function in spinal cord injured rats. Brain Res. Nov 17 2000;883(2):165-77). doi:10.1016 / s0006-8993(00)02778-5). The photographer and evaluator were different individuals, and the evaluator was blinded to the treatment details before behavioral evaluation. Body weight was also measured at the time of behavioral evaluation. The BBB motor function score used for evaluation is shown in Table 1.

[0043]

[0044] (4) Histopathological Evaluation Rats were sacrificed by isoflurane overdose, perfused intracardially with PBS, and then perfused with 4% PFA. The spinal cord was removed and immersion-fixed overnight at 4°C. The tissue was then sucrose-substituted with 15%, 20%, or 25% sucrose solution, embedded in Tissue-Tek O.C.T. compound (4583, SAKURA, Tokyo, Japan), and thin sections (up to 7 μm thick) were prepared. The sections were then resuspended in 20% BlockAce (UKB40, KAC Co., Ltd., Kyoto, Japan), 5% bovine serum albumin (01860-65, Nacalai Co., Ltd.), and 10% sucrose solution. After incubation in blocking solution containing 0.3% Triton-X-100 (168-11805, Fujifilm) and 0.5% Triton-X-100 (Tesque, Inc., Kyoto, Japan), the sections were incubated overnight at 4°C with one of the following primary antibodies: rabbit anti-NeuN (1:500; ab177487, Abcam, Cambridge, England), goat anti-Iba1 (1:500; ab5076, Abcam), mouse anti-GFAP (1:500; G3893, Sigma-Aldrich), rabbit anti-GST-pi (1:200; 312, MBL, Tokyo, Japan), or rabbit anti-endothelial cell antibody (RECA-1; 1:100; ab9774, Abcam).

[0045] After washing the sections with PBS, Alexa Fluor 488-labeled donkey anti-goat IgG, mouse IgG, or rabbit IgG (1:200, 705-545-003, 715-546-150, and 711-586-152, respectively, Jackson ImmunoResearch Laboratories Inc.) and 4′,6-diamidino-2-phenylindole (DAPI; 1:500, D9542, Sigma-Aldrich) were added and incubated at room temperature for 2 hours. The sections were washed with PBS, mounted with SlowFade Gold antifade reagent (S36937, Life Technology, Carlsbad, CA), and observed under a laser confocal microscope (Eclipse Ti, Nikon, Tokyo, Japan). For NeuN and TUNEL, 6 areas (200 × 200 μm per field) and 10 areas (150 × 150 μm per field) were randomly selected from the anterior and posterior horns, respectively. For RECA-1, 10 areas (150 × 150 μm per field) were randomly selected from the ventral and dorsal gray and white matter, respectively. Signal-positive cells were counted, and the number of cells per unit area (cells / mm) was calculated. ２ ) was calculated.

[0046] (5) Triphenyltetrazolium chloride (TTC) staining. Spinal cords harvested 7 days after local spinal injection of ET-1 were sliced ​​to a thickness of 3.0 mm, incubated in 2% TTC / PBS at 37°C for 30 minutes, and then fixed in 4% PFA. Spinal cords from normal rats (8 weeks old) were also stained.

[0047] (6) TdT-mediated dUTP nick-end labeling (TUNEL) assay. The TUNEL assay was performed using an in situ cell death detection kit (TMR Red, Roche Diagnostics, Basel, Switzerland). As a negative control, label solution was used instead of the TUNEL reaction mixture. As a positive control, normal spinal cord treated with DNase was used.

[0048] 2. Isolation of Muse cells Human bone marrow-derived MSCs (Lonza Japan, Tokyo, Japan) were used. According to previous literature (Kuroda Y, Wakao S, Kitada M, Murakami T, Nojima M, Dezawa M. Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells. Nature protocols. 2013;8(7):1391-415. doi:10.1038 / nprot.2013.076), the cells were cultured in a 10-cm dish at 37°C and 5% CO using Dulbecco's modified Eagle's medium (low glucose) (DMEM; Life Technologies, Carlsbad, CA), 10% fetal bovine serum (FBS) (Hyclone; Thermo-Fisher Scientific, Waltham, MA, USA), and 0.1 mg / mL kanamycin (Life Technologies). ２Muse cells were isolated from MSCs at passages 7 to 9 as follows: the primary antibody was a rat anti-SSEA-3 IgM antibody (1:1000; BioLegend, San Diego, CA), and the isotype control was a rat IgM κ chain isotype control (1:1000; BioLegend) for 1 hour. The cells were incubated with FITC-labeled anti-rat IgM antibody (1:100; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) as the secondary antibody for 1 hour, and anti-FITC microbeads (1:50; Miltenyi Biotec Inc., Auburn, CA) as the tertiary antibody for 30 minutes, and SSEA-3 positive cells were separated using MACS (autoMACS® Pro Separator; Miltenyi Biotec Inc.). After cell separation, the percentage of SSEA-3 positive cells among the MACS-enriched cells was analyzed using a BD FACS Aria (BD Biosciences, Franklin Lakes, NJ). Cells containing 70% or more SSEA-3 positive cells were defined as MACS-Muse cells and used as transplant cells. Akaluc / pcDNA3 was introduced into some MSCs using lentivirus, based on previous literature (see Shono Y, Kushida Y, Wakao S, et al. Protection of liver sinusoids by intravenous administration of human Muse cells in a rat extra-small partial liver transplantation model: American Journal of Transplantation. Jun 2021;21(6):2025-2039. doi: 10.1111 / ajt.16461). The MSCs were then incubated with rat anti-SSEA-3 IgM antibody (1:1000; BioLegend) as the primary antibody and allophycocyanin-labeled anti-rat IgM antibody (1:100; Jackson ImmunoResearch Laboratories Inc.) as the secondary antibody, and SSEA-3-positive and -negative cells were separated using FACS.To mimic Muse cells separated by MACS, the separated cells were mixed so that the proportion of SSEA-3 positive cells was 70%, and the mixture was designated as "Akaluc-Muse cells."

[0049] 3. Cell Transplantation by Intravenous Administration The day the spinal cord infarction model was created was defined as Day 0. Behavioral evaluation was performed 24 hours after surgery (Day 1), and animals with a BBB locomotor score of 0 were selected. The spinal cord infarction model was randomly divided into the following groups: a vehicle group administered with PBS, a group administered with 400,000 MSC cells, and a group administered with 400,000 Muse cells. All transplanted cells were suspended in 400 μL of PBS and administered via the penile vein. For the Muse cell group, MACS-enriched cells (MACS-Muse cells) were administered in behavioral evaluation experiments. In experiments to evaluate biodistribution, Akaluc-Muse cells or Akaluc-MSCs were administered, and immunohistochemical evaluation was also performed (see Iwano S, Sugiyama M, Hama H, et al. Single-cell bioluminescence imaging of deep tissue in freely moving animals. Science. Feb 23 2018;359(6378):935-939. doi:10.1126 / science.aaq1067).

[0050] 4. Behavioral Evaluation of Cell-Transplanted Rats The hindlimb movements of each rat were videotaped and recorded for 5 minutes, and then evaluated using the Basso, Beattie, and Bresnahan (BBB) ​​motor function scale (see Basso DM, et al., 1995 (supra); Metz GA, et al., 2000 (supra)). The rats were allowed to move freely in a circular pool with a diameter of 100 cm. The videotaper and the evaluator were different individuals, and the evaluator was blinded to the treatment details when evaluating the behavior. All animals were evaluated for behavior 2 hours after surgery and on Day 1. Animals with a BBB score of 0 on Day 1 were selected, and other animals were excluded from the analysis. The rat spinal cord infarction model was randomly divided into three groups, and various cells were administered intravenously: (1) MACS-Muse cell-administered group (MACS-Muse group), (2) MSC-administered group (MSC group), and (3) PBS-administered group (vehicle group). The rat model consisted of 13 rats in the MACS-Muse group, 12 rats in the MSC group, and 12 rats in the vehicle group. Hindlimb motor function was assessed on Days 2, 3, 5, and 7, and then once a week until Day 56, after which the rats were sacrificed on Day 56. Body weight was also measured at the time of behavioral assessment. Animals that died before Day 56 were excluded from the analysis.

[0051] 5. Evaluation of the Biodistribution of Transplanted Cells To observe the in vivo distribution of Muse cells after intravenous administration, Venus-Akaluc was introduced into MSCs using lentivirus. First, pcDNA3 Venus-Akaluc, provided by Iwano (Iwano S, et al., 2018 (ibid.)), was inserted into the vector plasmid pWPXL to construct pWPXL-Venus-Akaluc. Next, pWPXL-Venus-Akaluc was introduced into LentiX-293T cells (TaKaRa Bio Inc, Shiga, Japan), which are lentivirus packaging cells, together with the packaging plasmid pMD2G and the envelope plasmid pCMV deltaR8.74 using Lipofectamine 2000 (Thermo Fisher Scientific). After culturing for 3 days, the lentivirus supernatant was collected, centrifuged, and passed through a 0.45 μm filter before being introduced into MSCs. These were separated using FACS as Venus(+) cells (Akaluc-MSCs) and Venus(+) / SSEA-3(+) double-positive cells (Akaluc-Muse cells). To isolate Akaluc-Muse cells, rat anti-SSEA-3 IgM antibody (1:1000; BioLegend) was used as the primary antibody, and allophycocyanin-labeled anti-rat IgM antibody (1:100; Jackson ImmunoResearch Laboratories Inc.) was used as the secondary antibody. Based on the behavioral assessment, 24 hours after the spinal cord infarction model was created, the model animals were randomly assigned to three groups, and various cells were intravenously administered: (1) 400,0000 Akaluc-Muse cells / 400 μL PBS (Akaluc-Muse group), (2) 400,0000 Akaluc-MSC cells / 400 μL PBS (Akaluc-MSC group), and (3) 400 μL PBS (vehicle group). The rat models used were 3 in each group. The in vivo localization of Muse cells and MSCs was evaluated on Day 7 using an IVIS Spectrum CT (Perkin Elmer, Waltham, MA).The rat model was sacrificed using an overdose of isoflurane 5 minutes after intravenous administration of 0.5 ml of 15 mM AkaLumine-HCl, an artificial substrate for Akaluc®, and each organ was rapidly removed. Each organ was immersed in 0.5 mM AkaLumine-HCl and then imaged using an in vivo imaging system (IVIS). The localization of Muse cells and MSCs was assessed by the total flux (photons / s; light intensity) of the organ surface. Total flux in the spinal cord tissue was assessed within a 3 cm radius centered on the ET-1 injection site. The tissue was sliced ​​at 3 mm intervals, and the sum of the total flux in each section was defined as the individual value. The luminous intensity was quantified using Living Image software (ver. 4.5, PerkinElmer, Waltham, MA). The total flux of each organ in the Akaluc-Muse and Akaluc-MSC groups was evaluated after subtracting the total flux (autofluorescence) of each organ in the vehicle group.

[0052] 6. Histopathological evaluation of the spinal cord of cell-transplanted rats On day 56, rats were sacrificed by isoflurane overdose, and the spinal cord was subjected to histopathological evaluation in the same manner as in 1(4) above. The spinal cord tissue used in the in vivo evaluation experiment was also subjected to histopathological evaluation. The primary antibodies used were rabbit anti-human mitochondria (1:200, ab133789, Abcam), mouse anti-NeuN (1:200; MAB377, Sigma-Aldrich), mouse anti-MAP-2 (1:200; M1406, Sigma-Aldrich), mouse anti-β-TUBLINIII (1:200; T8660, Sigma-Aldrich), goat anti-Iba1 (1:500; ab5076, Abcam), mouse anti-GFAP (1:500; G3893, Sigma-Aldrich), goat anti-GST3 / GST-pi (1:50; ab53943, Abcam), and goat anti-CD31 (1:200; AF3628, RD). The secondary antibodies used were Alexa Fluor 488-labeled donkey anti-rabbit IgG and chicken IgG (1:200, 711-586-152 and 703-545-155, respectively, Jackson ImmunoResearch Laboratories Inc.), or Alexa Fluor 594-labeled donkey anti-mouse IgG and goat IgG (1:200, 715-586-150 and 705-585-003, respectively, Jackson ImmunoResearch Laboratories Inc.). For the animals on day 56 after the onset of spinal cord infarction, two 800 × 800 μm areas were selected on the left and right sides of the spinal cord ventral horn within a 9 mm rostral-caudal range from the spinal cord infarction site. Human mitochondria-positive cells were counted, and the number of cells per unit area (cells / mm) in each cross section was calculated. ２ ) was calculated.

[0053] 7. Statistical Analysis All statistical analyses were performed using the GraphPad Prism software package, version 10.0.2 (GraphPad Software, San Diego, CA). Results were expressed as mean ± standard error. Two-way analysis of variance and Tukey's honestly significant difference test were used for comparisons of two or more groups. A t-test was used for comparisons between two groups. The significance level was P<0.05 ( ＊ )

[0054] Example 1 Preparation of a Rat Spinal Cord Infarction Model A rat spinal cord infarction model was prepared by injecting 0.7 μL of enodocerin-1 (ET-1) (2.5 mg / ml) into the left and right spinal cord at a 40° angle to a depth of 1.5 mm (the fused silica tube was adjusted to a length of 1.5 mm) into the 13th thoracic spinal cord (hereinafter referred to as the "ET-1 group").

[0055] Example 2. Improvement of hindlimb motor function by intravenous administration of Muse cells in a rat spinal cord infarction model The experimental system scheme for the cell administration experiment is shown in Figure 1. 24 hours after model creation, Muse cells concentrated by MACS (MACS-Muse cells) (Figure 2), MSCs, or PBS were administered. Rats that died during observation were excluded from the graph. Figure 3 shows the time course of the BBB motor function score in each group. The BBB motor function score in the MACS-Muse group was statistically significantly higher than that in the MSC group and vehicle group from Day 35 onwards. The BBB motor function scores on Day 56 were 9.5±1.7 in the MACS-Muse group, 4.7±1.7 in the MSC group, and 4.5±1.3 in the vehicle group (MACS-Muse group vs. MSC group; p<0.01, MACS-Muse group vs. vehicle group; p<0.01, MSC group vs. vehicle group; not significant).

[0056] Example 3. Body weight changes in each group Body weight was monitored up to Day 56 for each group. The mean preoperative body weight was set to 1.0, and the weight change was calculated as a ratio at each time point (Figure 4). On Day 1, the weights were 0.93 for the MACS-Muse group, 0.93 for the MSC group, and 0.94 for the vehicle group. On Day 14, the weights were 1.00 for the MACS-Muse group, 0.96 for the MSC group, and 0.96 for the vehicle group. On Day 56, the weights were 1.25 for the MACS-Muse group, 1.22 for the MSC group, and 1.20 for the vehicle group. No statistically significant differences were observed in the preoperative weight ratios between the groups at each observation time point.

[0057] Example 4. Evaluation of biodistribution of transplanted cells In the Akaluc-Muse group and Akaluc-MSC group, Akaluc signals were observed in the spinal cord and lungs on Day 7 (Figures 5A and 5B). No signals were observed in other organs (Figure 5C). Total flux in the spinal cord was 3066.5±533.6 in the Akaluc-Muse group and 1079.7±219.4 in the Akaluc-MSC group, with statistically significant signal accumulation observed in the Akaluc-Muse group (p=0.049). Total flux in the lung was 819.5±411.8 in the Akaluc-Muse group and 1606.6±925.5 in the Akaluc-MSC group, and although signal accumulation was observed in Akaluc-MSCs, no statistically significant difference was observed (p=0.50, FIG. 5D).

[0058] Example 5. Histological evaluation of the spinal cord in cell-transplanted rats Histological evaluation was performed using spinal cord tissue from a rat spinal cord infarction model transplanted with Muse cells. Spinal cord tissue from the area surrounding the puncture site was used from the Akaluc-Muse group (Day 7) and the MACS-Muse group (Day 56) (Fig. 6). GFP-positive cells were observed in the Akaluc-Muse group, and human mitochondria-positive cells were observed in the MACS-Muse group. Positive and negative controls for each staining are shown in Fig. 7. The distribution of transplanted cells in the spinal cord tissue surrounding the spinal cord infarction site was analyzed (Fig. 8A). In the MACS-Muse group on Day 56, the number of human mitochondria-positive cells ( / mm ２) from the infarct center were 10.9 ± 2.8 mm rostral, 17.4 ± 2.2 mm rostral, 14.3 ± 4.2 mm rostral, 1.8 ± 1.2 mm caudal, 10.4 ± 5.0 mm caudal, 18.0 ± 4.0 mm caudal, and 13.3 ± 3.1 mm caudal (n = 3, infarct center vs. 6 mm rostral, 6 mm caudal, p < 0.05, Figure 8B).

[0059] Example 6. Differentiation of Muse Cells into Neurons and Vascular Cells Spinal cord tissue from the MACS-Muse group (Day 56) was double-stained with human mitochondrial antibodies and the neuronal markers NeuN, MAP-2, and TUJ-1, the oligodendrocyte marker GST3 / GST-pi, the astrocyte marker GFAP, the microglial marker Iba-1, and the vascular endothelial marker CD31 (Figure 9). Positive and negative controls for each staining are shown in Figure 10. Double positivity was observed with NeuN, MAP2, TUJ1, GST3 / GST-pi, and CD31. However, double positivity with GFAP or Iba-1 was not observed.

[0060] Example 7. Efficiency of Differentiation into Neural Cells The number of cells double-positive for NeuN, MAP-2, GST3 / GST-pi, GFAP, Iba-1, and human mitochondrial antibodies was counted to examine the efficiency of differentiation of Muse cells into neurons (n=3). The percentage of cells double-positive with human mitochondrial antibodies was 70.6±5.4% for NeuN, 68.0±11.4% for MAP-2, and 10.6±0.3% for GST3 / GST-pi (FIG. 11). No double-positive cells with GFAP or Iba-1 were confirmed.

[0061] Example 8. Efficiency of differentiation into vascular endothelial cells In the same area as above, 17.6±1.8% of cells were found to be double positive for human mitochondria and CD31 (data not shown).

[0062] In this study, we demonstrated that intravenously administered Muse cells migrated and engrafted into the infarcted spinal cord tissue in a rat spinal cord infarction model, improving hindlimb motor function for 8 weeks without the use of immunosuppressants. Furthermore, the improvement in hindlimb motor function was statistically significantly greater than that achieved with MSCs.

[0063] (1) Homing of human Muse cells to the infarcted spinal cord. IVIS analysis on Day 7 confirmed Akaluc signals in spinal cord tissue, and histological evaluation also confirmed Akaluc-GFP-positive cells. In addition, histological evaluation of the MACS-Muse group on Day 56 confirmed human mitochondria-positive cells. These results indicate that Muse cells recognized the infarcted spinal cord, which had suffered ischemic damage, and homed to the spinal cord. The phenomenon of selective migration of Muse cells to injured organs has been reported in the heart (Yamada Y, et al., 2018 (supra)), lung (Yabuki H, et al., 2018 (supra)), aorta (Hosoyama K, Wakao S, Kushida Y, et al. Intravenously injected human multilineage-differentiating stress-enduring cells selectively engraft into mouse aortic aneurysms and attenuate dilatation by differentiating into multiple cell types. J Thorac Cardiovasc Surg. Jun 2018;155(6):2301-2313 e4. doi:10.1016 / j.jtcvs.2018.01.098), liver (Iseki M, et al., 2017 (supra)), and kidney (Uchida N, et al., 2017 (ibid.)), and furthermore, this has been confirmed in a cerebral infarction model (Abe T, Aburakawa D, Niizuma K, et al. Intravenously Transplanted Human Multilineage-Differentiating Stress-Enduring Cells Afford Brain Repair in a Mouse Lacunar Stroke Model. Stroke. Feb 2020;51(2):601-611. doi:10.1161 / STROKEAHA.119.026589) and a traumatic spinal cord injury model (Kajitani T, Endo T, Iwabuchi N, et al.Association of intravenous administration of human Muse cells with deficit amelioration in a rat model of spinal cord injury. J Neurosurg Spine. Jan 1 2021;34(4):648-655. doi:10.3171 / 2020.7.SPINE20293), and ALS models (Yamashita T, Kushida Y, Wakao S, et al. Therapeutic benefit of Muse cells in a mouse model of amyotrophic lateral sclerosis. Sci Rep. Oct 13 2020;10(1):17102. doi:10.1038 / s41598-020-74216-4). The S1P-S1P receptor 2 (S1PR2) axis has been identified as the mechanism of migration of Muse cells (Yamada Y, et al., 2018 (cited above)), and Muse cells expressing S1PR2 have the ability to sense S1P produced by damaged tissue and migrate to the damaged tissue.

[0064] The tissue localization of Akaluc-Muse cells administered intravenously via IVIS was analyzed. No Akaluc signal was observed at the ET-1 injection site, i.e., the center of the spinal cord infarction, but signals were confirmed in the rostrocaudal area. Histological evaluation of the MACS-Muse spinal cord tissue on Day 56 also revealed that there were fewer human mitochondrial-positive cells in the center of the spinal cord infarction, and more in the area 3-9 mm rostrocaudal (Figure 8B). This result is consistent with the results from IVIS and indicates that the intravenously administered Muse cells continued to engraft from Day 7 to Day 56. This suggests that, compared to the infarct center, more Muse cells accumulated in the peri-infarct area of ​​the spinal cord, where blood flow remained and where infarction had not yet occurred.

[0065] (2) Spontaneous Differentiation of Human Muse Cells into Neuronal or Vascular Cells After homing to damaged tissue, Muse cells spontaneously differentiate into tissue-specific cells by phagocytosing apoptotic cells, replacing and repairing the damaged tissue (Iseki M, et al., 2017 (supra); Kajitani T, et al., 2021 (supra)). In a study in which Muse cells were administered to a mouse cerebral infarction model, approximately 65% ​​of the engrafted Muse cells were NeuN-positive, approximately 30% were MAP-2-positive, and approximately 10% were GST-pi-positive, with no GFAP- or Iba-1-positive cells (Uchida H, et al., 2017 (supra)). In a study in which Muse cells were administered to a rat traumatic spinal cord injury model, approximately 50% of cells were MAP-2 positive, approximately 25% were GFAP positive, and approximately 25% were GST-pi positive (Takahashi Y, Kajitani T, Endo T, et al. Intravenous Administration of Human Muse Cells Ameliorates Deficits in a Rat Model of Subacute Spinal Cord Injury. Int J Mol Sci. Sep 27 2023;24(19)doi:10.3390 / ijms241914603). Similar results were obtained in the present study, with the percentages of cells double-positive with human mitochondria being NeuN 70.6±9.4%, MAP-2 68.0±11.4%, and GST3 / GST-pi 10.6±0.3% (n=3, Figure 11). The percentage of cells double positive for human mitochondria and CD31 was 17.6±1.8%.

[0066] (3) Effect of human Muse cells on improving hindlimb motor function in a rat spinal cord infarction model In this study, a rat spinal cord infarction model administered with human Muse cells showed a statistically significant improvement in hindlimb motor function compared to the MSC-administered group and the vehicle group. The mechanisms of the therapeutic effect suggested by these experimental results are (i) differentiation of Muse cells into neurons, and (ii) improvement in blood flow due to differentiation of Muse cells into vascular endothelial cells. The prevailing view is that blood flow to the spinal cord is not only confined to the artery of Adamkiewicz, but also to collateral circulation, a concept known as the "collateral network concept" (Griepp RB, Griepp EB. Spinal cord perfusion and protection during descending thoracic and thoracoabdominal aortic surgery: the collateral network concept. Ann Thorac Surg. Feb 2007;83(2):S865-9; discussion S890-2. doi:10.1016 / j.athoracsur.2006.10.092). It is speculated that intravenously administered Muse cells not only differentiate into neurons but also contribute to the establishment of collateral circulation as part of the therapeutic mechanism.

[0067] On the other hand, the BBB motor function score at Day 56 was 4.7±1.7 in the MSC group and 4.5±1.3 in the vehicle group, with no statistically significant difference between the two groups. However, there are numerous prior publications on MSC administration for spinal cord ischemic injury, and it has been reported to be effective as a treatment (Takahashi S, et al., 2018 (ibid.); Kurose T, et al., 2019 (ibid.); Yasuda N, Sasaki M, Kataoka-Sasaki Y, et al. Intravenous delivery of mesenchymal stem cells protects both white and gray matter in spinal cord ischemia. Brain Res. Nov 15 2020;1747:147040. doi:10.1016 / j.brainres.2020.147040). The following are considered to be the reasons why MSCs did not have an effective therapeutic effect in this experiment. First, while many literature on MSC administration to rat spinal cord ischemia models uses a cell count of 1 million, the cell count in this experiment was 400,000, which differs from previous reports. Second, this study involved a long-term observation period of 56 days, which differs from the often-seen short-term treatment outcome reports. Third, because this experiment used a novel rat spinal cord infarction model, it may not be possible to make a general comparison with previously reported experiments using a spinal cord infarction model induced by aortic clamping.

[0068] (4) Optimal Administration Timing of Muse Cells In this experimental protocol, human Muse cells were administered the day after spinal cord infarction. This was set based on the expected clinical time from the onset of spinal cord infarction during surgery to the recognition of paraplegia after surgery. Although a therapeutic effect was observed when Muse cells were administered the day after the onset of spinal cord infarction, there may be an administration timing that can achieve even greater therapeutic effects. Optimization may be possible by measuring S1P concentrations in spinal cord tissue at each time point.

[0069] (5) Future prospects In this study, Muse cells were administered the day after the onset of spinal cord infarction, and improvement in hindlimb motor function was observed over a long-term observation period of 56 days without the use of immunosuppressants. This protocol is in line with actual clinical practice, and Muse cell therapy for spinal cord ischemic injury is expected to become a new treatment strategy in the future.

[0070] Conclusions: We established a novel rat spinal cord infarction model in which local injection of endothelin-1 into spinal cord tissue induces infarction centered on the ventral horn of the spinal cord, resulting in long-term spinal cord dysfunction and long-term survival. This study is the first to clarify the survival rate through behavioral and histological assessments over a 56-day period. Intravenous administration of human Muse cells without immunosuppressants to a rat spinal cord infarction model the day after the onset of spinal cord infarction improved hindlimb motor function. Furthermore, Muse cells homed to the infarcted spinal cord and differentiated into spinal cord neurons and vascular endothelial cells, which may have contributed to the recovery of neural function. These results suggest that administration of Muse cells by a simple intravenous method during the acute phase of spinal cord infarction may produce a long-term therapeutic effect of 56 days, even without the use of immunosuppressants.

[0071] The disclosures of all patents, patent applications, and other publications cited in this specification are incorporated herein by reference in their entireties to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A cell preparation for treating, preventing, alleviating and / or delaying the onset of spinal cord infarction in a subject, comprising SSEA-3-positive pluripotent stem cells isolated from biological mesenchymal tissue or cultured mesenchymal cells.

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, comprising a cell fraction enriched for SSEA-3-positive pluripotent stem cells by MACS and / or FACS.

4. A cell preparation according to any one of claims 1 to 3, wherein the pluripotent stem cells are CD105 positive.

5. A cell preparation according to any one of claims 1 to 3, wherein the pluripotent stem cells are CD117-negative and CD146-negative.

6. 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.

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

8. A cell preparation according to any one of claims 1 to 3, wherein the pluripotent stem cells have all of the following properties: (i) low or no telomerase activity; (ii) the ability to differentiate into cells of any of the three germ layers; (iii) the lack of neoplastic growth; and (iv) the ability to self-renew.

9. The cell preparation according to any one of claims 1 to 3, wherein the spinal cord infarction is motor dysfunction, sensory dysfunction, or bladder and rectum dysfunction.

10. A cell preparation according to any one of claims 1 to 3, in which the pluripotent stem cells have the ability to engraft into tissue at the site of spinal cord infarction.

11. A method for producing a cell preparation for treating, preventing, alleviating, and / or delaying the onset of spinal cord infarction in a subject, the cell preparation comprising SSEA-3-positive pluripotent stem cells, the method comprising a step of isolating the SSEA-3-positive pluripotent stem cells from mesenchymal tissue of a living organism or cultured mesenchymal cells.

12. The method according to claim 11, comprising a step of enriching SSEA-3-positive pluripotent stem cells by external stress stimulation and / or MACS.

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