Pluripotent stem cells for treatment of spinal cord infarction

Abstract

To provide a novel medical use of pluripotent stem cells (Muse cells) in regenerative therapy.SOLUTION: 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.SELECTED DRAWING: None

Description

[Technical Field]

[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. [Background technology]

[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 pathological manifestation. In addition to decreased motor function in the lower limbs, it can also cause sensory impairment and bladder-rectum dysfunction, significantly reducing quality of life and significantly impacting prognosis. The incidence of paraplegia following thoracoabdominal aortic surgery has been reported to be 8.3% for thoracoabdominal aortic artificial vascular replacement (Non-Patent Document 1) and 11% for thoracoabdominal 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 the administration of drugs 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 limited to short-term observations, and the therapeutic mechanisms are believed to be paracrine and anti-inflammatory (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 of 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 exist in vivo and therefore do not exhibit tumorigenicity. Furthermore, Muse cells, administered intravascularly alone, recognize sphingosine-1-phosphate (S1P) as a transmitter and migrate to damaged tissue (Non-Patent Document 13). They then phagocytose apoptotic cells and spontaneously differentiate into tissue-specific cells, resulting in tissue repair (Non-Patent Document 14). Previous studies of 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 1-4). Furthermore, because Muse cells express human leukocyte antigen (HLA)-G, which is expressed in the placenta, intravenously administered allogeneic donor Muse cells are less susceptible to immune rejection, selectively home to the recipient's damaged tissue, and survive as functionally differentiated cells for over six months. In a clinical trial (JapicCTI-184103) in which Muse cell therapy was administered to patients with cerebral infarction, it was found that the Muse cell 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 Muse cell-based treatment for spinal cord infarction as a novel, proactive treatment that goes beyond conventional conservative treatments and other cell therapies. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 5968442 [Patent Document 2] Patent No. 6604492 [Patent Document 3] Patent No. 7029729 [Patent Document 4] Patent No. 7072777 [Non-patent literature]

[0005] [Non-Patent Document 1] 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.2018.03.410 [Non-patent document 2] Pini 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.2021.10.015 [Non-Patent Document 3] Coselli 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.2002.122024 [Non-Patent Document 4] Yin 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.2014.02.047 [Non-Patent Document 5] Wang 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.2016.4753 [Non-patent Document 6] Fang 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.2012.11.087 [Non-patent Document 7] Yasuda 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 / ezaa476 [Non-patent Document 8] Nakai 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.2021.06.008

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[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. [Means for solving the problem]

[0007] The present inventors created a rat spinal cord infarction model and used this 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 biological mesenchymal tissue or cultured mesenchymal cells. [2] The cell preparation described in [1], 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], which contains a cell fraction enriched for SSEA-3-positive pluripotent stem cells by MACS and / or FACS. [4] The cell preparation according to any one of [1] to [3], wherein the pluripotent stem cells are CD105 positive. [5] The cell preparation according to any one 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 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. [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 containing SSEA-3-positive pluripotent stem cells for treating, preventing, alleviating, and / or delaying the onset of spinal cord infarction in a subject, the method comprising a step of isolating the SSEA-3-positive pluripotent stem cells from mesenchymal tissue of a living body or cultured mesenchymal cells.

[12] The method according to

[11] , which comprises 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 through a tissue regeneration mechanism in which Muse cells differentiate at the site of spinal cord infarction into cells that constitute the normal tissue surrounding the site. [Brief explanation of the drawings]

[0010] [Figure 1]The experimental system scheme is shown. The day the spinal cord infarction model was created was defined as Day 0. On Day 1, behavioral assessment was performed using the BBB Locomotor Scale, and animals with a BBB Locomotor Score of 0 were used for the experiment. Mice were randomly assigned to the MACS-Muse group, MSC group, or vehicle group, and behavioral assessment and weight measurement were performed on Days 2, 3, 5, and 7, and every week thereafter until Day 56. They were then sacrificed. Mice to be used in the in vivo imaging system (IVIS) were sacrificed on Day 7 and photographed. [Figure 2] SSEA-3-positive cells were isolated from MSCs using MACS. The SSEA-3 positivity rate in the final cell population was shown by FACS analysis. MSCs from passages 7 to 9 were used. SSEA-3-positive cells were enriched using MACS, and cell populations with an SSEA-3 positivity rate of >70% were defined as MACS-Muse cells. [Figure 3] The BBB motor function score over time for each group is shown. The BBB motor function score for the MACS-Muse group (n = 13), MSC group (n = 12), and vehicle group (n = 12) was shown. The BBB motor function score on 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 (p < 0.01 for the MACS-Muse group vs. the MSC group, p < 0.01 for the MACS-Muse group vs. the vehicle group, and not significant for the MSC group vs. the vehicle group). [Figure 4] The weight change 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 weight was set to 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 any observation time point. [Figure 5]This shows the in vivo localization of administered cells using IVIS. Accumulation of Muse cells and MSCs in (A) the spinal cord and (B) the lungs on day 7. (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. [Figure 6] This shows the localization of intravenously administered Muse cells in spinal cord cross sections. (A) Spinal cord tissue from the Akaluc-Muse group (Day 7). Akaluc-GFP-positive cells were observed mainly in the anterior horn of the spinal cord. The outline of the gray matter is indicated by a white dotted line. (B) Spinal cord tissue from the MACS-Muse group (Day 56). Human mitochondria-positive cells were observed mainly in the anterior horn of the spinal cord. The outline of the gray matter is indicated by a white dotted line. [Figure 7] Positive and negative controls are shown for each staining. In a and b, human umbilical cord was used. In c and d, GFP mouse cerebrum was used. [Figure 8] The localization of intravenously administered Muse cells in spinal cord cross sections is shown. (A) Seven cross sections, 3 mm, 6 mm, and 9 mm rostral to the spinal cord infarction (puncture site), were randomly selected from the left and right sides of the spinal cord, with 800 μm square fields centered on the ventral horn of the spinal cord. (B) The 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 that 6 mm rostral and 6 mm caudal (p < 0.05). [Figure 9] This shows the differentiation of Muse cells into neurons and vascular cells. Immunohistological staining of the spinal cord in the MACS-Muse group at day 56. Double positivity was observed for human mitochondria and the neuronal markers NeuN, MAP-2, and TUJ-1, the oligodendrocyte marker GST3 / GST-pi, and the vascular endothelial marker CD31. No double-positive cells were observed for the astrocyte marker GFAP or the microglial marker Iba-1. White arrows indicate double-positive cells. Bar = 50 μm. [Figure 10]Positive and negative controls are shown for each staining. af: rat spinal cord gray matter; g and h: rat spinal cord white matter; i and j: rat anterior spinal artery. [Figure 11] The efficiency of differentiation 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 double-positive cells with GFAP or Iba-1 were observed. DETAILED DESCRIPTION OF THE INVENTION

[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 mitochondria HLA: human leukocyte antigen Iba1: ionized calcium-binding adaptor molecule 1 iPS: induced pluripotent stem IVIS: In Vivo Imaging System MACS: Magnetic Activated Cell Sorting MAP-2: Microtubule-associated protein-2 MSC: Mesenchymal stem cell Muse:Multilineage-differentiating stress-enduring 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 relates to the use of cell preparations or pharmaceutical compositions containing SSEA-3-positive pluripotent stem cells (Muse cells) for the treatment, prevention, alleviation, and / or delay of the onset of spinal cord infarction (ischemic myelopathy). 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 preparations 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, spasticity (chronic phase), increased deep tendon reflexes (chronic phase), muscle rigidity, bradykinesia, involuntary movements (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 (such as temperature, pressure, and touch), deep sensations (such as position sense and vibration), and complex sensations (such as two-point discrimination and cutaneous writing sense) are not recognized normally due to damage to the spinal cord. Depending on the severity, symptoms can 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. These cells possess properties of both pluripotent stem cells and mesenchymal stem cells and are identified, for example, by double positivity 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, 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 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."In this specification, the term "pharmaceutical composition" is used to have 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 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.

[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 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.

[0019] 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.

[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 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.

[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® Pro Separator; Miltenyi Biotec Inc.) in addition to or instead of the above-mentioned external stress stimulus method.

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

[0024] (2) Preparation and use of cell preparations and pharmaceutical compositions 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.

[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.) 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.

[0026] The number of Muse cells contained in the cell preparation or pharmaceutical composition prepared above can be appropriately adjusted taking into account the subject's gender, age, weight, condition of the affected area, and the condition of the cells used, so as to achieve therapeutic effects 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 its associated symptoms or conditions, 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 × 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 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. However, if the recipient is a subject that is heterologous to the cells (e.g., mouse, 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 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 created 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 and neurological development). As described in Example 1 below, the model can be created by injecting, for example, 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 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 model of spinal cord infarction The cell preparations and pharmaceutical compositions 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, cat walk test, foot fault 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 the inside. The footprints are then videotaped from below, and images of the footprints, which are illuminated only at the contact surface using the principle of total internal reflection, are 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 subject 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 using 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. [Example]

[0039] Materials and Methods 1. Preparation of a 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. The rats were housed in cages in a room maintained at a temperature of 22–25°C and a 12-hour light / dark cycle (lights on from 8:00 AM to 8:00 PM). Food and water were available ad libitum.

[0040] (1) Convection-enhanced delivery (CED) technology To create a rat spinal cord infarction model, we used the convection-enhanced delivery (CED) technique, based on previous literature (see 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 using a 10 μL Hamilton syringe connected to a polyethylene tube (6010-35606; inner diameter 250 μm, GL Sciences, Tokyo, Japan), with a 27G needle and fused silica tube (TSP100170; inner diameter / outer diameter: 100 / 170 μm, Molex, Lisle, IL) at the end. Anesthesia was induced with 5.0% isoflurane and maintained at 2.0%. The rat was placed in a prone position in a stereotaxic apparatus (NARISHIGE, Tokyo, Japan), and 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 3 cm dorsal midline incision 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 lateral funiculus, slightly ventral to the level where the dorsal root originates, was selected as the puncture site. The dura mater and arachnoid mater were punctured with a 27G needle, and a fused silica tube adjusted to a 1.5mm 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 cords were 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 1–3 mg / ml and infused at a rate of 0.2 μL / min. The needle tip was left in place for 5 min and then withdrawn over 1 min. The muscles outside the puncture site were marked with 8-0 monofilament suture. After hemostasis was confirmed, the erector spinae muscles and skin were sutured closed. The animals received manual compression daily to assist voiding until voiding ability was restored.

[0042] (3) Behavioral assessment All animals were evaluated for hindlimb motor function preoperatively, 2 hours postoperatively, and on postoperative days 1, 3, 5, and 7, and then weekly thereafter until postoperative day 56, until death. Hindlimb movements of each rat were videotaped and recorded for 5 min and assessed using the Basso, Beattie, and Bresnahan (BBB) ​​locomotor scale (see 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 the behavioral evaluation. Body weight was also measured at the time of behavioral evaluation. The BBB motor function score used for the evaluation is shown in Table 1.

[0043] [Table 1]

[0044] (4) Histopathological evaluation Rats were sacrificed by isoflurane overdose and perfused intracardially with PBS followed by 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 solutions, embedded in Tissue-Tek OCT compound (4583, SAKURA, Tokyo, Japan), and thinly sectioned (~7 μm thick). The sections were prepared in 20% BlockAce (UKB40, KAC Co., Ltd., Kyoto, Japan) and 5% bovine serum albumin (01860-65, Nakarai). After incubation in blocking solution containing 0.3% Triton-X-100 (168-11805, Fujifilm) and 0.5% Teflon® (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 with PBS, sections were incubated with Alexa Fluor 488-conjugated 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) for 2 hours at room temperature. After washing with PBS, sections were mounted with SlowFade Gold antifade reagent (S36937, Life Technology, Carlsbad, CA) and observed under a laser confocal microscope (Eclipse Ti, Nikon, Tokyo, Japan). Six areas (200 × 200 μm per field) and ten areas (150 × 150 μm per field) were randomly selected from the anterior and posterior horns for NeuN and TUNEL, respectively, and ten areas (150 × 150 μm per field) were randomly selected from the ventral and dorsal gray and white matter for RECA-1. 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 were harvested 7 days after local injection of ET-1, 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). For negative controls, labeling solution was used instead of the TUNEL reaction mixture. Normal spinal cord treated with DNase was used as a positive control.

[0048] 2. Muse Cell Isolation Human bone marrow-derived MSCs (Lonza Japan, Tokyo, Japan) were used. They were cultured in 10-cm dishes at 37°C and 5% CO2 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) according to a previous publication (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). Muse cells were isolated from MSCs at passages 7–9 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 secondary antibody was an FITC-labeled anti-rat IgM antibody (1:100; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) for 1 hour, and the tertiary antibody was an anti-FITC microbead (1:50; Miltenyi Biotec Inc., Auburn, CA) for 30 minutes. SSEA-3 positive cells were then isolated by 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.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), some MSCs were transfected with Akaluc / pcDNA3 using lentivirus, and the cells were incubated with rat anti-SSEA-3 IgM antibody (1:1000; BioLegend) as the primary antibody and allophycocyanin-conjugated anti-rat IgM antibody (1:100; Jackson ImmunoResearch Laboratories Inc.) as the secondary antibody. SSEA-3-positive and SSEA-3-negative cells were separated using FACS. To mimic Muse cells isolated by MACS, the isolated cells were mixed so that the proportion of SSEA-3 positive cells was 70%, and this 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 mice with a BBB locomotor score of 0 were selected. The mice were randomly assigned to the following groups: a vehicle group administered PBS to the spinal cord infarction model; a group administered 400,000 MSCs; and a group administered 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 for behavioral evaluation experiments. In biodistribution experiments, 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 Assessment of Cell-transplanted Rats Hindlimb movement of each rat was videotaped and recorded for 5 minutes and evaluated using the Basso, Beattie, and Bresnahan (BBB) ​​locomotor function scale (see Basso DM, et al., 1995, supra; Metz GA, et al., 2000, supra). Animals were allowed to move freely in a circular pool with a diameter of 100 cm. The videotapering and the evaluator were different individuals, and the evaluators were blinded to the treatment. All animals were evaluated for behavioral performance 2 hours after surgery and on Day 1. Animals with a BBB score of 0 on Day 1 were selected, and all other animals were excluded from the analysis. Rat spinal cord infarction models were randomly assigned to three groups and intravenously administered various cells: (1) MACS-Muse cell-treated group (MACS-Muse group), (2) MSC-treated group (MSC group), and (3) PBS-treated group (vehicle group). The rat model consisted of 13 rats in the MACS-Muse group, 12 in the MSC group, and 12 in the vehicle group. Hindlimb motor function was assessed on days 2, 3, 5, and 7, and then once a week until day 56, at which point the rats were sacrificed. Body weight was also measured at each behavioral assessment. Animals that died before day 56 were excluded from the analysis.

[0051] 5. Assessment of the Biodistribution of Transplanted Cells To observe the in vivo distribution of Muse cells after intravenous administration, we transduced MSCs with Venus-Akaluc using lentivirus. First, we inserted pcDNA3 Venus-Akaluc, provided by Iwano (Iwano S, et al., 2018, supra), into the vector plasmid pWPXL to construct pWPXL-Venus-Akaluc. Next, we transfected pWPXL-Venus-Akaluc, along with the packaging plasmid pMD2G and the envelope plasmid pCMV deltaR8.74, into LentiX-293T cells (TaKaRa Bio Inc, Shiga, Japan) for lentivirus packaging using Lipofectamine 2000 (Thermo Fisher Scientific). After 3 days of culture, the lentiviral supernatant was collected, centrifuged, and passed through a 0.45 μm filter before being transfected into MSCs. These were then separated into Venus(+) cells (Akaluc-MSCs) and Venus(+) / SSEA-3(+) double-positive cells (Akaluc-Muse cells) using FACS. To separate Akaluc-Muse cells, rat anti-SSEA-3 IgM antibody (1:1000; BioLegend) was used as the primary antibody, and allophycocyanin-conjugated anti-rat IgM antibody (1:100; Jackson ImmunoResearch Laboratories Inc.) was used as the secondary antibody. Based on the behavioral assessments described above, 24 hours after spinal cord infarction model creation, rats were randomly assigned to one of three groups and intravenously administered the following cells: (1) 400,000 Akaluc-Muse cells / 400 μL PBS (Akaluc-Muse group), (2) 400,000 Akaluc-MSC cells / 400 μL PBS (Akaluc-MSC group), or (3) 400 μL PBS (vehicle group). Three rat models were used in each group. The in vivo localization of Muse cells and MSCs was assessed on Day 7 using an IVIS Spectrum CT (Perkin Elmer, Waltham, MA).The rat model was intravenously administered 0.5 ml of 15 mM AkaLumine-HCl, an artificial substrate for Akaluc, followed 5 minutes later by sacrifice with an overdose of isoflurane, 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. The total flux of the spinal cord tissue was assessed within a 3 cm area centered on the ET-1 injection site. The tissue was sectioned at 3 mm intervals, and the sum of the total flux of 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 in each organ of the Akaluc-Muse group and the Akaluc-MSC group was evaluated after subtracting the total flux (autofluorescence) in each organ of the vehicle group.

[0052] 6. Histopathological Evaluation of Cell-transplanted Rat Spinal Cords On day 56, rats were sacrificed by isoflurane overdose, and the spinal cord was subjected to histopathological evaluation as described 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-TUBLIN III (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), goat anti-CD31 (1:200; AF3628, RD SYSTEM, MN, USA), and chicken anti-GFP (1:1000; ab13970, Abcam). The secondary antibodies used were Alexa Fluor 488-conjugated 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-conjugated donkey anti-mouse IgG and goat IgG (1:200, 715-586-150 and 705-585-003, respectively, Jackson ImmunoResearch Laboratories Inc.). For animals 56 days after spinal cord infarction, two 800 × 800 μm areas were selected on the left and right sides, centered on the ventral horn of the spinal cord, 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 in each cross section (cells / mm) 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 are shown as mean ± standard error. For comparisons of two or more groups, two-way analysis of variance and Tukey's honestly significant difference test were used. For comparisons between two groups, a t-test was used. The significance level was P < 0.05 ( * ) was decided.

[0054] Example 1. Preparation of a rat spinal cord infarction model A rat spinal cord infarction model was created 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 and 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 (hereafter 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 for the cell administration experiment is shown in Figure 1. Twenty-four hours after model creation, Muse cells enriched by MACS (MACS-Muse cells) (Figure 2), MSCs, or PBS were administered. Rats that died during observation are 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 and vehicle groups from Day 35 onward. The BBB motor function scores at 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 transition in each group Body weight was monitored for each group up to Day 56. The mean preoperative weight was defined as 1.0, and weight changes at each time point were calculated as a ratio (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. There were no statistically significant differences in the preoperative weight ratios between groups at any observation time point.

[0057] Example 4. Evaluation of biodistribution of transplanted cells In both the Akaluc-Muse and Akaluc-MSC groups, Akaluc signals were detected in the spinal cord and lungs on Day 7 (Figures 5A and 5B). No signals were detected in other organs (Figure 5C). The 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, demonstrating statistically significant signal accumulation in the Akaluc-Muse group (p = 0.049). The total flux in the lungs was 819.5 ± 411.8 in the Akaluc-Muse group and 1606.6 ± 925.5 in the Akaluc-MSC group, demonstrating greater signal accumulation in the Akaluc-MSC group, but not statistically significant (p = 0.50, Figure 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) (Figure 6). GFP-positive cells were observed in the Akaluc-Muse group, and human mitochondrial-positive cells were observed in the MACS-Muse group. Positive and negative controls for each staining are shown in Figure 7. The distribution of transplanted cells in the spinal cord tissue surrounding the spinal cord infarction site was analyzed (Figure 8A). In the MACS-Muse group at Day 56, the number of human mitochondrial-positive cells ( / mm 2) were 10.9 ± 2.8 mm rostral, 17.4 ± 2.2 mm rostral, and 14.3 ± 4.2 mm rostral, 1.8 ± 1.2 mm rostral, 10.4 ± 5.0 mm rostral, 18.0 ± 4.0 mm rostral, and 13.3 ± 3.1 mm rostral, respectively (n = 3, 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. Differentiation efficiency 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 Muse cell differentiation 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 (Figure 11). No double-positive cells with GFAP or Iba-1 were confirmed.

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

[0062] Consideration 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 by 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 recognize the infarcted spinal cord, which has suffered ischemic damage, and home 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 (ibid.)), lung (Yabuki H, et al., 2018 (ibid.)), 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 (ibid.)), 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 Muse cell migration (Yamada Y, et al., 2018, supra), and Muse cells expressing S1PR2 have the ability to sense S1P produced by damaged tissue and migrate to the damaged tissue.

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

[0065] (2) Spontaneous differentiation of human Muse cells into neural or vascular cells Muse cells are known to home to damaged tissue, phagocytose apoptotic cells, and spontaneously differentiate into tissue-specific cells to replace and repair the damaged tissue (Iseki M, et al., 2017 (ibid.); Kajitani T, et al., 2021 (ibid.)). 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 in the observed cross-sections, but no GFAP- or Iba-1-positive cells were observed (Uchida H, et al., 2017 (ibid.)). 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, 25% were GFAP positive, and 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). Human mitochondria and CD31 double positive cells accounted for 17.6±1.8%.

[0066] (3) The effect of human Muse cells on hindlimb motor function in a rat spinal cord infarction model In this study, rats treated with human Muse cells showed statistically significant improvements in hindlimb motor function compared with MSC-treated and vehicle-treated groups. The therapeutic effects of these results may be due to (i) the differentiation of Muse cells into neurons and (ii) the improvement of blood flow due to the differentiation of Muse cells into vascular endothelial cells. The "collateral network concept," which considers that blood flow to the spinal cord is not only provided by the artery of Adamkiewicz but also by collateral circulation, is widely accepted (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 blood 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, showing no statistically significant difference between the two groups. However, there are numerous previous publications reporting effective treatments for spinal cord ischemic injury using MSCs (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 points may explain why MSCs did not have a significant therapeutic effect in this experiment. First, while many literature studies on MSC administration to rat spinal cord ischemia models use 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 new 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 caused 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 based on the clinical scenario of spinal cord infarction after aortic surgery, which is the 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 other administration timings that can provide even greater therapeutic benefits. Optimization may be possible by measuring S1P levels in spinal cord tissue at each time point.

[0069] (5) Future Outlook 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 clinical practice, and Muse cell therapy for spinal cord ischemic injury is expected to become a new therapeutic strategy in the future.

[0070] conclusion We established a novel rat spinal cord infarction model by locally injecting endothelin-1 into spinal cord tissue, resulting in infarction centered on the anterior 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 the rat spinal cord infarction model on 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 contribute to neurological recovery. These results suggest that intravenous administration of Muse cells during the acute phase of spinal cord infarction may have a long-term therapeutic effect lasting for 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. The cell preparation according to any one of claims 1 to 3, wherein the pluripotent stem cells are CD105 positive.

5. The 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. The cell preparation according to any one of claims 1 to 3, 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.

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. The cell preparation according to any one of claims 1 to 3, 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, 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 a 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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