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WO2025170054A1 - Cellules souches pluripotentes pour le traitement de l'infarctus médullaire spinal - Google Patents

Cellules souches pluripotentes pour le traitement de l'infarctus médullaire spinal

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Publication number
WO2025170054A1
WO2025170054A1 PCT/JP2025/004169 JP2025004169W WO2025170054A1 WO 2025170054 A1 WO2025170054 A1 WO 2025170054A1 JP 2025004169 W JP2025004169 W JP 2025004169W WO 2025170054 A1 WO2025170054 A1 WO 2025170054A1
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Prior art keywords
cells
spinal cord
negative
stem cells
muse
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English (en)
Japanese (ja)
Inventor
将之 大谷
佳克 齋木
真理 出澤
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Tohoku University NUC
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Tohoku University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • 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.
  • 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, reducing quality of life and significantly impacting prognosis.
  • the incidence of paraplegia following thoracoabdominal aortic surgery, even in facilities with extensive surgical experience, has been reported to be 8.3% for thoracoabdominal aortic artificial vascular replacement (Non-Patent Document 1) and 11% for thoracoabdominal aortic stent graft treatment (Non-Patent Document 2).
  • Non-Patent Document 3 Cerebrospinal fluid drainage
  • permissive hypertension and the administration of medications such as steroids and naloxone are currently used as treatments, but their effectiveness is limited, and no established treatment method exists.
  • 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).
  • SSEA-3 stage-specific embryonic antigen-3
  • Muse cells Unlike embryonic stem (ES) cells and induced-pluripotent stem (iPS) cells, Muse cells exist in vivo and are therefore non-tumorigenic. 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).
  • S1P sphingosine-1-phosphate
  • Non-Patent Document 13 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).
  • 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 periods of more than six months.
  • HLA human leukocyte antigen
  • Muse cells have an anti-apoptotic effect (Non-Patent Document 20), an inhibitory effect on fibrosis and fibrolysis (Non-Patent Document 17), and angiogenesis (Non-Patent Document 13). Furthermore, Muse cells possess stress resistance and anti-inflammatory properties (Non-Patent Documents 21 and 22), suggesting that they may persist even under the stressful environment of inflammation. Because Muse cells are endogenous stem cells, functional improvement and tissue repair of damaged tissue may occur as a biological response, but it is expected that the therapeutic effect can be further enhanced by administering Muse cells into the body from outside the body. Due to these unique properties, it is believed that treatment of spinal cord infarction with Muse cells could be established as a new proactive treatment method that goes beyond conventional conservative treatments and other cell therapies.
  • 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.117.311648 Wakao S, Oguma Y, Kushida Y, Kuroda Y, Tatsumi K, Dezawa M. Phagocytosing differentiated cell-fragments is a novel mechanism for controll ing somatic stem cell differentiation within a short time frame. Cell Mol Life Sci. Oct 6 2022;79(11):542.
  • Pluripotent Nontumorigenic Adipose Tissue-Derived Muse Cells have Immunomodulat ory Capacity Mediated by Transforming Growth Factor-beta1. Stem Cells Transl Med. Jan 2017;6(1):161-173. doi:10.5966/sctm.2016-0014
  • 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 in 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.
  • pluripotent stem cells e.g., Muse cells
  • the present invention provides cell preparations and/or pharmaceutical compositions containing Muse cells that are effective in 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.
  • the inventors created a rat spinal cord infarction model and used this rat model to examine the therapeutic effects of Muse cell administration on spinal cord infarction. 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.
  • the present invention is as follows.
  • [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.
  • 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 comprising a step of isolating the SSEA-3-positive pluripotent stem cells from mesenchymal tissue of a living body or cultured mesenchymal cells.
  • the method according to [11] comprising a step of enriching SSEA-3-positive pluripotent stem cells by external stress stimulation and/or MACS.
  • the experimental system scheme is shown. The day the spinal cord infarction model was created was defined as 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 week thereafter until Day 56. The animals 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 population was shown by FACS analysis. MSCs from passages 7 to 9 were used.
  • IVIS in vivo imaging system
  • SSEA-3-positive cells were enriched by MACS, and cell populations with an SSEA-3 positivity rate of >70% were defined as MACS-Muse cells.
  • the graph shows the change over time in the BBB motor function score for each group.
  • the BBB motor function scores on Day 56 were 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 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.
  • SSEA-3-positive pluripotent stem cells e.g., Muse cells
  • a detailed description of the present invention is provided below.
  • 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 cell Muse: Multilineage-differentiating stress-enduring
  • NeuN neuronal nucleus NO: nitric oxide
  • PBS
  • 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.
  • the cell preparations of the present invention can be used to treat disorders caused by spinal cord infarction, preferably motor dysfunction and sensory (functional) disorders.
  • motor dysfunction refers to a condition in which voluntary movement is difficult, impossible, or cannot be performed smoothly, and refers to motor paralysis and ataxia. Specific examples include disorders of fine motor skills, Babinski's sign, spasticity, spasm (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, due to damage to the spinal cord, 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. Depending on the severity, symptoms may include sensory loss (anesthesias), hypoesthesia (decreased sensation), hyperesthesia, or abnormal sensations (paresthesia).
  • Pluripotent stem 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.
  • 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. WO 2011/007900. Furthermore, as reported by Wakao et al. (S. Wakao, et al., Proc. Natl. Acad. Sci. USA, Vol. 108, pp.
  • pluripotent stem 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 simply referred to as "SSEA-3-positive cells.”
  • non-Muse cells refer to cells contained in biological mesenchymal tissue or cultured mesenchymal tissue, other than “SSEA-3-positive cells.”
  • pharmaceutical composition is used to have a broader concept than “cell preparation,” or may be used synonymously with "cell preparation.”
  • Muse cells or cell populations containing Muse cells can be isolated from biological tissue (e.g., mesenchymal tissue) using an antibody against the cell surface marker SSEA-3 alone, or using both antibodies against SSEA-3 and CD105.
  • biological tissue e.g., mesenchymal tissue
  • biological organism refers to a mammalian organism.
  • 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 isolated directly from biological tissue using markers.
  • ES cells embryonic stem cells
  • iPS cells embryonic stem cells
  • meenchymal tissue refers to tissues such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligaments, tendons, dental pulp, umbilical cord, and umbilical cord blood, as well as tissues present in various organs.
  • Muse cells can be obtained from bone marrow, skin, or adipose tissue.
  • mesenchymal tissue from a living body and isolate and use Muse cells from this tissue.
  • Muse cells may also be isolated from cultured mesenchymal cells such as fibroblasts or bone marrow mesenchymal stem cells using the above-mentioned isolation method.
  • the Muse cells used may be autologous or allogeneic to the recipient of the cell transplant.
  • 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.
  • adult human skin is known to contain various types of stem and progenitor cells.
  • 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.
  • 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).
  • 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
  • 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.
  • 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) not exhibiting neoplastic growth; and (iv) having self-renewal ability.
  • the Muse cells used in the cell preparation of the present invention have all of the above properties.
  • "low or no telomerase activity” refers to low or undetectable telomerase activity when detected using, for example, a TRAPEZE XL telomerase detection kit (Millipore).
  • telomere activity refers to, for example, telomerase activity equivalent to that of human fibroblasts, which are somatic cells, or telomerase activity that is 1/5 or less, preferably 1/10 or less, of that of HeLa cells.
  • 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.
  • Muse cells have the ability to self-renew (self-replicate).
  • self-renewal refers to the fact that 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 that cells from the embryoid-like cell mass can be brought back into suspension culture at a single cell level to form a next-generation embryoid-like cell mass, from which differentiation into three germ layers and the formation of an embryoid-like cell mass in suspension culture can again be confirmed.
  • the self-renewal may be repeated one or more times.
  • 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, which have at least one, and preferably all, of the following properties, and which 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 the three germ layers; (v) do not exhibit neoplastic growth; and (vi) have the ability to self-renew.
  • the external stress may be any one or a combination of protease treatment, culture at a low oxygen concentration, culture under low phosphate conditions, culture at a 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.
  • the protease treatment time is preferably 0.5 to 36 hours in total to impart external stress to the cells.
  • 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, it is preferable that the protease is trypsin, collagenase, or dispase.
  • 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.
  • MCS magnetically activated cell sorting
  • the Muse cells having the above 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 other means. It is believed that the Muse cells then differentiate into the cells that make up the tissue, thereby treating spinal cord infarction or related symptoms and diseases.
  • 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).
  • physiological saline or an appropriate buffer e.g., phosphate-buffered saline.
  • the cells may be cultured and expanded to a predetermined cell concentration before cell transplantation.
  • 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.
  • the culture of the recovered Muse cells can be carried out in a standard growth medium (e.g., ⁇ -minimal essential medium ( ⁇ -MEM) containing 10% fetal bovine serum).
  • a standard growth medium e.g., ⁇ -minimal essential medium ( ⁇ -MEM) containing 10% fetal bovine serum.
  • 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.
  • the cell preparation of the present invention 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.
  • 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.
  • the cell preparation may contain dimethyl sulfoxide (DMSO) or serum albumin to protect the cells, and antibiotics to prevent bacterial contamination and proliferation.
  • DMSO dimethyl sulfoxide
  • the cell preparation may contain other ingredients acceptable for the formulation (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.
  • ingredients acceptable for the formulation e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.
  • Those skilled in the art can add these factors and drugs to cell preparations at appropriate concentrations.
  • Muse cells can also be used as pharmaceutical compositions containing various additives.
  • the number of Muse cells contained in the cell preparation or pharmaceutical composition prepared as described above can be adjusted appropriately, taking into consideration the subject's gender, age, weight, condition of the affected area, and the condition of the cells used, 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.
  • "improvement” refers to the treatment, prevention, improvement, prevention of worsening, 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.
  • 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).
  • this model can be prepared by injecting, for example, 0.7 ⁇ L 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.
  • E-1 enodocerin-1
  • 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.
  • spinal cord ischemic disorders e.g., motor dysfunction, sensory impairment
  • 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.
  • motor function evaluation include, but are not limited to, the open field test, catwalk test, footfault test, and treadmill test.
  • sensory function evaluation include, but are not limited to, the von Frey test (pain sensation) and the hot plate test (temperature).
  • the "open field test” involves 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 animal's emotional behavior, such as exploratory behavior.
  • the observer actually records the test animal's behavior and also films 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).
  • 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, a “foot fault” is recorded, allowing the degree of impairment to be easily measured.
  • the "treadmill test” is a test in which the subject animal is made to walk at a constant speed on a treadmill and gradually increases the load to evaluate motor function.
  • the von Frey test is a physiological method for assessing pain sensitivity, measuring the subject's touch and pain thresholds through mechanical stimulation.
  • 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.
  • CED Convection-enhanced delivery
  • the rat was fixed in a prone position in a stereotaxic apparatus (NARISHIGÉ, 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 an approximately 3-cm dorsal midline incision was made from the 12th thoracic vertebra to the 1st lumbar vertebra.
  • the 13th thoracic arch was resected to widely expose the dorsal spinal cord.
  • 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.
  • 4.0% Evans Blue (056-04061, FUJIFILM, Tokyo, Japan) dissolved in phosphate-buffered saline (PBS) was injected to optimize the puncture angle.
  • PBS phosphate-buffered saline
  • the injection rate was 0.2 ⁇ L/min.
  • Rats were sacrificed with an overdose of isoflurane, 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).
  • 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.
  • Triton-X-100 168-11805, Fujifilm
  • Triton-X-100 Triton-X-100
  • 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).
  • 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).
  • TTC Triphenyltetrazolium chloride
  • 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).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • MSCs were cultured under conditions 2.
  • Muse cells were isolated from MSCs at passages 7 to 9 as follows: rat anti-SSEA-3 IgM antibody (1:1000; BioLegend, San Diego, CA) was used as the primary antibody, and rat IgM ⁇ chain isotype control (1:1000; BioLegend) was used as the isotype control, and the cells were incubated 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.
  • 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.
  • rat anti-SSEA-3 IgM antibody (1:1000; BioLegend) as the primary antibody
  • allophycocyanin-labeled anti-rat IgM antibody (1:100; Jackson ImmunoResearch Laboratories Inc.
  • SSEA-3-positive and -negative cells were separated using FACS.
  • 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.”
  • 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 ⁇ L 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.
  • 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.
  • the model animals were randomly assigned to three groups, and various cells were intravenously administered: (1) 400,0000 Akaluc-Muse cells/400 ⁇ L PBS (Akaluc-Muse group), (2) 400,0000 Akaluc-MSC cells/400 ⁇ L PBS (Akaluc-MSC group), and (3) 400 ⁇ L PBS (vehicle group).
  • Sections were cut at 3 mm intervals, and the sum of the total flux of each section was defined as the individual's value. Quantification of luminous intensity was performed 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.
  • 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- ⁇ -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).
  • Example 1 Preparation of a Rat Spinal Cord Infarction Model
  • a rat spinal cord infarction model was prepared by injecting 0.7 ⁇ L 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").
  • ET-1 group enodocerin-1
  • 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).
  • Example 3 Body weight transition 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).
  • the weights were 0.93 for the MACS-Muse group, 0.93 for the MSC group, and 0.94 for the vehicle group.
  • the weights were 1.00 for the MACS-Muse group, 0.96 for the MSC group, and 0.96 for the vehicle group.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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)).

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Abstract

Le but de la présente invention est de fournir une nouvelle utilisation médicale de cellules souches pluripotentes (cellules Muse) en thérapie régénérative. La présente invention concerne une préparation cellulaire et une composition pharmaceutique pour le traitement, la prévention, le soulagement et/ou le retardement de l'apparition d'un infarctus médullaire spinal, comprenant des cellules souches pluripotentes SSEA-3-positives isolées à partir de tissus mésenchymateux d'origine biologique ou de cellules mésenchymateuses cultivées. La présente invention est basée sur un mécanisme dans lequel des cellules Muse sont administrées à un sujet ayant un infarctus médullaire spinal, et les cellules sont greffées sur le tissu du site de l'infarctus médullaire spinal, ce qui permet de traiter l'infarctus médullaire spinal.
PCT/JP2025/004169 2024-02-09 2025-02-07 Cellules souches pluripotentes pour le traitement de l'infarctus médullaire spinal Pending WO2025170054A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011007900A1 (fr) * 2009-07-15 2011-01-20 Dezawa Mari Cellule souche pluripotente isolée à partir de tissue organique
JP2015159895A (ja) * 2014-02-26 2015-09-07 株式会社Ｃｌｉｏ 脳梗塞治療のための多能性幹細胞
WO2019216380A1 (fr) * 2018-05-09 2019-11-14 株式会社生命科学インスティテュート Agent thérapeutique pour lésion de la moelle épinière

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011007900A1 (fr) * 2009-07-15 2011-01-20 Dezawa Mari Cellule souche pluripotente isolée à partir de tissue organique
JP2015159895A (ja) * 2014-02-26 2015-09-07 株式会社Ｃｌｉｏ 脳梗塞治療のための多能性幹細胞
WO2019216380A1 (fr) * 2018-05-09 2019-11-14 株式会社生命科学インスティテュート Agent thérapeutique pour lésion de la moelle épinière

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
????????, Muse?????????????????????, ?23???????????, O-26-1, 21 February 2024, (OTANI, Masayuki et al., The 23rd Congress of the Japanese Society for Regenerative Medicine), non-official translation (Investigation of the Therapeutic Efficacy of Intravenous Muse Cell Administration in Spinal Cord Infarction) sections "Background", "Method", "Results", "Conclusion" *
HAYATSU, YUKIHIRO: "Novel cell therapy for spinal cord ischemic injury, Grants-in-Aid for Scientific Research: Final Research Report, Project number 19K09235", 30 January 2024 (2024-01-30), XP093342745, Retrieved from the Internet <URL:https://kaken.nii.ac.jp/ja/file/KAKENHI-PROJECT-19K09235/19K09235seika.pdf> *
OTANI, MASAYUKI, ?????: "Establishment of a novel rat model of spinal cord infarction and investigation of the therapeutic effect of multilineage-differentiating stress-enduring (Muse) cells for spinal cord infarction / ?????????????????Multilineage-differentiating stress-enduring (Muse)????????????????", 2024, pages 1 - 76 *

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