Canine placenta-derived mesenchymal stem cells and use thereof

Abstract

Dogs are the most familiar and old companion animals of humans. Recently, as the number of companion animals grows rapidly, the interest and need for health care of companion dogs are growing. The canine placenta-derived mesenchymal stem cells according to the present invention have a remarkable therapeutic effect on various neurological diseases, skin diseases, or endocrine diseases while maintaining an excellent cell proliferation rate, self-renewal ability, and adipose differentiation ability, and thus are expected to be widely used for animal welfare and health promotion of canine animals including companion dogs and wild dogs.

Description

Canine placental-derived mesenchymal stem cells and uses thereof

[0001] The present invention relates to canine placental-derived mesenchymal stem cells and their uses.

[0002] Dogs are one of humanity's most familiar and oldest companion animals. With the recent rapid increase in the number of households owning pets, interest in and need for their health are growing. While dogs are classified as vertebrates and mammals, like humans, dogs lack saliva amylase, which digests carbohydrates. Unlike humans, dogs have a pH of 6.5 within their mouths, while dogs have a pH of 7.5. Their ear canals are L-shaped, with both vertical and horizontal ear canals, while humans have a straight ear canal. Furthermore, dogs have a 3-5-layer epidermis, compared to 10-15 layers in humans, making them more susceptible to skin diseases. Therefore, despite the strong interest in canine health and disease treatment, it is difficult to apply treatment mechanisms or pharmaceutical compositions studied in humans to dogs.

[0003] Meanwhile, efforts are being made to apply stem cells to treat intractable diseases in companion dogs. However, unlike humans, canine stem cell research is still in its infancy. Therefore, active research and development is needed to determine how to optimally culture stem cells from different tissues and what diseases they can be used to treat. Furthermore, considering the limitations of stem cell passage, securing sufficient cell numbers for research and treatment is crucial.

[0004] Accordingly, the present invention was conceived to address the aforementioned issues, and relates to canine placental-derived stem cells, a method for culturing such stem cells, and the therapeutic use of stem cells cultured using the method of the present invention. It is expected that the present invention will be widely utilized to promote animal welfare and health in canines, including companion dogs and wild dogs.

[0005] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating diseases in animals other than humans.

[0006] To this end, in one aspect of the present invention, a pharmaceutical composition for preventing or treating diseases in animals other than humans is provided, which comprises mesenchymal stem cells derived from the placenta of a canine animal as an active ingredient.

[0007] In another aspect of the present invention, a food composition for preventing or improving diseases of animals other than humans is provided, which comprises mesenchymal stem cells derived from the placenta of a canine animal as an active ingredient.

[0008] In another aspect of the present invention, a composition for external use for preventing or improving diseases of animals other than humans is provided, which comprises mesenchymal stem cells derived from the placenta of a canine animal as an active ingredient.

[0009] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0010] Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques have not been described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0011] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0012] In one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a disease in an animal other than a human, comprising mesenchymal stem cells derived from the placenta of a canine animal as an active ingredient.

[0013] In the present invention, the canine family (Canidae) refers to an animal belonging to a family of mammalian carnivores, and broadly categorized into species such as foxes, raccoon dogs, wolves, wild dogs, and domestic dogs. The skull is slightly elongated, and the larger of the canines is sharp enough to bite and break prey. The upper molars and the first lower molars at the very back are differentiated to enable fine chewing of food. The body is of medium size among carnivores, and is generally slender rather than bulky like bears. The legs are long in wolves and short in raccoon dogs. The forelimbs have five toes, and the hind limbs have four toes. They are terrestrial, walking with only the metatarsals at the ends of their feet touching the ground, and they have hook-shaped claws, but they are not sharp and are exposed. The tail is of average length, has a tuft of hair in the shape of a brush, and may or may not droop downwards. The snout is long and pointed, and the ears are also pointed, but there are slight differences depending on the breed. In the present invention, the canine animal may preferably belong to the genus Vulpes vulpes, Arctic vulpes, Fenech vulpes, Vulpes genus, Raccoon dog, or Canis, and more preferably, the canine animal may belong to the genus Canis, such as Maltese, Pomeranian, English Bulldog, American Bulldog, Continental Bulldog, Serrano bulldog, French Bulldog, Campeiro Bulldog, Chihuahua, Beagle, Yorkshire terrier, Dachshund, Pug, Chichu, Terrier, Spitz, Japan Spitz, German Spitz, Poodle, Shiba Inu, Akita, Schipperke,English Cocker Spaniel, Lhasa Apso, Boxer, Dalmatian, Miniature Pinscher, Brussels Griffon, Jack Russell Terrier, Australian Silky Terrier, Pungsan, Sapsali, Utility dog, Boston Terrier, Tibetan Terrier, Miniature Schnauzer, Bichon Frise, Border Collie, Samoyed, Welsh corgi, Jindo, Chow Chow, Siberian Husky, Bull Terrier Terrier), American Pitbull Terrier, Great Pyrenees, Labrador Retriever, Golden Retriever, Chesapeake Bay Retriever, Flat Coated Retriever, Curly Coated Retriever, German Shepherd, Tosa, English Mastiff, Bullmastiff, Tibetan Mastiff, Malinois, Belgian Sheepdog, Doberman Pinscher, Kangal, Greyhound, Bloodhound,and may be any one or more species selected from, but not limited to, the Great Dane.

[0014] In the present invention, the mesenchymal stem cells derived from the placenta of the canine animal are characterized by excellent cell proliferation rate, self-renewal ability, fat differentiation ability, and antioxidant capacity. At this time, the antioxidant capacity may be evaluated by measuring the glutathione content using the method (stem cell quality measurement method) disclosed in the above inventions (Korean Patent Publication No. KR 10-2017-0101837 A, KR 10-2019-0021943 A, KR 10-2019-0062313 A, KR 10-2019-0062311 A, or KR 10-2021-0085330 A) using a compound (FreSH-tracer) disclosed in the above inventions. The glutathione is an antioxidant that plays an important role in the oxidation-reduction reaction in the body. If the intracellular glutathione content is high, the cell can be defined as a young, healthy cell with a high therapeutic effect. To briefly summarize the stem cell quality measurement methods disclosed in the above prior inventions of the researchers of the present invention, the intracellular glutathione content is Measuring glutathione levels can be performed using a probe that changes wavelength when bound to glutathione and when not bound to glutathione. For example, the probe can be a small molecule probe (FreSHtracer) that can easily enter cells and organelles and bind to the thiol (-SH) group of glutathione. When FreSHtrace binds to glutathione, it exhibits fluorescence in the wavelength range of 510 nm (F510), and when it is not bound to glutathione, it exhibits fluorescence in the wavelength range of 580 nm (F580). The amount of intracellular glutathione can be measured by the fluorescence value of F510 / F580 measured in this way, and the F510 / F580 is expressed as FR (FreSHtracer Ratio). The reaction between FreSHtracer and glutathione is reversible and does not consume intracellular glutathione during the measurement.The above FreSHtrace (Mitochondria Fluorescent Real-time SH group-Tracer) or FreSH is a fluorescent material for detecting thiol in cells or cell organelles. Using this, the antioxidant capacity (ORC, Oxidative Stress-Resistant Capacity) of the cell is measured. At this time, the glutathione activation effect is judged by one or more evaluation indicators selected from the group consisting of GM, GH, and ORC, wherein the GM may be the average or median glutathione value (Glutathione Mean Level, GM) of the cell, the GH may be the glutathione heterogeneity (GH) of the cell, and the ORC may be the antioxidant capacity (Oxidative Stress-Resistant Capacity, ORC) of the cell. In addition, the GM may be calculated as the average or median of cell FR (FreSHtracer Ratio) or F510, the GH may be calculated as the coefficient of variation or robust coefficient of variation of cell FR or F510, and the ORC may be calculated as a value obtained by monitoring F510 (GSH high cell ratio, %) and F580 (GSH low cell ratio, %) in real time after treatment of cells with a terapoptosis-inducing factor.

[0015] In the present invention, the disease may be a skin disease, a neurological disease, an eye disease, an endocrine disease, or other diseases, and specifically, the skin disease may be allergic dermatitis, fungal dermatitis, bacterial dermatitis, atopic dermatitis, mite infection, folliculitis, alopecia, frostbite, eczema, hives, psoriasis, ringworm, lichen, keratosis pilaris, nevus, warts, in situ neoplasms, fibroma, dermatofibrosarcoma, lipoma, epidermal cyst, It may be at least one selected from the group consisting of cutaneous abscess, pruritus, xerosis cutis, pediculosis pubis, scabies, skin defect, and burn, and the neurological disease may be hypertrophic ganglionitis, hyperventilation syndrome, glioblastoma, amyotrophic lateral sclerosis, dystonia, myasthenia gravis, cerebral arteriovenous malformation, cerebral infarction, brain abscess, cerebral aneurysm, epilepsy,It may be at least one selected from the group consisting of cerebral vascular disease, narcolepsy, multiple sclerosis, polyneuropathy, hydrocephalus, tremor, seizure, meningitis, meningioma, neuroma, and paralysis, and the eye disease may be corneal ulcer, corneal opacity and scar, keratitis, conjunctivitis, fungal corneal ulcers, uveitis, pollen allergy, glaucoma, cataract, stye, Marfan syndrome, optic neuritis, and choroidal melanoma, and the endocrine disease may be at least one selected from the group consisting of hypernatremia, hyponatremia, hypermagnesemia, hypomagnesemia, hyperkalemia, hypokalemia, hyperalbuminemia, hypoalbuminemia, azotemia, acute tubulo-interstitial nephritis, acute renal failure, chronic renal failure, pyelonephritis, hypothyroidism, hyperthyroidism, thyroiditis,It may be at least one selected from the group consisting of hyperfunction of pituitary gland, hypopituitarism, hypoparathyroidism, hyperparathyroidism, portosystemic shunt, Cushing's syndrome, and diabetes, and the other diseases include cancer, peritonitis, pancreatitis, otitis media, laryngitis, hepatitis, anemia, thrombocytopenia, osteoporosis, dislocation, Arnold-chiari malformation, dysplasia, and spinal cord. It may be any one or more selected from the group consisting of, but not limited to, Spinal cord myelopathy, Cervical HIVD, and Herniation of intervertebral disk.

[0016] However, in the present invention, the mesenchymal stem cells derived from the placenta of the canine are most preferably stem cells derived from the placenta isolated from a Shih Tzu or a bulldog among the genus Canis, and the bulldog may be at least one selected from the group consisting of an English bulldog, an American bulldog, a continental bulldog, a Serrano bulldog, a French bulldog, and a Campeiro bulldog, and the placenta naturally shed from the mother within 24 hours without a separate chemical treatment after giving birth in the dog is washed with sterile 1× PBS containing 100 units / ㎖ penicillin and 100 μg / ㎖ streptomycin, the PBS washing solution is removed, the tissue is immersed in 5㎖ of 0.25% trypsin solution and treated at 37℃ for 30 minutes, the trypsin action is stopped with Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS, the suspension is collected and centrifuged at 1,500 rpm for 7 minutes, and The supernatant was removed, and the pellet was treated with 5 ml of collagenase IA (1 mg / ml) at 37°C for 45 minutes, the collagenase reaction was stopped with DMEM containing 10% FBS, and the pellet was centrifuged to remove the supernatant. The pellet was resuspended in DMEM containing 10% FBS, seeded on a dish prepared for tissue culture, and then passaged as a monolayer culture for 2 weeks to obtain the cells migrated from the tissue. The cells obtained by the above method are set as passage 1, and after the cells are separated from the tissue (passage 1), the cells can be cultured using a medium as a base medium containing MEM Alpha (Welgene, LM 008-02) as a base medium supplemented with 10% FBS (Hyclone, SH30919.03) and 1% PS (Hyclone, SV30010).As an effective ingredient of the pharmaceutical composition of the present invention, the canine placental-derived mesenchymal stem cells may be cells within passage 20, cells within passage 18, cells within passage 15, cells within passage 13, cells within passage 10, cells from passage 2 to passage 18, cells from passage 4 to passage 18, cells from passage 6 to passage 18, cells from passage 2 to passage 15, cells from passage 4 to passage 15, cells from passage 6 to passage 15, cells from passage 2 to passage 12, cells from passage 4 to passage 12, cells from passage 6 to passage 12, cells from passage 2 to passage 10, cells from passage 4 to passage 10, cells from passage 6 to passage 10, cells from passage 2 to passage 8, cells from passage 4 to passage 8, or cells from passage 6 to passage 8.

[0017] In the present invention, the pharmaceutical composition is a composition administered for a specific purpose, and for the purpose of the present invention, the pharmaceutical composition of the present invention is used for the prevention or treatment of diseases of animals other than humans. The pharmaceutical composition comprising canine placental-derived mesenchymal stem cells as an effective ingredient of the present invention may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions, but is not limited thereto, according to conventional methods. In addition, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, and fragrances for oral administration, and buffers, preservatives, analgesics, solubilizers, isotonic agents, and stabilizers may be mixed and used for injections, and bases, excipients, lubricants, and preservatives may be used for topical administration. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing it with the pharmaceutically acceptable carriers described above. For example, the pharmaceutical composition of the present invention may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. for oral administration, and the injection may be prepared in the form of unit dose ampoules or multiple doses. In addition, the composition may be formulated as a solution, suspension, tablet, capsule, sustained-release preparation, etc. Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be additionally included.

[0018] In the present invention, the route of administration of the pharmaceutical composition includes, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.

[0019] Meanwhile, in the present invention, disease prevention includes all of blocking symptoms caused by a specific disease in advance, or suppressing or delaying the symptoms by using the pharmaceutical composition of the present invention.

[0020] In addition, in the present invention, treatment or improvement of a disease includes all improvements or benefits in symptoms caused by a specific symptom by using the pharmaceutical composition of the present invention.

[0021] In another aspect of the present invention, the present invention provides a food composition for preventing or improving diseases of animals other than humans, comprising mesenchymal stem cells derived from the placenta of a canine animal as an active ingredient.

[0022] The definitions of canids (Canidae), canine placental-derived mesenchymal stem cells, and diseases herein are the same as those described in the pharmaceutical composition above, and therefore are omitted to avoid excessive complexity of the specification.

[0023] In the present invention, the food composition is a composition administered for a specific purpose, and for the purpose of the present invention, the food composition of the present invention is used for the prevention or improvement of diseases in animals other than humans. The food composition comprising canine placental-derived mesenchymal stem cells as an effective ingredient of the present invention can be manufactured in the form of various foods, such as beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, jellies, gels, confectionery, rice cakes, bread, etc. Since the food composition of the present invention is derived from natural substances (organisms) with almost no toxicity or side effects, it can be used with confidence even when taken for a long period of time for preventive purposes. When the composition of the present invention is included in a food composition, the amount can be added at a ratio of 0.1 to 100% of the total weight. Here, when the food composition is manufactured in the form of a beverage, there are no special restrictions other than containing the food composition at the indicated ratio, and various flavoring agents or natural carbohydrates, etc., can be contained as additional ingredients like conventional beverages. That is, it may include natural carbohydrates such as monosaccharides such as glucose, disaccharides such as fructose, sucrose, polysaccharides, dextrin, cyclodextrin, and common sugars, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agent (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agent (saccharin, aspartame, etc.) can be mentioned. In addition, the food composition of the present invention can contain various nutrients, vitamins, minerals (electrolytes), flavoring agent such as synthetic flavoring agent and natural flavoring agent, coloring agent, pectic acid and its salt, alginic acid and its salt, organic acid, protective colloid thickener, pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonating agent used in carbonated beverage, etc. These components can be used independently or in combination. The ratio of such additives is typically 0.It is generally selected from the range of 1 to 100 parts by weight, but is not limited thereto.

[0024] In another aspect of the present invention, the present invention provides an external preparation composition for preventing or improving diseases of animals other than humans, comprising mesenchymal stem cells derived from the placenta of a canine animal as an active ingredient.

[0025] The definitions of canids (Canidae), canine placental-derived mesenchymal stem cells, and diseases herein are the same as those described in the pharmaceutical composition above, and therefore are omitted to avoid excessive complexity of the specification.

[0026] In the present invention, the external preparation composition is a composition administered for a specific purpose, and for the purpose of the present invention, the external preparation composition of the present invention is used for the prevention or improvement of diseases of animals other than humans. The external preparation composition comprising canine placental-derived mesenchymal stem cells as an effective ingredient of the present invention includes a cosmetic composition applied to the external tissue of an animal body, and a therapeutic preparation. When the external preparation composition is used as a therapeutic preparation, the most common form may be an ointment or artificial tears. In addition, the external preparation composition containing the active ingredient of the present invention can be manufactured in the form of a toner, a nutritional lotion, a nutritional essence, a massage cream, a cosmetic bath additive, a body lotion, a body milk, a bath oil, a baby oil, a baby powder, a shower gel, a shower cream, a sunscreen lotion, a sunscreen cream, a suntan cream, a skin lotion, a skin cream, a UV-blocking cosmetic, a cleansing milk, a hair loss agent {for cosmetic use}, a face and body lotion, a face and body cream, a skin whitening cream, a hand lotion, a hair lotion, a cosmetic cream, jasmine oil, a bath soap, a liquid soap, a cosmetic soap, a shampoo, a hand cleanser (hand cleaner), a medicated soap {for non-medical use}, a cream soap, a facial wash, a hair rinse, a cosmetic soap, a tooth whitening gel, a toothpaste, etc. To this end, the composition of the present invention may further include an appropriate carrier, excipient, or diluent commonly used in the manufacture of an external preparation composition. Carriers, excipients, or diluents that may be further added to the external preparation composition of the present invention include, but are not limited to, purified water, oil, wax, fatty acid, fatty acid alcohol, fatty acid ester, surfactant, humectant, thickener, antioxidant, viscosity stabilizer, chelating agent, buffer, lower alcohol, etc. In addition, whitening agents, moisturizers, vitamins, sunscreens, perfumes, dyes, antibiotics, antibacterial agents, and antifungal agents may be included as needed.Examples of the oils that can be used include hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, and avocado oil. Examples of the waxes that can be used include beeswax, spermaceti, carnauba, candelilla, montan, ceresin, liquid paraffin, and lanolin. Examples of the fatty acids that can be used include stearic acid, linoleic acid, linolenic acid, and oleic acid. Examples of the fatty acid alcohols that can be used include cetyl alcohol, octyldodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, and hexadecanol. Examples of the fatty acid esters that can be used include isopropyl myristate, isopropyl palmitate, and butyl stearate. Examples of the surfactant that can be used include cationic surfactants, anionic surfactants, and nonionic surfactants known in the art, and surfactants derived from natural products are preferable if possible. In addition, it may include absorbents, thickeners, antioxidants, etc., which are widely known in the external application field, and the types and amounts of these are as known in the art.

[0027] In another aspect of the present invention, the present invention relates to a dog, including a Maltese, a Pomeranian, an English Bulldog, an American Bulldog, a Continental Bulldog, a Serrano bulldog, a French Bulldog, a Campeiro Bulldog, a Chihuahua, a Beagle, a Yorkshire terrier, a Dachshund, a Pug, a Chichu, a Terrier, a Spitz, a Japan Spitz, a German Spitz, a Poodle, a Shiba, an Akita, a Schipperke, an English Cocker Spaniel, Lhasa Apso, Boxer, Dalmatian, Miniature Pinscher, Brussels Griffon, Jack Russell Terrier, Australian Silky Terrier, Pungsan, Sapsali, Utility dog, Boston Terrier, Tibetan Terrier, Miniature Schnauzer, Bichon Frise, Border Collie, Samoyed, Welsh Corgi, Jindo, Chow Chow, Siberian Husky, Bull Terrier, American Pitbull Terrier, Great Dane Great Pyrenees,A step of selecting one of the following species: Labrador Retriever, Golden Retriever, Chesapeake Bay Retriever, Flat Coated Retriever, Curly Coated Retriever, German Shepherd, Tosa, English Mastiff, Bullmastiff, Tibetan Mastiff, Malinois, Belgian Sheepdog, Doberman Pinscher, Kangal, Greyhound, Bloodhound, and Great Dane; And, a method for producing a pharmaceutical composition, food composition, or external preparation composition for preventing or improving / treating a disease of an animal other than a human, comprising a step of isolating mesenchymal stem cells derived from the placenta of the animal, is provided.

[0028] In the above manufacturing method, the definitions of canines (Canidae), canine placental-derived mesenchymal stem cells, and diseases are the same as those described above in the pharmaceutical composition, and therefore are omitted to avoid excessive complexity of the specification.

[0029] In another aspect of the present invention, a method for preventing or treating a disease in an animal other than a human is provided, comprising the step of administering the pharmaceutical composition of the present invention to an animal other than a human in need of treatment for a skin disease, a neurological disease, an eye disease, an endocrine disease, or other disease.

[0030] In the above prevention or treatment method, the definitions of skin disease, nervous disease, eye disease, endocrine disease, or other diseases are the same as those described in the above pharmaceutical composition, and therefore are omitted to avoid excessive complexity of the specification.

[0031] In another aspect of the present invention, the pharmaceutical composition of the present invention is provided for use in preventing or treating skin diseases, neurological diseases, eye diseases, endocrine diseases, or other diseases in animals other than humans.

[0032] The definitions of skin diseases, neurological diseases, eye diseases, endocrine diseases, or other diseases for the above preventive or therapeutic purposes are the same as those described above in the pharmaceutical composition, and therefore are omitted to avoid excessive complexity of the specification.

[0033] The canine placenta-derived mesenchymal stem cells according to the present invention have a remarkable therapeutic effect on various neurological diseases, skin diseases, or endocrine diseases while maintaining excellent cell proliferation rate, self-renewal ability, and fat differentiation ability.

[0034] Figure 1 shows the results of analyzing the cell proliferation ability of stem cells derived from various types of canine placental tissues using PDL (Population Doubling Level) according to one embodiment of the present invention.

[0035] Figure 2 shows the results of analyzing the cell proliferation ability of stem cells derived from various types of canine placental tissues using DT (Doubling Time) according to one embodiment of the present invention.

[0036] FIG. 3 shows the results of analyzing the self-renewal ability of stem cells derived from various types of canine placental tissues in terms of colony forming units (CFU) according to one embodiment of the present invention.

[0037] Figure 4 shows the results of analyzing the fat differentiation ability of stem cells derived from various types of canine placental tissue according to one embodiment of the present invention.

[0038] Figure 5 shows the results of analyzing the antioxidant capacity of stem cells derived from various types of canine placental tissue according to one embodiment of the present invention.

[0039] Figure 6 shows the results of testing the therapeutic effect of the canine placental stem cells of the present invention in dogs suffering from hypothyroidism, chronic renal failure, and atopic dermatitis, according to one embodiment of the present invention.

[0040] Figure 7 shows the results of testing the therapeutic effect of the canine placental stem cells of the present invention on a dog suffering from epilepsy, according to one embodiment of the present invention.

[0041] Figure 8 shows the results of testing the therapeutic effect of the canine placenta-derived stem cells of the present invention on a dog suffering from cataracts, according to one embodiment of the present invention.

[0042] FIG. 9 shows the results of testing the therapeutic effect of the canine placental stem cells of the present invention in dogs suffering from chronic renal failure, peritonitis, pancreatitis, hypothyroidism, hepatitis, azotemia, anemia, hypoalbuminemia, and thrombocytopenia, according to one embodiment of the present invention.

[0043] FIG. 10a and FIG. 10b show the results of testing the therapeutic effect of the canine placental stem cells of the present invention in dogs suffering from hyponatremia, Cushing's syndrome, hypothyroidism, and FH dislocation, according to one embodiment of the present invention.

[0044] Figures 11a to 11d show the results of testing the therapeutic effect of the canine placental stem cells of the present invention on a dog suffering from epilepsy, according to one embodiment of the present invention.

[0045] FIG. 12 shows the results of testing the therapeutic effect of the canine placental stem cell of the present invention according to one embodiment of the present invention on dogs suffering from idiopathic epilepsy, paroxysmal dyskinesia, hydrocephalus (ventricular hypertrophy, Lt. 41%, Rt. 33%), Chiari malformation, laryngeal dysplasia, spinal cord myelopathy (moderate), and bilateral otitis media.

[0046] FIGS. 13a to 13d show the results of testing the therapeutic effect of the canine placental stem cells of the present invention in dogs suffering from seizures, paraplegia, spinal cord myelopathy, and multiple cervical IVDD, according to one embodiment of the present invention.

[0047] FIG. 14 shows the results of testing the therapeutic effect of the canine placental stem cells of the present invention according to one embodiment of the present invention on dogs suffering from C4-5 IVDD nucleus pulposus extrusion and acute nucleus pulposus protrusion, C1-2 hypertrophic ganglionitis, and annulus fibrosus protrusion at C2-3 and C5-6.

[0048] Figures 15a and 15b show the results of testing the therapeutic effect of the canine placental stem cells of the present invention on a dog suffering from laryngeal paralysis, according to one embodiment of the present invention.

[0049] Figures 16a to 16d show the results of testing the therapeutic effect of the canine placental stem cells of the present invention on dogs suffering from seizures and paralysis of the lower body, according to one embodiment of the present invention.

[0050] Figure 17 shows the results of testing the therapeutic effect of the canine placenta-derived stem cells of the present invention on a dog suffering from alopecia, according to one embodiment of the present invention.

[0051] Figures 18a to 18d show the results of testing the therapeutic effect of the canine placental stem cells of the present invention on a dog suffering from thrombocytopenia, according to one embodiment of the present invention.

[0052] We isolated and cultured placental stem cells from four different dog breeds (Maltese, Shih Tzu, Poodle, or French Bulldog) and analyzed their cell proliferation rates. Surprisingly, the Population Doubling Level (PDL) and Doubling Time (DT) showed significant differences depending on the dog breed. At passage 3, Shih Tzu and French Bulldog-derived stem cells maintained high PDL and low DT and still maintained active proliferation, but Maltese-derived stem cells showed low PDL and high DT, indicating a loss of proliferation. This means that it is difficult to secure a sufficient number of cells for research and treatment using Maltese-derived stem cells. On the other hand, French Bulldog-derived stem cells are most desirable for use in research and therapeutic development.

[0053] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0054] Example

[0055] Example 1. Culturing of canine placental stem cells

[0056] Placental tissues from four different dog breeds (Maltese, Shih Tzu, Poodle, or French Bulldog) were obtained within 24 hours and washed with sterile 1× PBS containing 100 units / mL penicillin and 100 μg / mL streptomycin (Thermo Fisher). The PBS wash was removed, and the tissues were treated with 5 mL of a 0.25% trypsin solution at 37°C for 30 minutes. The trypsin action was stopped with Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS, the collected suspension was centrifuged at 1,500 rpm for 7 minutes, and the supernatant was removed. The pellet was then treated with 5 mL of collagenase IA (1 mg / mL) at 37°C for 45 minutes, the collagenase reaction was stopped with DMEM containing 10% FBS, and the tissues were centrifuged again and the supernatant was removed. The pellet was resuspended in DMEM containing 10% FBS and seeded onto dishes prepared for tissue culture. Cells migrated from the tissue were passaged in monolayer culture for 2 weeks and designated as passage 1. Cells were then cultured in a basal medium containing MEM Alpha (Welgene, LM 008-02) supplemented with 10% FBS (Hyclone, SH30919.03) and 1% PS (Hyclone, SV30010). Subculture was performed each time the cells reached 80% of the culture dish confluent. Specifically, the culture medium was washed twice with PBS, 1 ml of TrypLE was added, and the reaction was performed for 2 minutes at 37°C and 5% CO2. This was neutralized with culture medium, centrifuged at 1500 rpm and 4°C for 5 minutes, the supernatant was removed, the pellet was resuspended, and the cell number was counted using Trypan blue. An appropriate amount of cells was inoculated into a new culture vessel and cultured again at 37°C and 5% CO2.

[0057] Example 2. Cell proliferation rate analysis (Cumulative Population Doubling Level, CPDL)

[0058] When the canine placental stem cells were cultured at 80-90% density of the cell culture vessel, the cells were centrifuged using the method of Example 1, and the pellet was resuspended to count the cell number. Each cell was seeded into 3 wells of a 6-well plate, and the cells were collected every 3 days to count the cell number. The PDL (Population Doubling Level) and DT (Doubling Time) values ​​were calculated using the following methods of Equations 1 to 3.

[0059] [Formula 1]

[0060] PDL = Ln (cell number I / cell number F) / ln2

[0061] In the above formula, cell number I: number of inoculated cells, cell number F: number of final collected cells.

[0062] [Formula 2]

[0063]

[0064] [Formula 3]

[0065] CPDL = cumulative PDL value

[0066] The measurement results surprisingly showed a large difference in PDL and DT depending on the dog species. In the early stage of culture (passage 4), the difference between species was not statistically significant, but after passage 5, the difference in PDL and DT values ​​between species began to widen. At passage 3, Shih Tzu- and French Bulldog-derived stem cells maintained high PDL and low DT and still maintained active proliferation, but Maltese-derived stem cells showed low PDL and high DT, indicating a loss of proliferation (Fig. 1 and Fig. 2). This means that it is difficult to secure a sufficient number of cells for research and treatment using Maltese-derived stem cells. On the other hand, French Bulldog-derived stem cells are most desirable for use in research and therapeutic development.

[0067] Example 3. Measurement of stem cell self-renewal capacity

[0068] To determine the self-renewal ability of canine placental stem cells in terms of colony forming units (CFU), passage 3 cells were seeded in 3 wells per well of a 6-well plate (200 cells per well) and cultured while changing the medium every 2-3 days. After 7 days, the cells were washed twice with DPBS and fixed with cold methanol for 5 minutes. After removing the methanol, 1 ml of 0.1% Crystal violet was added and the staining reaction was performed for 20 minutes. After removing the dye, the cells were washed three times with distilled water, the culture dish was dried, and a photograph was taken of the cells to count the number of stained colonies. The test results showed that the self-renewal ability of canine placental stem cells, like PDL and DT, showed significant differences depending on the dog species. At passage 3, Maltese and Poodle-derived stem cells showed minimal CFU formation, indicating a near-total loss of self-renewal capacity. However, French Bulldog-derived stem cells maintained a very high level of self-renewal capacity (Fig. 3). This suggests that French Bulldog-derived stem cells are highly desirable for research and therapeutic development, both quantitatively and qualitatively.

[0069] Example 4. Measurement of stem cell adipogenic differentiation capacity

[0070] To confirm the adipogenic ability of canine placental stem cells, passage 3 cells were seeded in 3 wells per well in a 48-well plate (25,000 cells per well) and replaced with differentiation medium the next day. The differentiation medium was MEM Alpha based and supplemented with 10% FBS, 10 μg / ml insulin, 500 μM IBMX, 1 μM dexamethasone, and 1 μM rosiglitazone. The cells were cultured while replacing the medium every 2-3 days, and after 7 days, the cells were washed twice with DPBS and fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. After removing the PFA, 0.2% Oil Red O solution was added and the staining reaction was performed with gentle shaking for 20 minutes. After removing the dye, the cells were washed 4-5 times with distilled water, and photographs were taken to observe the stained cells. The test results showed that the adipogenic capacity of canine placental stem cells also showed significant differences depending on the dog species. In Passage 3, Maltese and Shih Tzu-derived stem cells did not maintain the spherical shape characteristic of stem cells, and fat formation was very poor. Poodle-derived stem cells maintained the spherical shape characteristic of stem cells, but the number was very small. On the other hand, French bulldog-derived stem cells maintained the spherical shape characteristic of stem cells in a significantly large number of cells, and the degree of fat formation was also very excellent (Fig. 4). This means that French bulldog-derived stem cells have a much superior adipogenic capacity compared to stem cells from other canine species.

[0071] Example 5. Measurement of stem cell antioxidant capacity

[0072] Glutathione (GSH), an antioxidant substance in canine placental stem cells, was measured. To this end, passage 3 cells were seeded in 3 wells per cell in a 96-well plate (25,000 cells per well), cultured for 24 hours, replaced with fresh medium, treated with 2 μM RSL3 ((1S,3R)-RSL3), and incubated for 2 hours. The medium was then replaced with HBSS (Hank's Balanced Salt Solution) and treated with 10 μM MitoFreSHtracer. The MitoFreSHtracer was a compound disclosed in our research team's prior invention KR 10-2019-0062313 A. After 1 hour, the HBSS was replaced with fresh HBSS, and the F510 and F580 values ​​were measured using Operetta. The test results showed that French bulldog-derived stem cells had significantly higher antioxidant capacity (Oxidative Stress-Resistant Capacity, ORC) and glutathione mean level (GM), and lower glutathione heterogeneity (GH) than stem cells derived from other canine species (Fig. 5). This indicates that French bulldog-derived stem cells have significantly superior antioxidant capacity compared to stem cells derived from other canine species. Hereinafter, French bulldog-derived stem cells will be named PI-MSC, and their disease treatment potential will be tested preclinically in canines.

[0073] Example 6. Confirmation of the therapeutic effect of canine placental stem cells.

[0074] (1) Seol-ha, a 17-year-old Maltese weighing 1.54 kg, suffered from hypothyroidism, chronic renal failure, and atopic dermatitis. Seol-ha was administered PI-MSC injections at approximately weekly intervals and followed for nine months. Results showed a decrease in BUN and creatinine levels, and a significant improvement in her atopic dermatitis (Fig. 6).

[0075] (2) Snowflake, a 3.5 kg, 3-year-old Maltese, suffered from epilepsy. After administering PI-MSCs three times at approximately one-week intervals, the MRI findings of the epilepsy were confirmed to be alleviated (Fig. 7).

[0076] (3) Tashua, a 14-year-old poodle weighing 2.8 kg, suffered from cataracts. PI-MSC intravenous injections into Tashua confirmed improvement in his cataracts (Fig. 8).

[0077] (4) Nako, a 9-year-old Pomeranian weighing 3.8 kg, suffered from chronic renal failure, peritonitis, pancreatitis, hypothyroidism, hepatitis, azotemia, anemia, hypoalbuminemia, and thrombocytopenia. Nako was administered 20 injections of PI-MSCs over approximately 18 months and monitored for progression. Results showed improvement in hematological findings (Fig. 9).

[0078] (5) Coco, a 17-year-old Yorkshire Terrier weighing 5.7 kg, suffered from hyponatremia, Cushing's syndrome, hypothyroidism, and FH dislocation. Administering PI-MSC injections to Coco resulted in improvement in hyponatremia (Fig. 10).

[0079] (6) Mong, a 5-year-old, 3.4 kg, long-haired Chihuahua, suffers from epilepsy. After administering PI-MSCs three times at approximately one-week intervals to Mong, the CT findings of epilepsy were confirmed to be alleviated (Fig. 11).

[0080] (7) Princess, a 6-year-old companion dog weighing 3.3 kg, suffered from idiopathic epilepsy, paroxysmal dyskinesia, hydrocephalus (ventriculomegaly, Lt. 41%, Rt. 33%), Chiari malformation, laryngeal dysplasia, spinal cord myelopathy (moderate), and bilateral otitis media. After 10 injections of PI-MSCs administered to Princess at approximately 3-day intervals, MRI showed a significant improvement in hydrocephalus (Fig. 12).

[0081] (8) Minnie, a 10-year-old Yorkshire Terrier weighing 2.23 kg, suffered from seizures, paraplegia, spinal cord myelopathy, and multiple cervical intracranial lesions (IVDD). She was followed for approximately 14 months, receiving 17 injections of PI-MSCs. As a result, MRI showed that the right frontal lobe lesion observed before PI-MSC administration had disappeared, and the left frontal lobe lesion had changed. MRI also showed that the lesion in the C3-4 spinal cord observed before PI-MSC administration had disappeared (Fig. 13).

[0082] (9) A 13-year-old, 9.1 kg, pug-type dog, Tansun, suffered from C4-5 IVDD nucleus pulposus extrusion and acute nucleus pulposus protrusion, C1-2 hypertrophic ganglionitis, and C2-3, C5-6 annulus fibrosus protrusion. Tansun was administered 7 injections of PI-MSCs at 3-day intervals and monitored for progression. As a result, improvement in nucleus pulposus extrusion and nucleus pulposus protrusion was confirmed on MRI (Fig. 14).

[0083] (10) Danbi, a 5-year-old Maltese weighing 3.5 kg, fell from a table and suffered laryngeal paralysis, rendering her completely unable to use her hind legs. Danbi received nine injections of PI-MSC over approximately eight months, and her progress was monitored. As a result, MRI revealed remarkable recovery of her spinal cord, and she was able to stand with her hind legs straight (Fig. 15).

[0084] (11) Bolt, a 5-year-old Maltese weighing 2.23 kg, suffered from seizures and paraplegia. He was treated with PI-MSC injections 17 times over approximately 20 months and his progress was tracked. As a result, MRI revealed parenchymal changes in the ventral aspect of the thoracic spinal cord, dilatation of the central canal at T3-4, and a decrease in the size of the C3-4 spinal foramen compared to before. In addition, the left ventral spinal cord compression by the L2-3 intervertebral disc was found to be reduced compared to before. Ultimately, Bolt, who had been paraplegic, was able to walk normally again (Fig. 16).

[0085] (12) A 1.5 kg, 8-year-old Pomeranian dog suffered from alopecia. The dog was injected subcutaneously with PI-MSCs along the spine on its back six times over approximately one month, and the dog's progress was monitored. The results clearly demonstrated that hair growth occurred only in the areas where PI-MSCs were injected. Specifically, after the third PI-MSC injection, darkened hair follicles formed at the injection site and hair growth began. Furthermore, the hair growth became more distinct and thicker after the third PI-MSC injection (Fig. 17).

[0086] (13) A 6-year-old patient, weighing 7.7 kg, of the Bichon Frise breed, presented with purpura, corneal hemorrhage, bloody stool, hematuria, and bleeding tendency, and a blood analysis revealed a platelet count of 10×10 9 / L or less. The patient was a repeat patient who had previously visited our hospital due to recurrent immune-mediated thrombocytopenia (IMT), and hepatomegaly was confirmed on X-ray. Although he responded well to intravenous immunoglobulin infusion treatment supplemented with vincristine, steroids, and mycophenolate mofetil, thrombocytopenia recurred six times at 2-11 month intervals over 2.5 years. However, the development of steroid-induced hepatitis (ALT > 2,036 u / L; ALKP > 3500 u / L) and panleukopenia limited further immunosuppressive treatment for IMT. Therefore, PI-MSC therapy was performed as a second-line treatment for anti-inflammatory and immunomodulatory effects. PI-MSC (1 × 10 in normal saline) 6 After administering PI-MSCs (cells / kg) every other week for five times, platelet counts were confirmed to return to normal levels. After continuing to administer PI-MSCs once a week for approximately 3 months, platelet counts were maintained within the normal range (286×10 9 / L - 561×10 9Platelet counts were maintained within / L. Steroid administration was discontinued after three PI-MSC administrations, but serum levels of alanine transaminase (ALT) and alkaline phosphatase (ALKP) remained within the normal range. No side effects were observed (Fig. 18).

[0087] Although the present invention has been described in detail above, the scope of the present invention is not limited thereto, and it will be apparent to those skilled in the art that various modifications and variations are possible within a scope that does not depart from the technical spirit of the present invention described in the claims.

[0088] Efforts are being made to apply stem cells to treat intractable diseases in companion dogs. However, unlike humans, research on stem cells in dogs is still in its infancy. Therefore, active research and development is required to determine which tissue-derived stem cells can be optimally cultured and which diseases can be treated. The present invention relates to canine placental-derived stem cells, a method for culturing the stem cells, and the therapeutic use of stem cells cultured using the method of the present invention. It is expected that the present invention will be greatly utilized to promote animal welfare and health in canines, including companion dogs and wild dogs.

Claims

1. A pharmaceutical composition for the prevention or treatment of animal diseases other than humans, containing mesenchymal stem cells derived from the placenta of canines as an active ingredient.

2. In paragraph 1, A pharmaceutical composition wherein the above disease is a skin disease, a nervous disease, an eye disease, an endocrine disease, or other disease.

3. In paragraph 1, The above canine animals are Maltese, Pomeranian, English Bulldog, American Bulldog, Continental Bulldog, Serrano bulldog, French Bulldog, Campeiro Bulldog, Chihuahua, Beagle, Yorkshire terrier, Dachshund, Pug, Chichu, Terrier, Spitz, Japan Spitz, German Spitz, Poodle, Shiba, Akita, Schipperke, English Cocker Spaniel, Lhasa Apso, Boxer, Dalmatian, Miniature Pinscher, Brussels Griffon, Jack Russell Terrier, Australian Silky Terrier, Pungsan, Sapsali, Utility dog, Boston Terrier, Tibetan Terrier, Miniature Schnauzer, Bichon Frise, Border Collie, Samoyed, Welsh Corgi, Jindo, Chow Chow, Siberian Husky, Bull Terrier, American Pitbull Terrier, Great Pyrenees,A pharmaceutical composition comprising at least one species selected from the group consisting of Labrador Retriever, Golden Retriever, Chesapeake Bay Retriever, Flat Coated Retriever, Curly Coated Retriever, German Shepherd, Tosa, English Mastiff, Bullmastiff, Tibetan Mastiff, Malinois, Belgian Sheepdog, Doberman Pinscher, Kangal, Greyhound, Bloodhound, and Great Dane.

4. In paragraph 1, The above skin diseases are allergic dermatitis, fungal dermatitis, bacterial dermatitis, atopic dermatitis, mite infection, folliculitis, alopecia, frostbite, eczema, hives, psoriasis, ringworm, lichen, keratosis pilaris, nevus, warts, in situ neoplasms, fibroma, dermatofibrosarcoma, lipoma, epidermal cyst, cutaneous abscess, pruritus, xerosis A pharmaceutical composition comprising at least one selected from the group consisting of cutis, pediculosis pubis, scabies, skin defect, and burn.

5. In paragraph 1, The above neurological diseases include hypertrophic ganglionitis, hyperventilation syndrome, glioblastoma, amyotrophic lateral sclerosis, dystonia, myasthenia gravis, cerebral arteriovenous malformation, cerebral infarction, brain abscess, cerebral aneurysm, epilepsy, cerebral stroke, narcolepsy, multiple sclerosis, polyneuropathy, hydrocephalus, tremor, seizure, meningitis, meningioma, A pharmaceutical composition comprising at least one selected from the group consisting of neuroma, and paralysis.

6. In paragraph 1, A pharmaceutical composition, wherein the above eye disease is at least one selected from the group consisting of corneal ulcer, corneal opacity and scar, keratitis, conjunctivitis, fungal corneal ulcers, uveitis, pollen allergy, glaucoma, cataract, stye, Marfan syndrome, optic neuritis, and choroidal melanoma.

7. In paragraph 1, The above endocrine diseases are hypernatremia, hyponatremia, hypermagnesemia, hypomagnesemia, hyperkalemia, hypokalemia, hyperalbuminemia, hypoalbuminemia, azotemia, acute tubulo-interstitial nephritis, acute renal failure, chronic renal failure, pyelonephritis, hypothyroidism, hyperthyroidism, thyroiditis, hyperfunction of pituitary gland, hypopituitarism, hypoparathyroidism, A pharmaceutical composition comprising at least one selected from the group consisting of hyperparathyroidism, portosystemic shunt, Cushing's syndrome, and diabetes.

8. In paragraph 1, A pharmaceutical composition, wherein the above other diseases are at least one selected from the group consisting of cancer, peritonitis, pancreatitis, otitis media, laryngitis, hepatitis, anemia, thrombocytopenia, osteoporosis, dislocation, Arnold-chiari malformation, dysplasia, spinal cord myelopathy, cervical HIVD, and herniation of intervertebral disk.

9. A food composition for preventing or improving diseases in animals other than humans, containing mesenchymal stem cells derived from the placenta of canines as an active ingredient.

10. An external preparation composition for preventing or improving diseases in animals other than humans, containing mesenchymal stem cells derived from the placenta of a canine animal as an active ingredient.

11. A method for producing a pharmaceutical composition for preventing or treating an animal disease other than human, comprising a step of isolating mesenchymal stem cells from the placenta of a canine animal.

12. In paragraph 11, The above canine animals are Maltese, Pomeranian, English Bulldog, American Bulldog, Continental Bulldog, Serrano bulldog, French Bulldog, Campeiro Bulldog, Chihuahua, Beagle, Yorkshire terrier, Dachshund, Pug, Chichu, Terrier, Spitz, Japan Spitz, German Spitz, Poodle, Shiba, Akita, Schipperke, English Cocker Spaniel, Lhasa Apso, Boxer, Dalmatian, Miniature Pinscher, Brussels Griffon, Jack Russell Terrier, Australian Silky Terrier, Pungsan, Sapsali, Utility dog, Boston Terrier, Tibetan Terrier, Miniature Schnauzer, Bichon Frise, Border Collie, Samoyed, Welsh Corgi, Jindo, Chow Chow, Siberian Husky, Bull Terrier, American Pitbull Terrier, Great Pyrenees,A manufacturing method, wherein the product is at least one selected from the following species: Labrador Retriever, Golden Retriever, Chesapeake Bay Retriever, Flat Coated Retriever, Curly Coated Retriever, German Shepherd, Tosa, English Mastiff, Bullmastiff, Tibetan Mastiff, Malinois, Belgian Sheepdog, Doberman Pinscher, Kangal, Greyhound, Bloodhound, and Great Dane.

13. A method for preventing or treating a disease in an animal other than a human, comprising the step of administering the pharmaceutical composition of paragraph 1 to an animal other than a human in need of treatment for a skin disease, neurological disease, eye disease, endocrine disease, or other disease.

14. Use of the pharmaceutical composition of paragraph 1 for the prevention or treatment of skin diseases, neurological diseases, eye diseases, endocrine diseases, or other diseases in animals other than humans.

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