WO2021020720A1 - Cellules souches dérivées de liquide amniotique équin, et agent thérapeutique cellulaire pour régénérer le système musculo-squelettique les comprenant - Google Patents

Cellules souches dérivées de liquide amniotique équin, et agent thérapeutique cellulaire pour régénérer le système musculo-squelettique les comprenant Download PDF

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WO2021020720A1
WO2021020720A1 PCT/KR2020/007476 KR2020007476W WO2021020720A1 WO 2021020720 A1 WO2021020720 A1 WO 2021020720A1 KR 2020007476 W KR2020007476 W KR 2020007476W WO 2021020720 A1 WO2021020720 A1 WO 2021020720A1
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amniotic fluid
stem cells
derived
cells
horse
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Korean (ko)
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김민규
김은영
이은지
김동언
남윤석
길태영
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주식회사 엠케이바이오텍
충남대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • 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
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the present invention relates to equine fetal tissue derived stem cells and a cell therapy for musculoskeletal regeneration containing the same, and more specifically, to equine amniotic fluid derived stem cells (Equine amniotic fluid stem cells), analysis of the characteristics of amniotic stem cells prepared by the above method, stem cells derived from horse amniotic fluid containing the stem cells as an active ingredient, and a cell therapeutic agent for musculoskeletal regeneration containing the same.
  • Equine amniotic fluid stem cells equine amniotic fluid stem cells
  • Stem cells are cells that are capable of self-replicating and differentiate into cells of various tissues, and have the ability to regenerate damaged body tissues and cells.Therefore, there is no clear treatment for degenerative diseases or severe trauma. It is recognized and known as a next-generation technology with very high medical use for injuries that are difficult to treat.
  • Types of stem cells include embryonic stem cells using embryos in the early stages of development, adult stem cells using cells that make up the tissue after development, and pluripotent induction stem cells using gene editing technology. There are (induced Pluripotent stem cells), and adult stem cells exist in each tissue of the body and provide an important role in maintaining the homeostasis of the body, and are classified into tissue stem cells derived from endoderm, mesoderm, and ectoderm depending on the origin of the tissue. .
  • mesenchymal stem cells a type of adult stem cells, have stable differentiation ability and are less likely to generate ethical issues and tumor cells. Therefore, clinical trials have been conducted the most in cell therapy research and are already applicable. Entered.
  • mesenchymal stem cells have the ability to regenerate damaged tissues and cells by moving directly to the area where the body has been damaged. They can easily proliferate outside the body, and are multipotent cells that can differentiate into various cell types. It is being used as a useful target in cell therapy.
  • mesenchymal stem cells mainly include bone marrow, adipose tissue, and umbilical cord blood, and many studies have been conducted. Although it has the multipotential ability to differentiate into cells, studies are being conducted to find a source with similar differentiation ability to embryonic stem cells among mesenchymal stem cell-derived tissues because it has a limited aspect compared to that of embryonic stem cells. .
  • amniotic fluid-derived stem cells are the most recently discovered and studied mesenchymal stem cell-derived tissues, and research results show that they have excellent proliferation ability and have many cell-specific markers characteristic of embryonic stem cells.
  • Amniotic fluid-derived stem cells can be obtained during pregnancy by fetal examination through amniotic puncture or during birth through cesarean section, so they can be collected by a non-invasive method that does not harm the mother and fetus at all, and self-proliferate like other mesenchymal stem cells. The differentiation ability to differentiate into various types of cells was confirmed.
  • the above results show the possibility of amniotic fluid-derived mesenchymal stem cells as cell therapeutics as the tissues that are produced with the fetus and can be collected quickly among adult stem cell-derived tissues.
  • stem cell therapeutics in the treatment of human diseases has led to the development of animal stem cell therapeutics, and clinical trials and treatments are progressing at an alarming rate in horses and animals, mainly due to the development of companion animals such as dogs and cats and the racehorse industry.
  • companion animals such as dogs and cats and the racehorse industry.
  • the development of stem cell therapy for the treatment of periostitis which is a major injury factor for racehorses and roadsters, is expected to play a very effective role in shortening the recovery period of more than 6 months and promoting health, through which the racehorse industry, which occupies a major portion of the horse industry. It is expected to be a very important study in the development of
  • Korean Patent Application Publication No. 10-2019-0037504 (a cell therapy for treating and preventing bone diseases in horses and animals, and a manufacturing method thereof using a three-dimensional environment) describes "Initially, the mesenchymal stem cells of horses and animals were converted into bone cells. Invention for producing cells having cartilage and bone regeneration ability by inducing differentiation, and in particular, by creating a three-dimensional cell culture and differentiation environment using a hydrogel, and producing cells having cartilage and bone regeneration ability in a large amount quickly Has been presented.
  • the present invention is based on amniotic fluid-derived mesenchymal stem cells isolated from stem cells derived from fetal tissues of horses and animals, and embryonic stem cell-specific genes such as Oct4, c-Myc, and Klf4, and PAX6, ALCAM, Integrin ⁇ 1, Endoglin, HCAM It simultaneously expresses a specific gene in mesenchymal stem cells such as E. et al., has an excellent proliferation rate that is cultured for more than 50 passages, exhibits the ability to differentiate into mesoderm-derived cells and features multipotent stem cells, and is a marker related to the equine leukocyte immune response (ELAII). antigen), which shows a negative immunological characteristic for the musculoskeletal system, has been confirmed to be effective in treating the musculoskeletal system.
  • ELAII equine leukocyte immune response
  • the present invention is easier to collect than conventional bone marrow, adipose, cord blood-derived stem cells for treatment, has superior proliferation and differentiation ability, and thus has utility as a new cell therapy agent for musculoskeletal damage and diseases, and the musculoskeletal system containing the same. It is intended to provide cell therapy for regeneration.
  • the present invention is to provide a horse amniotic fluid-derived stem cell having a rapid and excellent growth rate (regeneration ability) compared to the conventional mesenchymal stem cells and a regeneration ability through the musculoskeletal treatment of horses, and a cell therapy for musculoskeletal regeneration containing the same. .
  • the present invention in order to solve the above problems and needs,
  • mesenchymal stem cells derived from amniotic fluid in horses and animals.
  • the present invention provides a mesenchymal stem cell derived from amniotic horse and animal characterized in that the horse and animal are selected from the group consisting of horses, donkeys, zebras, or hybrids born from hybrids thereof.
  • the present invention is a process of collecting amniotic fluid of a pregnant horse (step 1),
  • step 2 The process of separating mononuclear cells from the collected amniotic water (step 2),
  • It provides a method of producing mesenchymal stem cells derived from amniotic fluid from horses and animals, including the process of initially culturing the isolated amniotic fluid-derived mononuclear cells (step 3).
  • the present invention is a process of collecting amniotic fluid of a pregnant horse (step 1),
  • step 2 The process of separating mononuclear cells from the collected amniotic water (step 2),
  • step 3 The process of initial culture of the isolated amniotic fluid-derived mononuclear cells (step 3),
  • DMEM Dulbecco Modified Eagle Medium
  • Horse and animal mesenchymal stems including the process (step 5) of separating cells from the basal surface using 0.01 to 0.1% Typsin-EDTA after removing the medium in the culture dish and washing with PBS (Phosphate buffered saline, PH 7.2) It provides a method of making a cell.
  • PBS Phosphate buffered saline, PH 7.2
  • the present invention is the process of initially culturing the isolated amniotic fluid-derived mononuclear cells (step 3),
  • Horse and animal medium characterized by culturing in Roswell Park Memorial Institute (RPMI) 1640 medium culture medium containing 10-20% FBS (Fetal Bovine Serum), 0.5-1.5% Glutamax and 0.5-1.5% penicillin/streptomycin. It provides a method for producing mesenchymal stem cells.
  • RPMI Roswell Park Memorial Institute
  • the present invention provides a method for producing horse and animal mesenchymal stem cells, characterized in that the cell adhesion is increased using a culture dish coated with 0.05 to 0.15% gelatin in all culture processes.
  • the present invention is a method for producing horse and animal mesenchymal stem cells, characterized in that when more than 80% of the cells are attached to the bottom of the culture medium in order to maintain the undifferentiated state of cells in all culture processes, they are passaged or frozen and stored. Provides.
  • the present invention provides a method for producing mesenchymal stem cells derived from amniotic fluid in horses and animals, which exhibit the following characteristics.
  • the present invention provides a mesenchymal stem cell derived from amniotic fluid for horses and animals, characterized in that it has a musculoskeletal treatment function.
  • the present invention provides a cell therapy for treating and recovering musculoskeletal fractures, micro fractures, periostitis, tendon and ligament damage, muscle atrophy muscle atrophy, muscle fiber shunt, including mesenchymal stem cells derived from horse and animal amniotic fluid.
  • the present invention shows an action and effect capable of producing mesenchymal stem cells from amniotic fluid of horses and animals.
  • stem cells derived from the amniotic fluid of horses and animals of the present invention have the function and effect of confirming the ability to self-proliferate when cultured for more than 50 passages while maintaining the characteristics of stem cells.
  • the function and effect of confirming that the stem cells derived from the amniotic fluid of horses and animals of the present invention become mesenchymal stem cells whose differentiation capabilities such as adipocyte differentiation, osteoblast differentiation, and chondrocyte differentiation are confirmed are exhibited.
  • stem cells derived from amniotic fluid of horses and animals of the present invention exhibit pluripotent functions and effects as mesenchymal stem cells, including the characteristics of embryonic stem cells.
  • mesenchymal stem cells derived from amniotic fluid of horses and animals of the present invention are effective in treating the musculoskeletal system.
  • mesenchymal stem cells derived from amniotic fluid of horses and animals of the present invention are effective in treating musculoskeletal fractures, micro fractures, and periostitis.
  • mesenchymal stem cells derived from amniotic fluid of horses and animals according to the present invention have (a) self-proliferative ability that is cultured for more than 50 passages while maintaining the characteristics of stem cells, and (b) fat, cartilage, bone cells, etc. Has the ability to differentiate into mesodermal-derived cells of (c) embryonic stem cell-specific genes Oct4, c-Myc, Klf4 and mesenchymal stem cell-related genes PAX6, ALCAM, Integrin ⁇ 1, Endoglin, and HCAM. Expression characteristics are shown, and (d) cell surface markers CD44, CD90, and CD105 have a positive immunological phenotype, while CD14, CD45, CD79 ⁇ , and ELA-DR have a negative immunological phenotype.
  • FIG. 1 shows the initial culture of horse amniotic fluid-derived cells.
  • A shows a pattern in which stem cells derived from amniotic fluid adhere to the surface of a culture dish and grow in the form of fibroblasts (X100).
  • B is the result of dyeing with Diff-quick (X100).
  • FIG. 3 shows an adipocyte differentiation experiment of horse amniotic fluid-derived stem cells.
  • A represents a negative oil red O staining reaction of horse amniotic stem cells before differentiation induction (X100)
  • (B) when staining with Oil Red O after culture in differentiation induction medium Shows positive staining (X100).
  • C is (X200).
  • Figure 4 shows the osteoblast differentiation experiment of the stem cells derived from horse amniotic fluid.
  • A shows a negative reaction of Alizarin red S staining of equine amniotic stem cells before differentiation induction (X100)
  • B when staining with Alizarin red S after culture in differentiation induction medium , Shows positive staining (X100).
  • C is (X200).
  • FIG. 5 shows a chondrocyte differentiation experiment of horse amniotic fluid-derived stem cells.
  • A shows a negative Alcian blue staining reaction of equine amniotic stem cells before differentiation induction (X100)
  • B is positive when stained with Alcian blue after culture in differentiation induction medium. It shows that it was stained with (X100).
  • C is (X200).
  • RT-PCR reverse transcription polymerase chain reaction
  • Figure 8 shows the results of a flow cytometry (Flow Analysis Cytometry System) to confirm the characteristics of stem cells in amniotic fluid-derived cells.
  • flow cytometry Flow Analysis Cytometry System
  • cells derived from equine amniotic fluid showed positive immunological characteristics in the positive expression markers CD44, CD90, and C105 of mesenchymal stem cells, and negative immunity in the negative expression markers CD14, CD45, CD79 ⁇ and the immune-related specific marker ELAII. Showed the academic aspect.
  • FIG. 9 is an X-ray result showing the recovery of damaged tissues after transplantation of the stem cells of the present invention to the damaged site of a horse with a musculoskeletal system injury using horse amniotic fluid derived mesenchymal stem cells.
  • A, D shows the left cartilage damage site
  • B, E the third humerus fracture and cartilage damage site
  • C, F shows the condition before and after stem cell treatment at the radial fracture site.
  • the present invention provides a method for producing stem cells derived from amniotic fluid of horses and animals, and stem cells derived from amniotic fluid of horses and animals according to the method.
  • the horse and animal of the present invention is meant to include an animal composed of a horse, a donkey, a zebra or a hybrid born from a hybrid thereof.
  • the present invention provides a cell therapy for regeneration of the musculoskeletal system containing stem cells derived from horse and animal amniotic fluid.
  • the present invention is a technical feature of separating and culturing a stem cell line from amniotic fluid collected during childbirth of a pregnant horse.
  • the present invention performs a process of collecting amniotic fluid from a pregnant horse. (Step 1)
  • the present invention performs a process of collecting amniotic fluid in a non-invasive method during the birth process of a pregnant horse.
  • the present invention collects amniotic fluid during childbirth of a pregnant horse, but such amniotic fluid is obtained with a 30-70 cc syringe equipped with 18-22 G before rupture of the amniotic sac during delivery of a pregnant mare. After transferring to a ml test tube, the preparation process is carried out by transporting it to the laboratory within an average of about 3-4 hours while maintaining about 3 ⁇ 5°C.
  • G means a lake
  • 18 means a diameter of 1.270 mm
  • 22 means a size of 0.711 mm.
  • the present invention collects amniotic fluid during childbirth of a pregnant horse, but such amniotic fluid is obtained with a 50 cc syringe equipped with 18 G before rupture of the amniotic sac during delivery of a pregnant mare and After the transfer, the preparation process is carried out by carrying it to the laboratory within an average of about 3 hours while maintaining at about 4°C.
  • the present invention performs the process of separating mononuclear cells from the collected amniotic fluid. (Step 2)
  • the amniotic water collected above using a cell strainer After filtering the amniotic water collected above using a cell strainer, it is mixed with PBS (Phosphate Buffered Saline) containing 3 to 7% penicillin/streptomycin in a ratio of 1:0.5 to 1.5, and then 2,000 to 4,000. Centrifuge at rpm for 8-12 minutes to recover the pellet.
  • PBS Phosphate Buffered Saline
  • the amniotic water collected above is filtered using a 90-110 ⁇ M, preferably 100 ⁇ M cell strainer, and then mixed 1:1 with PBS (Phosphate Buffered Saline) containing 5% penicillin/streptomycin at 3,000 rpm. , Centrifugation for 10 minutes performs a process of recovering the pellet.
  • PBS Phosphate Buffered Saline
  • the recovered pellet is washed and centrifuged 1 to 3 times using PBS containing 3 to 7% penicillin/streptomycin to separate mononuclear cells.
  • the recovered pellet is washed and centrifuged twice using PBS containing 5% penicillin/streptomycin to separate monocytes.
  • the present invention performs a process of initial culturing the isolated amniotic fluid-derived mononuclear cells. (Step 3)
  • the present invention is a Roswell Park Memorial Institute containing 10-20% FBS (Fetal Bovine Serum), 0.5-1.5% Glutamax, and 0.5-1.5% penicillin/streptomycin for initial culture of the isolated amniotic fluid-derived mononuclear cells ( RPMI) 1640 medium culture medium is used.
  • FBS Fetal Bovine Serum
  • Glutamax 0.5-1.5% Glutamax
  • penicillin/streptomycin for initial culture of the isolated amniotic fluid-derived mononuclear cells (RPMI) 1640 medium culture medium is used.
  • RPMI Roswell Park Memorial Institute 1640 medium culture medium containing 15% FBS (Fetal Bovine Serum), 1% Glutamax, and 1% penicillin/streptomycin.
  • the present invention was dispensed into a 6well culture dish coated with 0.05 ⁇ 0.15% gelatin in order to increase cell adhesion, and cultured in an incubator in a 4.5 ⁇ 5.5% CO 2 environment at 38.5°C at the same body temperature as horses and animals.
  • the present invention performs a process of removing dead tissues and cells in the initial culture process.
  • the culture medium was replaced within 48h of the initial culture, and even when cells were observed to adhere to the bottom of the culture dish, the process of replacing the culture medium was performed.
  • passage or freezing is performed to obtain and store stem cells derived from amniotic fluid.
  • the growth shape of the cells in the amniotic fluid-derived stem cells cultured by the above-described process was observed as a spindle shape similar to that of fibroblasts. This is similar to the shape seen in other mesenchymal stem cells, and it is confirmed that they are mesenchymal stem cells derived from amniotic fluid through Diff-quick staining reagent for detailed observation of the cell morphology.
  • amniotic fluid-derived mesenchymal stem cells are prepared by the above process, and the amniotic fluid-derived mesenchymal stem cells thus prepared have excellent self-proliferation ability, It has a high differentiation ability, including differentiation into adipocytes, chondrocytes, and bone cells, and possesses the pluripotency of stem cells.
  • the mesenchymal stem cells derived from amniotic fluid prepared as described above have a function of treating the musculoskeletal system.
  • the production of horse and animal mesenchymal stem cells for musculoskeletal treatment of the present invention can be prepared by the following method.
  • the present invention contains 10-20% FBS (Fetal Bovine Serum), 0.5-1.5% Glutamax, and 0.5-1.5% penicillin/streptomycin for the cells derived from horse amniotic fluid between passages 3-5, initially cultured (manufactured) above. Perform the process of culturing using the Dulbecco Modified Eagle Medium (DMEM) culture medium (step 4).
  • FBS Fetal Bovine Serum
  • Glutamax Glutamax
  • penicillin/streptomycin penicillin/streptomycin
  • the cells derived from horse amniotic fluid between passages 3 to 5 prepared above were prepared in Dulbecco Modified Eagle Medium (DMEM) culture medium containing 15% FBS (Fetal Bovine Serum), 1% Glutamax, and 1% penicillin/streptomycin. To incubate.
  • DMEM Dulbecco Modified Eagle Medium
  • the cell is separated from the basal surface using 0.01 to 0.1% Typsin-EDTA (step 5),
  • a process of obtaining horse and animal mesenchymal stem cells is performed.
  • the medium in the culture dish is removed, washed with PBS (Phosphate buffered saline, PH 7.2), and the cells are separated from the basal surface using 0.05% Typsin-EDTA, and then 5 x 10 6 ⁇ 1 x 10 7 depending on the injection site. Veterinary cells are obtained.
  • PBS Phosphate buffered saline, PH 7.2
  • the present invention performs the process of manufacturing horse and animal mesenchymal stem cells for musculoskeletal therapy for injection by diluting the cells prepared with PBS containing 0.1 to 1.0% FBS and injecting them into a syringe. (Step 6)
  • the present invention prepares horse and animal mesenchymal stem cells for musculoskeletal therapy at a temperature of 4° C. in a state in which sunlight is blocked in a 3 ml syringe after diluting the cells prepared with PBS containing 0.5% FBS.
  • the present invention provides a cell therapy for musculoskeletal regeneration containing mesenchymal stem cells derived from amniotic fluid prepared as described above.
  • the present invention prepares mesenchymal stem cells derived from amniotic fluid of horses and animals in the following examples, and the mesenchymal stem cells derived from amniotic fluid have excellent self-proliferation ability, and adipocytes, cartilage cells, and bone cells It has a high differentiation ability, such as Rho differentiation, and has the characteristics of retaining the pluripotency of stem cells, and it is confirmed by a test that the function of musculoskeletal therapy appears.
  • Amniotic fluid samples were obtained with a 50 cc syringe equipped with 18 G and transferred to a 50 ml test tube before rupture of the amniotic sac at the time of delivery of a pregnant mare, and then transferred to the laboratory within an average of about 3 hours while maintaining at about 4°C. .
  • the collected amniotic water was filtered using a 100 ⁇ M cell strainer, mixed 1:1 with PBS (Phosphate Buffered Saline) containing 5% penicillin/streptomycin, and centrifuged at 3,000 rpm for 10 minutes to collect a pellet. , The recovered pellet was washed twice with PBS containing 5% penicillin/streptomycin and centrifuged to separate monocytes.
  • PBS Phosphate Buffered Saline
  • RPMI Roswell Park Memorial Institute
  • FBS Fetal Bovine Serum
  • Glutamax 1% penicillin/streptomycin
  • the culture medium was replaced within 48h of the initial culture start, and the culture medium was replaced even when cells were observed to adhere to the bottom of the culture dish.
  • the culture medium bottom When more than 80% of the cells were attached, passaged or frozen and stored. The amniotic fluid-derived cells were observed in an attached form after an average of about 5 days.
  • the growth shape of the cells was observed as a spindle shape similar to that of fibroblasts. This is similar to the shape seen in other mesenchymal stem cells, and it was confirmed with Diff-quick staining reagent for detailed observation of the cell morphology.
  • DMEM Dulbecco Modified Eagle Medium
  • the growth and proliferation efficiency of the amniotic stem cells obtained by the above-described method was confirmed through a doubbling time analysis.
  • Doubling time analysis is expressed as an index representing the proliferation rate of cells through time analysis in which cells are doubled from the initial number, and is expressed as follows.
  • T-T0 Cell culture time (hr)
  • N0 number of initially dispensed cells
  • N number of final cultured cells
  • equine amniotic mesenchymal stem cells were dispensed in a six-well dish at a density of 200,000 cells/well, and then cultured in an incubator in a 5% CO 2 environment at 38.5°C in a way that no new medium was added. The culture time and the total number of active cells were observed.
  • the medium in the six-well culture dish was removed, washed with PBS (Phosphate buffered saline, PH 7.2), and the cells were separated from the basal surface using 0.05% Typsin-EDTA. Thereafter, the action of Trypsin was stopped with a new culture medium containing 5% FBS, and the cells isolated from the cell culture dish were used for cell counting.
  • PBS Phosphate buffered saline, PH 7.2
  • the Doubling time curve shows that amniotic stem cells are growing up to passage 50 or more. This suggests excellent self-renewal ability of the horse amniotic fluid-derived mesenchymal stem cells obtained in the present invention as compared with the 14th generation of the existing horse amniotic fluid-derived mesenchymal stem cells.
  • the differentiation into adipocytes, cartilage cells, and bone cells was induced in a medium suitable for each lineage using the StemPro Differentiation Kit of Gibco.
  • the medium was changed once every 2 days, and after about 1 week, it was confirmed that both amniotic fluid-derived mesenchymal stem cells gradually changed from fibroblast shape to round shape during the differentiation induction process.
  • the StemPro Osteogenesis Differentiation Kit of Gibco was used in a six-well dish at a density of 200,000 cells/well and cultured in an incubator in a 5% CO2 environment at 38.5°C.
  • the medium was changed once every 2 days, and after about 1 week, it was confirmed that both amniotic fluid-derived mesenchymal stem cells gradually changed from fibroblasts to cubic cells during the differentiation induction process.
  • the medium was added or exchanged once every 3 days. As a result of staining with Alcian blue after about 14 days, staining due to chondrocyte differentiation could be observed (FIG. 5).
  • mesenchymal stem cells have specific expression patterns that are distinguished by specific cell surface markers, and are positively expressed in horse and animal mesenchymal stem cells presented by the International Stem Cell Committee (ISCT)
  • ISCT International Stem Cell Committee
  • CD44, CD90, CD105 showing negative expression, CD14, CD45, CD79 ⁇ showing negative expression, and ELA-II of immune-related markers were performed through flow cytometry analysis and reverse transcription polymerase chain reaction (RT-PCR). .
  • RNA sample (1 ⁇ l of Total RNA) was synthesized using iScript reverse transcriptase (BIO-RAD) with oligo dT primers and random primers. Primers are shown in Table 1 below.
  • the synthesized cDNA was reacted a total of 34 times at 95°C for 4 minutes, 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds, and finally PCR was performed at 73°C for 5 minutes.
  • PCR was performed at an appropriate annealing temperature, and the PCR product was confirmed for gene expression through agarose gel electrophoresis capable of comparing the amplification size (FIG. 2).
  • the cDNA used for real-time quantification was synthesized in the same manner as the cDNA synthesis method used for RT-PCR. Realtime PCR was performed 39 times under the conditions of 95°C for 10 minutes, 94°C for 15 seconds, 58°C for 30 seconds, and 72°C for 30 seconds.
  • a gene from horse skin-derived fibroblasts was used, and the expression amount of a stem cell-specific marker was compared in horse amniotic fluid-derived mesenchymal stem cells by comparing the relative expression amount with the beta-actin gene.
  • Equine amniotic fluid-derived cells are flow cells that detect the markers of fluidly flowing cells based on a laser after attaching various combinations of antibodies conjugated to each cell surface-specific antibody and fluorescent IgG such as FITC (fluorescein isothiocyanate) or PE (phycoerythrin). Cellular marker expression and expression levels were analyzed through analysis.
  • fluorescent IgG such as FITC (fluorescein isothiocyanate) or PE (phycoerythrin).
  • the antibodies used were as follows: CD14, CD44-FITC, CD45, CD79 ⁇ -FITC, CD90-PE, CD105-FITC, ELA-DR-FITC and IgG-FITC. About 5-8x10 5 cells per sample were used to analyze one antibody and flow cytometry (FACScan, BD Biosciences, USA) was performed using Cellquest software.
  • the present invention was tested for the effect of treating the musculoskeletal system of the horse and animal mesenchymal stem cells prepared in the above (2) Preparation Example of Horse and Animal Mesenchymal Stem Cells for Musculoskeletal Treatment.
  • Stem cell therapy for musculoskeletal treatment was performed by preparing cells within 3 hours. After finding the exact damage that needs treatment using ultrasound, the cells diluted with PBS were sterilized using a syringe with an 18222 gauge needle, and after being injected into the wound, the progress was observed using ultrasound at intervals of 2 months. When it was confirmed that the recovery rate of the wound was remarkably fast (Fig. 9).
  • FIG. 9 is an X-ray result showing the recovery of damaged tissue after transplantation to a treatment site of a horse with a musculoskeletal injury disease using horse amniotic fluid-derived mesenchymal stem cells.
  • A, D shows the left cartilage damage site
  • B, E the third humerus fracture and cartilage damage site
  • C, F shows the condition before and after stem cell treatment at the radial fracture site.
  • the horse and animal amniotic fluid-derived mesenchymal stem cells prepared according to the present invention are very effective in musculoskeletal injury diseases.
  • the present invention is useful in the production, manufacturing, sales, distribution, and research industries related to the production of stem cells of horses and animals and cell therapy for regeneration of the musculoskeletal system using the same.
  • the present invention is useful in the production, manufacturing, sales, distribution, and research industries related to the production of mesenchymal stem cells extracted from amniotic fluid of horses and animals, and cell therapy for musculoskeletal regeneration using the same.

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Abstract

La présente invention concerne des cellules souches issues d'un tissu foetal équin, et un agent thérapeutique cellulaire pour la régénération d'un système musculo-squelettique les comprenant, et plus particulièrement, un procédé de préparation de cellules souches dérivées du liquide amniotique équin; l'analyse de caractéristiques de cellules souches de liquide amniotique préparées par le procédé; des cellules souches dérivées de liquide amniotique équin comprenant les cellules souches en tant que principe actif; et un agent thérapeutique cellulaire pour régénérer un système musculo-squelettique comprenant lesdites cellules. La présente invention concerne des cellules souches dérivées du liquide amniotique équin. De plus, la présente invention concerne un procédé de préparation de cellules souches mésenchymateuses dérivées d'un liquide amniotique équin, comprenant les étapes suivantes: (Étape 1) collecte du liquide amniotique provenant d'une jument enceinte; (étape 2) isolement des monocytes du liquide amniotique collecté; et (étape 3) culture dans un premier temps des monocytes dérivés de liquide amniotique isolés. De plus, la présente invention concerne un procédé de préparation de cellules souches mésenchymateuses équines, comprenant les étapes consistant à : (Étape 1) collecte du liquide amniotique provenant d'une jument enceinte; (étape 2) isolement des monocytes du liquide amniotique collecté; (étape 3) culture dans un premier temps des monocytes dérivés de liquide amniotique isolés; (étape 4) culture des cellules issues du liquide amniotique équin, initialement cultivées, de passages 3 à 5 en utilisant le milieu de culture Dulbecco's Modified Eagle (DMEM), contienant de 10 à 20 % de sérum bovin foetal (FBS), 0,5 à 1,5 % de glutamax, et 0,5 à 1,5 % de pénicilline/streptomycine; et (étape 5) élimination du milieu dans un récipient de culture, son lavage avec une solution saline tamponnée au phosphate (PBS, pH 7,2), puis la séparation des cellules de la surface de base à l'aide de 0,01 à 0,1 % de Typsin-EDTA. La présente invention concerne également des cellules souches dérivées du liquide amniotique équin ayant une fonction de traitement du système musculo-squelettique. En outre, la présente invention concerne un agent thérapeutique cellulaire pour la régénération d'un système musculo-squelettique, comprenant les cellules souches dérivées du liquide amniotique équin.
PCT/KR2020/007476 2019-07-26 2020-06-10 Cellules souches dérivées de liquide amniotique équin, et agent thérapeutique cellulaire pour régénérer le système musculo-squelettique les comprenant WO2021020720A1 (fr)

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KR10-2019-0091193 2019-07-26

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KR20120026014A (ko) * 2010-09-08 2012-03-16 주식회사 강스템홀딩스 말과동물 양수 유래 다분화능 줄기세포 및 그 제조방법
KR20130056782A (ko) * 2011-11-22 2013-05-30 주식회사 강스템홀딩스 말과동물 양막-유래 중간엽 줄기세포
KR20190037504A (ko) * 2017-09-29 2019-04-08 대한민국(농림축산식품부 농림축산검역본부장) 말과 동물의 골질환 치료 및 예방용 세포 치료제 및 3차원 환경을 이용한 이것의 제조방법

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