Pharmaceutical Composition

Abstract

The purpose of the present invention is to provide a pharmaceutical composition that contains mesenchymal stem cells and that has an excellent therapeutic effect against various diseases, injuries, wounds, and bedsores. This invention relates to a pharmaceutical composition for treating a non-porcine animal, the pharmaceutical composition containing juvenile porcine-derived mesenchymal stem cells that produce at least one humoral factor selected from TGF-β1, TGF-β2, VEGF-A and VEGF-C.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for treating non - porcine animals, and more particularly to a pharmaceutical composition for treating non - porcine animals containing mesenchymal stem cells derived from young pigs.

Background Art

[0002] With the recent progress of research on somatic stem cells including mesenchymal stem cells, the clinical application of somatic stem cells has already shifted from the basic research stage to the development stage. Somatic stem cells have three major functions (pluripotency, immunomodulatory ability, and remodeling ability of the extracellular environment), and are expected as therapeutic cells for intractable diseases.

[0003] Regarding the first pluripotency, it is the ability of somatic stem cells to directly differentiate into bone or cartilage, etc., and the administered somatic stem cells exert a therapeutic effect by complementing lost cells or replacing cells with insufficient functions.

[0004] The second immunomodulatory ability exerts a therapeutic effect by acting on the patient's immune cells through the secretion of anti - inflammatory cytokines, chemokines, or exosomes from somatic stem cells, or through cell - adhesion factors between cells, and suppressing immune reactions such as inflammation or graft - versus - host disease.

[0005] Regarding the third remodeling ability of the extracellular environment, it exerts a therapeutic effect on infarcted sites in ischemic diseases, fibrotic sites caused by inflammation, etc., through the secretion of angiogenesis - promoting factors, vascular - inductive factors, growth factors, or anti - fibrotic factors from somatic stem cells.

[0006] Mesenchymal stem cells exist in mammalian bone marrow, adipose tissue, islets, umbilical cord blood, etc., and are somatic stem cells derived from mesodermal tissues (mesenchyme), having the ability to differentiate into cells belonging to the mesenchymal lineage. In recent years, clinical trials have been conducted for diseases such as graft-versus-host disease, cardiovascular disorders, autoimmune diseases, osteoarthritis, osteogenesis imperfecta, liver disorders, respiratory diseases, spinal cord injuries, cerebral infarctions, or renal failure (Non-Patent Document 1), and various clinical applications are expected (Non-Patent Document 2). However, the effects in these clinical applications cannot be said to be sufficient.

[0007] In addition, in the clinical application of mesenchymal stem cells, there are issues such as ensuring the safety of donor procurement, the invasion to the donor, and virus negative testing for each donor. Since the efficacy of mesenchymal stem cells varies greatly depending on conditions such as the donor and their age, ensuring stable quality is also a major issue (Non-Patent Document 3).

[0008] Angiogenesis is complexly regulated under the balance of various angiogenesis promoting factors, inhibitory factors, metalloproteases, or other enzymes, and is deeply involved in wound healing or various diseases. Angiogenesis includes physiological phenomena observed in granulation formation during wound healing, and pathological phenomena in angiogenesis diseases such as inflammatory diseases or arteriosclerosis (Non-Patent Document 4).

[0009] Lymphatic vessels form a wide network in the living body together with blood vessels, absorb interstitial fluid, proteins, fats, immune cells, etc. that leak from blood vessels in peripheral tissues, and maintain the closed circulatory system of blood vessels by circulating back to the blood vascular system through collecting lymphatic vessels. Induction of lymphangiogenesis has been observed together with angiogenesis in the healing of wounds and various pathological inflammations (Non-Patent Document 5).

Prior Art Documents

Non-Patent Documents

[0010]

Non-Patent Document 1

[0011] In view of the above situation, an object of the present invention is to provide a pharmaceutical composition containing mesenchymal stem cells that exhibit excellent therapeutic effects against various diseases, damaged parts, wounds, and pressure ulcers. [Means for Solving the Problems]

[0012] The present inventors have found that mesenchymal stem cells prepared from young pigs highly express specific humoral factors, have a smaller cell size compared to conventional mesenchymal stem cells, and are excellent in proliferation ability, and have completed the present invention.

[0013] That is, the present invention relates to the following. 1. A pharmaceutical composition for treating non-pig animals, A pharmaceutical composition containing mesenchymal stem cells derived from neonatal pigs that produce at least one humoral factor selected from transforming growth factor-β (hereinafter TGF-β) 1, TGF-β2, vascular endothelial growth factor (hereinafter VEGF)-A, and VEGF-C. 2. The pharmaceutical composition according to 1 above, which treats the non-pig animal by promoting angiogenesis and / or lymphangiogenesis. 3. The pharmaceutical composition according to 1 or 2 above, which treats at least one selected from peripheral arterial disease, cerebral infarction, myocardial infarction, acute lung injury, wound, skin injury, and pressure ulcer. 4. The pharmaceutical composition according to any one of 1 to 3 above, wherein the mesenchymal stem cells derived from neonatal pigs are derived from pigs from fetal to less than 1 month old after birth. 5. The pharmaceutical composition according to any one of 1 to 4 above, wherein the mesenchymal stem cells derived from neonatal pigs are derived from pigs from fetal to less than 25 days old after birth. 6. The pharmaceutical composition according to any one of 1 to 5 above, wherein the non-pig animal is a human.

Advantages of the Invention

[0014] The pharmaceutical composition of the present invention contains mesenchymal stem cells derived from neonatal pigs, and due to the action of humoral factors produced by the mesenchymal stem cells derived from neonatal pigs, it has excellent therapeutic effects on various diseases, injury sites, wounds, and pressure ulcers.

Brief Description of the Drawings

[0015]

Figure 1

Figure 2

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Figure 5

Mode for Carrying Out the Invention

[0016] Mesenchymal stem cells are somatic stem cells derived from mesodermal tissues (mesenchyme), and refer to cells that have the ability to differentiate into cells belonging to the mesenchymal system such as osteocytes, cardiomyocytes, chondrocytes, tendon cells, and adipocytes, and can proliferate while maintaining this differentiation ability. The mesenchymal stem cells derived from young pigs in the present invention may be mesenchymal stem cells isolated from young pigs, and include, for example, mesenchymal stem cells derived from the bone marrow, pancreas, skin, or fat of young pigs.

[0017] In the present invention, "young pig" refers to a pig from fetus to less than 1 month old after birth, preferably less than 25 days old after birth. The young pig is preferably for medical use, and more preferably a young pig that can be used for cell transplantation into humans. The type of pig is not particularly limited, and examples include Landrace (e.g., Danish Landrace, American Landrace, British Landrace, Dutch Landrace, Swedish Landrace), Large Yorkshire, Berkshire, Duroc, Hampshire, Middle Yorkshire, and mini pigs, and among them, Landrace is preferred.

[0018] The mesenchymal stem cells derived from neonatal pigs in the present invention may be mesenchymal stem cells isolated from neonatal pigs, including their primary cultured cells and cells obtained by subculturing the primary cultured cells, which may also be mesenchymal stem cells capable of generating various cells expressing various differentiation markers.

[0019] The mesenchymal stem cells derived from neonatal pigs in the present invention produce at least one humoral factor selected from TGF-β1, TGF-β2, VEGF-A, and VEGF-C, and preferably produce at least TGF-β1, TGF-β2, and VEGF-C among them.

[0020] TGF-β is a cytokine family with biological activity in the latter half, and there are three highly structurally homologous isoforms, TGF-β1, 2, and 3, in mammals. TGF-β has the effects of promoting angiogenesis and promoting lymphangiogenesis.

[0021] VEGF is a cytokine family that acts specifically on vascular endothelial cells, and there are seven types, namely VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PlGF (placental growth factor)-1, and PlGF-2. VEGF has the effects of promoting angiogenesis and promoting lymphangiogenesis.

[0022] The pharmaceutical composition of the present invention contains mesenchymal stem cells derived from neonatal pigs that produce at least one humoral factor selected from TGF-β1, TGF-β2, VEGF-A, and VEGF-C. By the action of the humoral factor produced by these cells, it can promote at least one of angiogenesis and lymphangiogenesis, and can treat non-pig animals.

[0023] Examples of treatments that promote at least one of angiogenesis and lymphangiogenesis include at least one selected from disease treatment, injury site treatment, and wound healing, preferably at least one treatment selected from peripheral arterial disease, cerebral infarction, myocardial infarction, acute lung injury, trauma, skin injury, and pressure ulcers.

[0024] The non-pig animal is not particularly limited as long as it is an animal other than a pig, and is preferably a mammal other than a pig. Examples include humans, mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cows, sheep, goats, marmosets, monkeys, etc.

[0025] In the present invention, the production of the above-mentioned humoral factors by the mesenchymal stem cells derived from young pigs is preferably highly expressed. Here, "highly expressed" means that the expression level of the humoral factor is equal to or higher than that of conventional mesenchymal stem cells. Here, as the conventional mesenchymal stem cells, the mouse bone marrow-derived mesenchymal stem cells described later in the examples can be cited as an example.

[0026] In the present invention, the mesenchymal stem cells derived from young pigs have a significantly higher expression level of the above-mentioned humoral factors compared to mouse bone marrow-derived mesenchymal stem cells. Preferably, the protein expression intensity is 1.1 or more, more preferably 1.2 or more, and even more preferably 1.3 or more, compared to mouse bone marrow-derived mesenchymal stem cells. The protein expression intensity can be confirmed, for example, by FACS analysis using a specific antibody, ELISA, or the like.

[0027] As a preferred combination of humoral factors highly expressed by mesenchymal stem cells derived from neonatal pigs in the present invention, TGF-β1, TGF-β2 and VEGF-C can be mentioned. Specifically, for example, the expression levels of TGF-β1 and TGF-β2 after culturing the mesenchymal stem cells derived from neonatal pigs in the present invention in the MSC medium described below for 3 days are preferably 1.1 times or more, more preferably 1.5 times or more, and even more preferably 2 times or more, compared with the mesenchymal stem cells derived from mouse bone marrow cultured under the same conditions. Also, for example, the expression level of VEGF-C after culturing the mesenchymal stem cells derived from neonatal pigs in the present invention in the MSC basal medium described below for 3 days is preferably 1.1 times or more, more preferably 1.2 times or more, and even more preferably 1.3 times or more, compared with the mesenchymal stem cells derived from mouse bone marrow cultured under the same conditions.

[0028] In the present invention, the mesenchymal stem cells derived from neonatal pigs preferably have a positive rate of both CD44 and CD90, which are cell markers, of 60% or more, more preferably 70% or more, and even more preferably 80% or more. Also, the cell marker CD29 preferably has a positive rate of 60% or more, more preferably 70% or more, and even more preferably 80% or more. As described later in the examples, the positive rate of the cell marker can be confirmed by a method such as using flow cytometry.

[0029] In the present invention, the mesenchymal stem cells derived from neonatal pigs preferably have a doubling time in the logarithmic growth phase of 36 hours or less, more preferably 32 hours or less, even more preferably 28 hours or less, particularly preferably 24 hours or less, and most preferably 20 hours or less. Also, the doubling time in the logarithmic growth phase is preferably 14 hours or more, and more preferably 16 hours or more.

[0030] For culturing the mesenchymal stem cells derived from neonatal pigs in the present invention in the logarithmic growth phase, for example, the stem cells of the present invention are seeded in a medium containing vitamin C described below (for example, MSC medium), and cultured at 37°C in 5% CO 2It can be carried out by culturing in a culture incubator in the presence of [relevant substances]. The shorter the doubling time in the logarithmic growth phase, the more possible it is to prepare a large amount of mesenchymal stem cells derived from young pigs in a short time and at low cost.

[0031] The mesenchymal stem cells derived from young pigs in the present invention preferably have an average diameter of 17 μm or less, more preferably 16.5 μm or less, still more preferably 16 μm or less, particularly preferably 15.5 μm or less, and most preferably 15 μm or less. The average diameter is preferably 10 μm or more, and more preferably 12 μm or more. The smaller the average diameter, the more possible it is to prevent the formation of pulmonary embolism by administering the mesenchymal stem cells derived from young pigs. The average diameter can be measured, for example, using Nucleo Counter NC-200 (trademark). Here, "average" means arithmetic mean.

[0032] The differentiation of the mesenchymal stem cells derived from young pigs in the present invention into adipocytes can be induced by culturing the mesenchymal stem cells derived from young pigs in the present invention, for example, in the presence of insulin, MCGS (serum component, Mesenchymal Cell Growth Supplement), dexamethasone, indomethacin, isobutylmethylxanthine, etc.

[0033] For the differentiation into and maintenance of adipocytes, commercially available kits or media etc. may be used. For example, hMSC differentiation BulletKit (trademark)-adipogeni (PT-3004) manufactured by Lonza Walkersville, hMSC adipogenic induction medium (PT-3102B) manufactured by Lonza Walkersville, hMSC adipogenic maintenance medium (PT-3102B) manufactured by Lonza Walkersville, etc. can be mentioned. The differentiation of the mesenchymal stem cells derived from young pigs into adipocytes can be confirmed using a commercially available kit. For example, Adipo Red (trademark) assay reagent manufactured by Lonza can be mentioned.

[0034] The differentiation of the mesenchymal stem cells derived from neonatal pigs in the present invention into osteocytes can be induced by culturing the mesenchymal stem cells derived from neonatal pigs of the present invention, for example, in the presence of dexamethasone, ascorbate, MCGS, β-glycerophosphate, etc. A commercially available kit may also be used, for example, hMSC differentiation BulletKit (trademark)-osteogenic, PT-3004, etc. manufactured by Lonza Walkersville. The differentiation of the mesenchymal stem cells derived from neonatal pigs into osteocytes can be confirmed by a commercially available alkaline phosphatase staining kit (for example, manufactured by Cosmo Bio Co., Ltd.), a commercially available calcification staining kit (for example, manufactured by Cosmo Bio Co., Ltd.), etc.

[0035] The differentiation of the mesenchymal stem cells derived from neonatal pigs in the present invention into chondrocytes can be induced by culturing the mesenchymal stem cells derived from neonatal pigs of the present invention, for example, in the presence of TGF-β3, dexamethasone, insulin-transferrin-selenious acid (ITS), sodium pyruvate, proline, ascorbate. A commercially available kit may also be used, for example, hMSC differentiation BulletKit (trademark)-chondrogenic, PT-3003, etc. manufactured by Lonza Walkersville. The differentiation of the mesenchymal stem cells derived from neonatal pigs into chondrocytes can be confirmed by Alcian blue staining, etc.

[0036] The method for producing the pharmaceutical composition according to the present invention includes a step of preparing mesenchymal stem cells derived from neonatal pigs. As one aspect of the method for preparing mesenchymal stem cells derived from neonatal pigs, for example, a method including the following steps can be mentioned. (1) Step of collecting cells from neonatal pigs (2) Step of culturing the cells collected in step (1) to prepare mesenchymal stem cells derived from neonatal pigs Hereinafter, each step will be described.

[0037] (1) Step of collecting cells from neonatal pigs In step (1), cells are collected from the bone marrow, fat, skin, pancreas, etc. of young pigs. Specifically, for example, when collecting cells from the bone marrow of young pigs, bone marrow cells can be collected from the femur, iliac crest, sternum, etc. of young pigs. For example, the femur of a young pig is recovered, both ends are cut, a needle is inserted, and it is rinsed with a physiological buffer solution (e.g., phosphate buffer solution, hereinafter also referred to as PBS) added with heparin, and the effluent is recovered as bone marrow fluid from the opposite side. When the amount of the effluent decreases, the bone is turned over, the needle is inserted into the opposite side, and it is rinsed again with PBS to prepare bone marrow fluid, which is a cell-containing solution.

[0038] Furthermore, a mononuclear cell fraction derived from young pigs may be isolated by generally centrifuging the cell-containing solution prepared above. The cell-containing solution prepared above is diluted with PBS or the like, and the diluted cell-containing solution is placed on the medium layer in a tube containing a medium for human lymphocyte separation (e.g., Ficoll-Paque PLUS manufactured by GE Healthcare Life Sciences).

[0039] The tube is centrifuged to form layers, and the layer containing mononuclear cells derived from young pigs is recovered. The recovered solution is further centrifuged, the supernatant is removed, diluted with PBS or the like, and centrifuged again to isolate the mononuclear cell fraction. The cells of the mononuclear cell fraction isolated in this way may be cryopreserved before culturing. By freezing the isolated mononuclear cell fraction derived from young pigs, cells that are less affected by freeze-thaw can be selectively prepared. When cryopreserving before culturing, the temperature is preferably -80°C or lower, more preferably -150°C or lower.

[0040] Also, for example, when collecting cells from the pancreas of young pigs, islets are recovered from young pigs, and in some cases, the islets are suspended and cultured to prepare cell masses for adherent culture for the purpose of preparing stem cells.

[0041] Also, for example, when collecting cells from the fat of a young pig, the fat is collected from the young pig, minced finely with scissors, and then subjected to enzyme treatment. It is filtered through a cell strainer and centrifuged at low speed. The cells sedimented at the bottom of the tube are used for culture. Also, for example, when collecting cells from the skin (including hair) of a young pig, the skin is collected from the young pig and subjected to enzyme treatment. After the enzyme treatment, the hair is removed from the skin, and the Bulge part is collected and used for culture. When culturing, 3T3 feeder cells are used.

[0042] (2) A step of culturing the cells collected in step (1) to prepare mesenchymal stem cells derived from a young pig The cells, cell fractions, or cell aggregates collected in the above step (1) contain many unintended cells other than stem cells. Usually, a culture method of removing these cells is used by using a basal medium without vitamin C (for example, the MSC basal medium described later), which is essential for the survival of these unintended cells.

[0043] In step (2) of the present invention, the cells, cell fractions, or cell aggregates collected in the above step (1) are preferably at 35 - 39 °C, more preferably at 36 - 38 °C, most preferably at 37 °C, preferably in 4 - 6%, more preferably in 4.5 - 5.5%, most preferably in 5% CO 2 By culturing in a culture incubator in the presence, unintended cells other than mesenchymal stem cells are removed, and the mesenchymal stem cells derived from a young pig in the present invention are proliferated.

[0044] Since the mesenchymal stem cells derived from a young pig in the present invention have a significantly high growth rate, for the culture of removing the above-mentioned unintended cells, even if only a medium containing vitamin C (for example, the MSC medium described later) is used without using a basal medium without vitamin C, the mesenchymal stem cells derived from a young pig in the present invention can be prepared. In addition, in order to remove the above-mentioned unintended cells, after culturing using a basal medium without vitamin C, the medium is replaced with a medium containing vitamin C to proliferate the mesenchymal stem cells derived from a young pig in the present invention, whereby the mesenchymal stem cells derived from a young pig in the present invention can also be prepared.

[0045] The mesenchymal stem cells derived from young pigs in the present invention are cultured, for example, by the following method. Using a culture vessel coated with gelatin (for example, a plate coated with 0.1% gelatin) or a culture vessel without gelatin coating (for example, a plate), a basal medium without vitamin C (for example, the MSC basal medium described later) or a medium containing vitamin C (for example, the MSC medium described later) is used, preferably 5.0×10 5 cells to 5.0×10 7 cells / 9.6 cm 2 are seeded and incubated, for example, at 37°C under 5% CO 2 , 90% humidity conditions to obtain primary cultured cells.

[0046] The culture period for obtaining primary cultured cells is preferably 3 to 12 days, more preferably 3 to 11 days, and most preferably 3 to 10 days after seeding. The primary cultured cells may be passaged. The stem cells obtained by passaging are also referred to as passaged cultured cells.

[0047] The passage of primary cultured cells or passaged cultured cells is preferably 2 to 6 days, more preferably 2 to 5 days, even more preferably 2 to 4 days, and most preferably 3 days after seeding the stem cells, after the stem cells reach 30% to 100% confluent, preferably 50% to 95% confluent, more preferably 60% to 90% confluent, and most preferably 70% to 85% confluent.

[0048] The seeding of stem cells is carried out using a culture vessel coated with gelatin (for example, a plate coated with 0.1% gelatin) or a culture vessel without gelatin coating (for example, a plate) and a medium containing vitamin C (for example, the MSC medium described later), preferably 5.0×10 5 cells to 5.0×10 7 cells / 9.6 cm 2 are seeded. The culture of stem cells is, for example, at 37°C under 5% CO 2、Cultivate under the condition of 90% humidity. During the cultivation of stem cells, change the medium as needed to proliferate the mesenchymal stem cells derived from young pigs in the present invention.

[0049] As the MSC basal medium and MSC medium, conventionally known ones can be used, or commercially available ones can also be used. Examples of the MSC basal medium include a medium prepared by adding 55 mL of Gibco Fetal bovine serum (FBS) and 5.5 mL of Sigma-Aldorich Penicillin-Streptomycin to 500 mL of Gibco MEMα (Nucleosides, no Ascorbic acid). Examples of the MSC medium include a medium prepared by adding 55 mL of Gibco Fetal bovine serum (FBS), 5.5 mL of Sigma-Aldorich Penicillin-Streptomycin, and 22.2 μL of Sigma-Aldorich FGF-Basic, recombinant, expressed in E.coli, suitable for cell culture (final concentration: 1 ng / mL) to 500 mL of Gibco MEMα (Nucleosides).

[0050] Subculture is preferably carried out at least once or more. The number of subcultures is not particularly limited as long as the mesenchymal stem cells derived from young pigs in the present invention can be obtained, but it is preferably 1 to 3 times, more preferably 1 to 20 times.

[0051] The mesenchymal stem cells derived from young pigs in the present invention can be cryopreserved. The timing of cryopreservation is not particularly limited, but it is preferably after 1 to 20 subcultures, more preferably after 2 to 10 subcultures. Conventional methods can be used for cryopreservation and thawing.

[0052] As a method for cryopreserving mesenchymal stem cells derived from young pigs in the present invention, specifically, for example, they can be dispersed in a cryopreservation solution and cryopreserved at -80°C or lower in a freezer or in liquid nitrogen until needed. Examples of the cryopreservation solution include a solution obtained by mixing OPF-301 [Ringer's lactate solution containing 3% trehalose and 5% dextran (International Publication No. WO2014 / 208053)] and dimethyl sulfoxide (DMSO) at a ratio of 9:1, a serum-containing or serum-free preservation solution that can be used for cryopreserving animal cells, or a commercially available reagent for cryopreserving cells [preferably, CELLBANKER (registered trademark) manufactured by Takara Bio Inc. or the like].

[0053] The pharmaceutical composition of the present invention may contain other components other than mesenchymal stem cells derived from young pigs, provided that the expected therapeutic effect is maintained. Examples of the components that can be used in the pharmaceutical composition of the present invention include organic biodegradable materials such as hyaluronic acid, collagen, or fibrinogen, gelling materials such as hyaluronic acid, collagen (for example, soluble collagen such as acid-soluble collagen, alkali-soluble collagen, enzyme-soluble collagen, etc.), or fibrin glue, and aqueous solvents such as buffer solutions such as sterilized water, physiological saline, or phosphate solution. In addition to these components, it may also contain antibiotics, stabilizers, preservatives, pH adjusters, humoral factors, etc.

[0054] The administration method when using the pharmaceutical composition of the present invention as a pharmaceutical product is not particularly limited, but intramuscular administration, subcutaneous administration, intravascular administration (preferably intravenous administration), intraperitoneal administration, enteral administration, etc. are preferred. Among them, intramuscular administration, subcutaneous administration, and intravascular administration are more preferred.

[0055] The dosage (administered amount) of the pharmaceutical composition of the present invention may vary depending on the patient's condition (for example, body weight, age, symptoms, physical condition, etc.) and the dosage form of the pharmaceutical composition of the present invention. From the viewpoint of achieving a sufficient preventive or therapeutic effect, a larger amount is preferably used. On the other hand, from the viewpoint of suppressing side effects, a smaller amount tends to be preferred.

[0056] Generally, when administered to adults, the number of mesenchymal stem cells derived from neonatal pigs is 5×10 2 ~1×10 12 cells per administration, preferably 1×10 4 ~1×10 11 cells per administration, more preferably 1×10 5 ~1×10 10 cells per administration. Note that this dosage can be administered multiple times as a single dose, or it can be divided and administered multiple times.

[0057] Also, generally, when administered to adults, the number of mesenchymal stem cells derived from neonatal pigs per kilogram of body weight is 1×10 - 5×1010 cells / kg, preferably 1×10 2 ~5×10 9 cells / kg, more preferably 1×10 3 ~5×10 8 cells / kg. Note that this dosage can be administered multiple times as a single dose, or it can be divided and administered multiple times.

Example

[0058] Reference Example 1 〔Recovery of bone marrow cells derived from neonatal pigs〕 Bone marrow was collected from the femurs of neonatal pigs. The femurs were recovered from neonatal pigs (23-day-old medical Landrace pigs), both ends were cut, a 12G needle was inserted, and it was rinsed with 50 mL of heparinized PBS [3 mL of heparin (1000 U / mL), 47 mL of PBS], and 50 mL of bone marrow effluent (hereinafter also abbreviated as bone marrow fluid) was collected from the opposite side. When the amount of the effluent decreased, the bone was reversed, the needle was inserted on the opposite side, and it was rinsed again with PBS to collect the bone marrow fluid. 50 μL of the sample was taken in a 15 mL conical tube for counting with 1950 μL of PBS (40-fold dilution), and the cell count was measured with a cell counter.

[0059] 〔Isolation of the mononuclear cell (npMNC) fraction derived from neonatal pigs〕 The bone marrow fluid obtained by the above procedure was gently resuspended. The entire bone marrow fluid was divided into four 50 mL tubes, 10 mL each, diluted to 30 mL with PBS in each tube, and after confirming that the cells did not adhere to the tubes, they were mixed well. 10 mL of Ficoll-Paque PLUS (manufactured by GE Healthcare Life Sciences) was added to four new 50 mL tubes, and 30 mL of the bone marrow fluid mixed with PBS was placed on top of the Ficoll-Paque PLUS layer.

[0060] The tubes were centrifuged at 400×g for 30 minutes at 20 °C, accelerated slowly without brakes (1 / 3 of full speed), and three different layers were formed. Since the mononuclear cell fraction was located in the floating white ring, the entire white ring was collected into 50 mL tubes (×4) containing 25 mL of PBS. Centrifuged at 400×g for 7 minutes at room temperature, and the supernatant was removed. PBS was added up to 40 mL, and centrifuged again at 400×g for 7 minutes at room temperature. When the cell count was measured in the same manner as above, 25 - 30% of the cells among the total bone marrow cells were isolated as the mononuclear cell fraction, each (20 - 30)×10 6 cells were isolated.

[0061] 〔Cryopreservation of cells in the npMNC fraction〕 The cells of the isolated mononuclear cell fraction were placed in cryovials containing FBS (90% FBS and 10% DMSO) mixed with 10 7 cells / mL of DMSO, and the total volume of the cell suspension was made 1 ml [cell count / 10×10 6 = volume (mL) of FBS mixed with DMSO]. The cryovials were stored at -20 °C for 1 hour, then at -80 °C for 24 hours, and finally transferred to a liquid nitrogen tank for long-term storage.

[0062] 〔Culture of cells in the npMNC fraction and preparation of npBM-MSC〕 Quickly thaw the cell suspension containing the cells of the npMNC fraction cryopreserved in a cryovial in a 37°C water bath. Using a micropipette, gently add the thawed cell suspension to 30 mL of MSC basal medium adjusted to a temperature equilibrium (37°C) [medium prepared by adding 55 mL of Gibco Fetal bovine serum (FBS) and 5.5 mL of Sigma-Aldorich Penicillin-Streptomycin to 500 mL of Gibco MEMα (Nucleosides, no Ascorbic acid); the same applies hereinafter]. Centrifuge at 500×g for 5 minutes at room temperature, resuspend the pellet in 4 mL of MSC basal medium equilibrated to the temperature, and gently pipette up and down. As a result of measuring the total cell count and viable cell count, the total cell count was 4.18×10 6 cells, the viable cell count was 6.6×10 5 cells, and the survival rate was 15.8%.

[0063] Coat a 6-well plate with 0.1% gelatin, let it stand in an incubator (37°C, 5% CO 2 ) for 10 - 15 minutes, and remove the gelatin before use. Add the cell suspension to each prepared 0.1% gelatin-coated 6-well plate, gently rock to disperse the cell suspension on the growth surface (gelatin coat), and seed 2.09×10 6 cells / well in 2 mL of MSC basal medium. In a CO 2 incubator, at 37°C with 5% CO 2, Cultured under the condition of 90% humidity, and after 3 days, the cells were exchanged to MSC medium [a medium prepared by adding 55 mL of Gibco Fetal bovine serum (FBS), 5.5 mL of Sigma-Aldorich Penicillin-Streptomycin, and 22.2 μL of Sigma-Aldorich FGF-Basic, recombinant, expressed in E. coli, suitable for cell culture (final concentration: 1 ng / mL) to 500 mL of Gibco MEMα (nucleosides), the same hereinafter] to proliferate the cells, and thereafter, the MSC medium was exchanged once every 3 days. After 10 days, npBM-MSC became confluent. Note that when using a plate without gelatin coating, npBM-MSC also became confluent after 10 days in the same manner.

[0064] [Subculture] After npBM-MSC reached almost 100% confluence, cells were collected from 2 wells and re-seeded into T75 flasks with or without 0.1% gelatin coating.

[0065] The cells were washed with 2 mL of PBS (calcium and magnesium free), 320 μL of 0.25% trypsin was added per well and left in the incubator for several minutes. When the cells detached, they were neutralized with 1680 μL of MSC medium. The cell suspension was collected into a 50 mL tube using a 1 mL pipette, 16 mL (8 mL × 2 wells) of MSC medium was added, and then centrifuged at 500 × g for 5 minutes at room temperature. Using a pipette, the obtained pellet was gently resuspended in temperature-equilibrated MSC medium (2 mL). As a result of measuring the total cell number and viable cell number, the total cell number was 2.05×10 6 cells, the viable cell number was 2.02×10 6 cells, and the survival rate was 98.5%.

[0066] MSC medium was added to T75 flasks with and without 0.1% gelatin coating, and re-seeded to 4.5×10 5 viable cells / flask T75 flasks, and CO2 In an incubator, cultured at 37 °C under conditions of 5% CO 2 , 90% humidity. These cells were used as the first passage. Three days after seeding the first passage, regardless of the presence or absence of a 0.1% gelatin coat, 100% confluence was reached.

[0067] 〔Preparation of npBM-MSC〕 After npBM-MSC reached almost 100% confluence, cells were harvested from two T75 flasks with or without a 0.1% gelatin coat. The cells were washed with 8 mL of PBS(-), 2.4 mL of 0.25% trypsin per well was added, left in the incubator for several minutes, and neutralized with 12.6 mL of MSC medium when the cells detached. The cell suspension was collected in a 50 mL tube and centrifuged at 500 × g for 5 minutes at room temperature.

[0068] Temperature-equilibrated MSC medium (10 mL) was added to the obtained pellet, gently resuspended up and down with a pipette, and the results of measuring the total cell number and viable cell number are shown below. Cells from 0.1% gelatin-coated flasks (×2): total cell number 1.62×10 7 cells, viable cell number 1.60×10 7 cells, survival rate: 98.8% Cells from flasks without gelatin coat (×2): total cell number 1.48×10 7 cells, viable cell number 1.46×10 7 cells, survival rate: 98.6%

[0069] 〔Cryopreservation of npBM-MSC〕 Separate from the above culture, early passage npBM-MSC was frozen to prepare a cell stock. The npBM-MSC pellet treated with trypsin was resuspended in a solution prepared by mixing CELLBANKER (registered trademark) 1 or OPF-301 [Ringer's lactate solution containing 3% trehalose and 5% dextran (International Publication No. WO2014 / 208053)] and DMSO at a ratio of 9:1 at the desired concentration, and 1.5×10 6Cells were prepared at a concentration of 1 mL / vial. The vials were placed in a bicell and stored at -80°C for 24 hours. After that, the cells were transferred from -80°C to liquid nitrogen for long-term storage.

[0070] [[CFU Assay]] 630 npBM-MSC (P2) cells were seeded at a density of 30 cells / cm 2 in a 21 cm culture dish (uncoated with gelatin or coated with 0.1% gelatin) and cultured in MSC medium. The MSC medium was changed every 3 days. After 6 days of culture, the adherent cells were washed twice with 4 mL of PBS and fixed with 4 mL of ice-cold methanol at 4°C for 15 minutes. To visualize the colonies, the cells were stained with 4 mL of Giemsa diluted 1:19 with phosphate buffer for 30 minutes, washed at room temperature (RT), and washed twice with H 2 2O. 2

[0071] Next, the number of colonies with more than 50 cells was counted, and the colony formation efficiency of the cells was calculated. The colony formation efficiency of the cells was calculated by dividing the number of colonies per dish by the number of cells seeded per dish (630). The results are shown in Table 1. The values in Table 1 represent the mean ± SD (n = 3).

[0072] [[Table 1]]

[0073] As shown in Table 1, as a result of the CFU assay, it was found that the obtained npBM-MSC could form colonies regardless of the presence or absence of gelatin coating.

[0074] [[Average Cell Diameter]] Table 2 shows the results of measuring the average cell diameter of hBM-MSC (P4) and the obtained npBM-MSC. The average cell diameter was measured using a Nucleo Counter NC-200 (trademark), and the mean value (n = 3) was calculated.

[0075] [[Table 2]]

[0076] As shown in Table 2, it was found that the obtained neonatal porcine bone marrow-derived mesenchymal stem cells had a smaller average diameter compared to human bone marrow-derived mesenchymal stem cells.

[0077] 〔Evaluation of Proliferation Rate〕 For hBM-MSC and npBM-MSC, the cells were seeded at a density of 5000 cells / cm 2 (1.25×10 5 cells / flask) in a T25 flask and cultured using MSC medium. The MSC medium was changed every 3 days. The total number of viable and dead cells was counted 1, 2, 4, and 8 days after the start of culture. The results are shown in Table 3 and Table 4, and Figures 1(a) and 1(b). The values in Table 3 and Table 4 are mean ± SD (n = 4).

[0078]

Table 3

[0079]

Table 4

[0080] As shown in Table 3 and Table 4, and Figures 1(a) and 1(b), it was found that the obtained neonatal porcine bone marrow-derived mesenchymal stem cells had a significantly faster cell proliferation rate compared to human bone marrow-derived mesenchymal stem cells.

[0081] 〔Differentiation into Adipocytes〕 For hBM-MSC and npBM-MSC, differentiation into adipocytes was induced according to the protocol using hMSC differentiation BulletKit (trademark)-adipogeni, PT-3004 (manufactured by Lonza Walkersville). On the 17th day after the start of induction, staining was performed using Oil Red manufactured by Sigma-Aldorich. As a result, it was found that the obtained neonatal porcine bone marrow-derived mesenchymal stem cells could differentiate into adipocytes, similar to human bone marrow-derived mesenchymal stem cells.

[0082] [Differentiation into osteocytes] For hBM-MSC and npBM-MSC, differentiation into osteocytes was induced according to the protocol using hMSC differentiation BulletKit (trademark)-osteogenic, PT-3002 (manufactured by Lonza Walkersville). On the 14th day after the start of induction, staining was performed using an alkaline phosphatase staining kit manufactured by Cosmo Bio to confirm differentiation into osteocytes. As a result, it was found that the obtained neonatal porcine bone marrow-derived mesenchymal stem cells could differentiate into osteocytes, similar to human bone marrow-derived mesenchymal stem cells.

[0083] [Differentiation into chondrocytes] For npBM-MSC, differentiation into osteocytes was induced according to the protocol using hMSC differentiation BulletKit (trademark)-chondrogenic, PT-3003 (manufactured by Lonza Walkersville). On the 19th day after the start of induction, HE staining was performed. As a result, it was found that the obtained npBM-MSC could differentiate into chondrocytes.

[0084] Reference Example 2 [Culture of cells in the npMNC fraction and preparation of npBM-MSC] The MSC basal medium or MSC medium was incubated in an incubator (37°C, 5% CO 2) It was left standing for 10 to 15 minutes. Similar to Test Example 1, a cell suspension containing the cells of the npMNC fraction cryopreserved in a cryovial in a 37°C water bath was quickly thawed. Using a micropipette, the thawed cell suspension was gently added to 30 mL of temperature-equilibrated (37°C) MSC basal medium and dispensed into two 50 mL tubes, 15 mL each.

[0085] Centrifuged at 500×g for 5 minutes at room temperature, the pellet was resuspended in 2 mL of temperature-equilibrated MSC basal medium or MSC medium and gently pipetted up and down. The results of measuring the total cell number and viable cell number are shown below. 2 mL of MSC basal medium: Total cell number 2.60×10 6 cells, viable cell number 4.8×10 5 cells, viability 18.5% 2 mL of MSC medium: Total cell number 2.55×10 cells, viable cell number 4.5×10 5 cells, viability 17.6%

[0086] An amount of cell suspension calculated so that the number of seeded cells was as follows was added to a 6-well plate (without gelatin coating) containing the following medium for each well, and gently shaken to disperse the cell suspension on the growth surface. 2 mL of MSC basal medium: 2.60×10 6 cells / 1 well were seeded 2 mL of MSC medium: 2.55×10 6 cells / 1 well were seeded

[0087] CO 2 It was placed in an incubator and incubated at 37°C under conditions of 5% CO 2 , 90% humidity. The medium was changed with MSC medium 3 days and 6 days after seeding to grow the cells, and subcultured on the 8th day after seeding.

[0088] 〔Subculture〕 After the npBM-MSC reached approximately 50 - 60% confluence, the cells were recovered from one well and re-seeded into a T75 flask without gelatin coating.

[0089] The cells were washed with 2 mL of PBS(-), 320 μL of 0.25% trypsin was added per well, and the mixture was left in an incubator for several minutes. Once the cells detached, they were neutralized with 1680 μL of MSC medium. The cell suspension was collected in a 50 mL tube, 8 mL of MSC medium was added, and the mixture was centrifuged at 500×g for 5 minutes at room temperature.

[0090] The obtained pellet was added with temperature-equilibrated MSC medium (2 mL), gently resuspended up and down with a pipette, and the results of measuring the total cell number and viable cell number are shown below. Group of MSC basal medium at P0 seeding: Total cell number 5.0×10 5 cells, viable cell number 5.0×10 5 cells, survival rate: 100% Group of MSC medium at P0 seeding: Total cell number 3.3×10 5 cells, viable cell number 3.3×10 5 cells, survival rate: 100%

[0091] 15 mL of MSC medium was added to a T75 flask (without gelatin coating), and npBM-MSC was reseeded to reach the following cell numbers and cultured in an incubator. These cells were designated as the first passage. Group of MSC basal medium at P0 seeding: Viable cell number 5.0×10 5 cells / flask Group of MSC medium at P0 seeding: Viable cell number 3.3×10 5 cells / flask

[0092] [Preparation of npBM-MSC] After the cells reseeded by the above procedure reached approximately 80 - 90% confluence, cells were collected from one T75 flask (without gelatin coating). The cells were washed with 8 mL of PBS(-), 2.4 mL of 0.25 mL / well trypsin was added, and the mixture was left in an incubator for several minutes. Once the cells detached, they were neutralized with 12.6 mL of MSC medium. The cell suspension was collected in a 50 mL tube and centrifuged at 500×g for 5 minutes at room temperature.

[0093] To the obtained pellets, MSC medium (5 mL) equilibrated to room temperature was added, and the mixture was gently resuspended up and down with a pipette. The results of measuring the total cell number and viable cell number are shown below. Cells from one flask (MSC basal medium for 3 days after seeding of P0): total cell number 5.12×10 6 cells, viable cell number 5.09×10 6 cells, viability: 99.5% Cells from one flask (from seeding of P0 to MSC medium): total cell number 4.76×10 6 cells, viable cell number 4.73×10 6 cells, viability: 99.4%

[0094] 〔Cryopreservation of npBM-MSC〕 Separate from the above culture, early passage cells were frozen in the same manner as in Test Example 1 to prepare a cell stock.

[0095] Reference Example 3 The cell surface antigens of the npMNC prepared in Reference Example 1 and Reference Example 2 were analyzed. The preparation methods of each sample used for the analysis are shown in Table 5. In Table 5, "Switch" indicates that MSC basal medium (vitamin C-free) was used at the initial culture and the culture was changed to MSC medium (vitamin C-containing), which is the growth medium, during the growth culture.

[0096]

Table 5

[0097] 〔Analysis of cell surface antigens〕 Each cell sample was taken out from the liquid nitrogen tank, the lid was loosened to release the pressure, then the lid was closed again, and it was thawed with gentle stirring in a thermostatic bath pre-warmed to 37°C for 1 - 2 minutes. Each thawed cell was transferred to a 15 mL centrifuge tube containing 5 mL of Stain Buffer (manufactured by BD), centrifuged at 500×g for 5 minutes at 4°C, and the supernatant was removed. 5 mL of Stain Buffer was added, centrifuged at 500×g for 5 minutes at 4°C, and washed twice.

[0098] Resuspended with 2 mL of Stain Buffer (manufactured by BD) and counted the number of live cells. Centrifuged again (500×g, 5 minutes, 4°C), and resuspended with Stain Buffer (manufactured by BD) to a cell density of 1×10 7 cells / mL. Aliquoted 20 μL (2×10 5 cells) into each 1.5 mL tube, and prepared 4 tubes each for unstained control, CD44, CD90, and Isotype Control.

[0099] Added 4 μL of Anti-CD44, Mouse (MEM-263), PE (manufactured by GeneTex), 1 μL of PE Mouse Anti-Human CD90 (manufactured by BD) (cross-reactive with pigs), and 4 μL of PE Mouse IgG1, κ Isotype Control (manufactured by BD) to their respective tubes, and incubated in the dark on ice for 45 minutes. The unstained control was also stored on ice.

[0100] Added 1 mL of Stain Buffer (manufactured by BD) to each tube, centrifuged at 500×g for 5 minutes at 4°C, and washed twice. Tapped the cell pellet to loosen, resuspended with 500 μL of Stain Buffer (manufactured by BD), and transferred to a test tube for flow cytometry through a filter immediately before analysis. Stored on ice in the dark until analysis and analyzed using flow cytometry.

[0101] As a result, in all samples, CD44 and CD90, which are markers of mesenchymal stem cells, were positive. Also, the target mesenchymal stem cells could be established without coating with gelatin during initial culture. In all cases, no non-specific reaction was observed in the measurement of Isotype Control.

[0102] Reference Example 4 [Preparation of Mesenchymal Stem Cells Derived from Neonatal Pig Pancreas] Islets were collected from neonatal pigs and cultured in suspension to prepare cell aggregates, which were then cryopreserved in the same manner as in Reference Example 1. The cryopreserved neonatal pig islets in cryovials were quickly thawed in a 37°C water bath.

[0103] Using a micropipette, the thawed islet suspension was gently added to 30 mL of MSC basal medium adjusted to a temperature equilibrium (37°C). Centrifugation was performed at 210×g for 1 minute at 4°C. When the islets were not frozen, at room temperature, after the islets settled by natural sedimentation, the supernatant was removed. The pellet was resuspended in 4 mL of MSC basal medium equilibrated at the temperature, and gently pipetted up and down.

[0104] The islet suspension was added to a 6-well plate, gently shaken to disperse the cell suspension on the growth surface (without gelatin coating), and seeded with 1650 IEQ to 2125 IEQ of islets / 1 well in 2 mL of MSC basal medium.

[0105] CO 2 In an incubator, culture was carried out at 37°C under conditions of 5% CO 2 , 90% humidity. After 3 days, the cells were replaced with MSC medium to allow cell growth, and thereafter, the MSC medium was replaced once every 3 days. Table 6 shows the sample preparation conditions. Regardless of the presence or absence of initial freezing, 100% confluence was reached 6 days after seeding.

[0106]

Table 6

[0107] 〔Subculture〕 After neonatal pig islet-derived mesenchymal stem cells (npISLET-MSC) reached approximately 80% to almost 95% confluence, the cells were collected from 2 wells and re-seeded into a T75 flask without gelatin coating.

[0108] The cells were washed with 2 mL of PBS (calcium- and magnesium-free), 320 μL of 0.25% trypsin per well was added, and the cells were left in the incubator for several minutes. Once the cells detached, they were neutralized with 1680 μL of MSC medium. The cell suspension was collected into a 50 mL tube using a 1 mL pipette. After adding 16 mL (8 mL × 2 wells) of MSC medium, the cells were centrifuged at 500 × g for 5 minutes at room temperature. The obtained pellet was gently resuspended in temperature-equilibrated MSC medium (2 mL) using a pipette.

[0109] 〔Average cell diameter〕 20 mL of the MSC medium was added to a non-gelatin-coated T75 flask for reseeding, and CO 2 In the incubator, it was cultured at 37 °C under 5% CO 2 , 90% humidity conditions. These cells were designated as the first passage. Three days after seeding the first passage, regardless of the presence or absence of initial freezing, they reached 100% confluence. From this, it was found that the proliferation rate of mesenchymal stem cells prepared from the islets of young pigs was comparable to that of mesenchymal stem cells prepared from the bone marrow of young pigs. The average diameter of the obtained mesenchymal stem cells derived from young pig islets is shown in Table 7.

[0110]

Table 7

[0111] As shown in Table 7, regardless of the freezing conditions in the preparation of islets, it was possible to prepare mesenchymal stem cells derived from young pig islets, and the average diameter was found to be comparable whether there was freezing or not.

[0112] 〔Analysis of cell surface antigens〕 Each cell sample was taken out from the liquid nitrogen tank, the lid was loosened to release the pressure, and then the lid was closed again. The sample was thawed with gentle stirring in a thermostatic bath pre-warmed to 37°C for 1 - 2 minutes. Each thawed cell was transferred to a 15 mL centrifuge tube containing 5 mL of Stain Buffer (manufactured by BD), and centrifuged at 500×g for 5 minutes at 4°C. The supernatant was removed. 5 mL of Stain Buffer was added, and the sample was centrifuged at 500×g for 5 minutes at 4°C and washed twice.

[0113] It was resuspended in 2 mL of Stain Buffer (manufactured by BD), and the number of viable cells was counted. Re-centrifugation (500×g, 5 minutes, 4°C) was performed, and it was resuspended in Stain Buffer (manufactured by BD) to a cell density of 1×10 7 cells / mL, and 20 μL (2×10 5 cells) were dispensed into each 1.5 mL tube, and a total of 4 tubes each for unstained control, CD29, CD44, and CD90 were prepared.

[0114] 1 μL of Mouse Alexa Fluor 647 Mouse Anti-Pig CD29 (manufactured by BD), 4 μL of Anti-CD44, Mouse (MEM-263), PE (manufactured by GeneTex), and 1 μL of PE Mouse Anti-Human CD90 (manufactured by BD) (cross-reactive with pigs) were added to each tube, and incubated in the dark on ice for 45 minutes. The unstained control was also stored on ice.

[0115] 1 mL of Stain Buffer (manufactured by BD) was added to each tube, and centrifuged at 500×g for 5 minutes at 4°C and washed twice. The cell pellet was tapped to loosen it, resuspended in 500 μL of Stain Buffer (manufactured by BD), and transferred to a test tube for flow cytometry through a filter immediately before analysis. It was stored on ice in the dark until analysis and analyzed using flow cytometry.

[0116] As a result, high positive rates were observed for CD29, CD44, and CD90, which are markers of mesenchymal stem cells, in all samples. In addition, the desired mesenchymal stem cells could be established regardless of the presence or absence of freezing during the initial culture.

[0117] Test Example 1 npBM-MSC prepared in the same manner as in Reference Example 1 was added to a 6-well plate at a cell count of 5 × 10 4 cells / 2 mL / well or mBM-MSC (OriCellTM strain C57BL / 6 mouse, catalog number MUBMX-01001, lot number 170221I31, Cyagen Biosciences Inc.) at a cell count of 1 × 10 5Seeds were sown at a density of cells / 2 mL / well and cultured using MSC medium. After 3 days of culture, the supernatant was collected and the concentrations of TGF-β1, TGF-β2, VEGF-A, and VEGF-C were measured. The TGF-β1 and TGF-β2 concentrations were measured using an ELISA kit and corrected by the number of cells at the time of supernatant collection. The results are shown in Figs. 2(a) and (b), Figs. 3(a) and (b). The TGF-β1 concentrations in pigs and mice were measured using R&D SYSTEMS® Quantikine® ELISA Mouse / Rat / Porcine / Canine TGF-β1 (MB100B, Bio-Techne Corporation, Minneapolis, MN, USA). The TGF-β2 concentrations in pigs and mice were measured using R&D SYSTEMS® Quantikine® ELISA Mouse / Rat / Canine / Porcine TGF-β2 (MB200, Bio-Techne Corporation). The VEGF-A concentrations in pigs and mice were measured using Swine VEGF-A Do-It-Yourself ELISA (KFS-DIY0751S-003, Kingfisher Biotech, Inc., St. Paul, MN, USA) and Mouse VEGF-A Do-It-Yourself ELISA (KFS-DIY0746M-003, Kingfisher Biotech, Inc.), respectively. The VEGF-C concentrations in pigs and mice were measured using Porcine VEGF-C ELISA kit (MBS2512025, MyBioSource, Inc., San Diego, CA, USA) and Mouse VEGF-C ELISA kit (MBS2503462, MyBioSource, Inc.), respectively.

[0118] As shown in FIGS. 2(a) and (b) and FIGS. 3(a) and (b), it was found that mesenchymal stem cells derived from juvenile pig bone marrow produce TGF-β1, TGF-β2, VEGF-A, and VEGF-C. In addition, it was found that mesenchymal stem cells derived from juvenile pig bone marrow highly express TGF-β1, TGF-β2, and VEGF-C compared with mesenchymal stem cells derived from mouse bone marrow.

[0119] Test Example 2 According to the method described in the literature (Motohiro Nishida, et al: J Vasc Surg: 2016:64:219-226), after ligating the left femoral artery of 12-week-old male C57BL / 6J mice, it was transected to create an ischemic limb. Mesenchymal stem cells derived from juvenile pig bone marrow prepared in the same manner as in Reference Example 1 were suspended in PBS at a cell count of 1×10 5 cells, 5×10 5 cells, 1×10 6 cells or 2.5×10 6 cells / 0.1 mL, and 0.1 mL was injected intramuscularly.

[0120] Using a laser Doppler flowmeter (DS2 manufactured by Moor Instruments Ltd, UK), the blood flow in the lower limbs was measured over time until 4 weeks after the surgery, and the affected side was compared with the healthy (control) side. The results are shown in FIG. 4.

[0121] As shown in FIG. 4, administration of mesenchymal stem cells derived from juvenile pigs resulted in a significant blood flow improvement effect.

[0122] Test Example 3 Mesenchymal stem cells derived from juvenile pig bone marrow prepared in the same manner as in Reference Example 1, or mesenchymal stem cells derived from mouse bone marrow, were suspended in PBS at a cell count of 1×10 5 cells, or 1×10 6 cells / 0.1 mL, and 0.1 mL was injected intramuscularly into the thigh muscle tissue of the ischemic limb prepared in the same manner as in Test Example 2.

[0123] Using a laser Doppler flowmeter (DS2 manufactured by Moor Instruments Ltd, UK), blood flow in the lower limbs was measured over time until 4 weeks after surgery, and the affected side was compared with the healthy (control) side. The results are shown in Fig. 5.

[0124] As shown in Fig. 5, it was found that the blood flow improvement effect by mesenchymal stem cells derived from young pigs was significantly higher compared to that by mouse bone marrow-derived mesenchymal stem cells.

[0125] Although the present invention has been described in detail using specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the intention and scope of the present invention. This application is based on a Japanese patent application (Japanese Patent Application No. 2019-82768) filed on April 24, 2019, the entire contents of which are incorporated herein by reference.

Claims

1. 1. A pharmaceutical composition for treating a non-porcine animal, comprising: The present invention comprises mesenchymal stem cells derived from young pigs, which are derived from the bone marrow of pigs from fetuses to those less than one month old, have an average diameter of 17 μm or less, and a doubling time in the logarithmic growth phase of 36 hours or less, and produce at least the humoral factors transforming growth factor-β (hereinafter referred to as TGF-β) 1, TGF-β2, and vascular endothelial growth factor (hereinafter referred to as VEGF)-C, the expression levels of TGF-β1, TGF-β2 and VEGF-C in the immature pig-derived mesenchymal stem cells after 3 days of culture in an MSC medium are 1.1 times or more higher than the expression levels of TGF-β1, TGF-β2 and VEGF-C in mouse bone marrow-derived mesenchymal stem cells cultured under the same conditions; A pharmaceutical composition for treating said non-porcine animal by promoting angiogenesis and / or lymphangiogenesis.

2. The pharmaceutical composition according to claim 1, wherein the immature pig-derived mesenchymal stem cells produce at least TGF-β1, TGF-β2, VEGF-A and humoral factors of VEGF-A.

3. The pharmaceutical composition according to claim 1 or 2, wherein the mesenchymal stem cells derived from young pigs are derived from fetuses or pigs less than 25 days old.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the non-pig animal is a human.

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