A novel population of dental pulp stem cells

Abstract

To provide safe and high-performance stem cells appropriate for clinical application, and a method for producing the stem cells.SOLUTION: Provided are a stem cell population derived from human deciduous tooth dental pulp, wherein 90% or more of the stem cell population is characterized by being CD117-negative, CD73-positive, CD90-positive, and CD105-positive, and a method for producing a stem cell population derived from human deciduous tooth dental pulp, the method comprising a step of culturing cells isolated from human deciduous tooth dental pulp in a medium that does not contain FBS (fetal bovine serum) in the presence of human platelet lysate (hPL).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications: This specification includes the contents described in the specifications of PCT / JP2022 / 036892 (filed October 3, 2022) and Japanese Patent Application No. 2023-080181 (filed May 15, 2023), which are the priority documents of this application. Technical fields: The present invention relates to a stem cell population derived from human deciduous dental pulp and its culture supernatant, a pharmaceutical composition containing the stem cell population, and a method for producing the same. [Background technology]

[0002] Stem cells derived from human dental pulp (dental pulp stem cells) have been reported to be effective in treating spinal cord injury, cerebral palsy (perinatal hypoxic-ischemic encephalopathy), and lower limb ischemia in animal model experiments. However, in previous reports, cells were administered directly to the spinal cord immediately after spinal cord injury (Non-Patent Document 1) or to the brain several hours after brain injury in a cerebral palsy model (Non-Patent Document 2), which is not a realistic administration method for clinical application. In another cerebral palsy model, cells were administered intravenously 24 hours after brain injury, but improvement in motor function was observed more than five months later (Non-Patent Document 3). Therefore, a treatment method that provides earlier effects is desirable. Regarding lower limb ischemia, cells were administered to model animals with moderate symptoms immediately after vascular ligation (Non-Patent Document 4) or several hours after vascular ligation (Non-Patent Document 5), which is not in line with the actual treatment of severe lower limb ischemia, the primary target of stem cell therapy.

[0003] Traditionally, fetal bovine serum (FBS) has been used to isolate dental pulp stem cells from dental pulp tissue (Non-Patent Documents 1-6). However, the possibility of xenogeneic serum components remaining in the administered formulation cannot be completely ruled out, making the use of FBS undesirable for clinical application. When using cells isolated from a patient's own dental pulp tissue, it is possible to prepare the cells using the patient's own autologous serum, but this would impose a burden on the patient and would increase the time and cost required for customization. To make dental pulp stem cell therapy available to more patients, it is necessary to mass-produce allogeneic cells and provide them off-the-shelf for treatment.

[0004] The cellular characteristics of dental pulp stem cells isolated by conventional methods using FBS have been analyzed, and it has been reported that they contain, for example, 50% or more, or 96.2%, of CD117 (c-kit)-positive cells (Patent Document 1, Non-Patent Document 7). Some previous studies have isolated CD117-positive cells and reported that they are multipotent (Non-Patent Document 8). Other reports of dental pulp stem cells include CD325 (N-cadherin) positivity (Non-Patent Document 9) and the existence of both CD51 (integrin αV) and CD49d (integrin α4)-positive and -negative cells (Non-Patent Documents 10 and 11).

[0005] It has been reported that pluripotent stem cells can be obtained by trypsinizing human dental pulp tissue and then culturing the tissue in a serum-free medium containing human platelet lysate (hPL). However, the obtained cell population is positive for both the hematopoietic stem cell markers CD34 and CD45, and is therefore considered to be a heterogeneous cell population (Non-Patent Document 12). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2014 / 141210A2(JP6491606B) [Non-patent literature]

[0007] [Non-licensed document 1] Sakai et al., J Clin Invest. 2012 Jan;122(1):80-90. doi:10.1172 / JCI59251. [Non-licensed document 2] Yamagata et al., Stroke. 2013 Feb;44(2):551-4. doi:10.1161 / STROKEAHA.112.676759. [Non-licensed document 3] Kitase et al., Stem Cells Dev. 2020 Jan 15;29(2):63-74. doi:10.1089 / scd.2019.0221. PMID: 31801412.

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

[0008] An object of the present invention is to provide safe and highly functional stem cells suitable for clinical application, and a method for producing the same. [Means for solving the problem]

[0009] The inventors have found that a stem cell population obtained by culturing cells isolated from the dental pulp of human deciduous teeth in a medium containing human platelet lysate (hPL) has structural characteristics (surface marker expression) different from previously reported dental pulp stem cells. Furthermore, the inventors have found that the stem cell population obtained by this method produces higher amounts of cytokines useful for tissue regeneration and exhibits superior tissue regeneration ability than dental pulp stem cells derived from human deciduous teeth obtained by the conventional method using FBS.

[0010] The present invention is based on the above findings, and in a first aspect, provides the following [1] to

[30] . [1] A (genetically unmodified) stem cell population derived from human dental pulp, 90% or more of which are characterized as CD117-negative, CD73-positive, CD90-positive, and CD105-positive, and the human dental pulp is human deciduous dental pulp. [2] The stem cell population according to [1], wherein 90% or more of the stem cells are CD325-negative or CD51-positive; preferably, 90% or more of the stem cells are CD325-negative and CD49d-positive and / or CD51-positive; more preferably, 90%, 95%, 97%, 98%, or 99% or more of the stem cells are CD325-negative and CD49d-positive and / or CD51-positive. [3] The stem cell population according to [1] or [2], wherein the stem cell population is CD31 negative. [4] The stem cell population according to any one of [1] to [3], wherein the stem cell population is CD34-negative and CD45-negative. [5] The stem cell population according to any one of [1] to [4], wherein the stem cell population produces SCF (stem cell factor) at a weight ratio of at least 1 / 20, preferably at least 1 / 10 of IL-6. [6] A stem cell population according to any one of [1] to [5], wherein the stem cell population produces SCF (stem cell factor) at a weight ratio of 1 / 10 to 1 / 2, preferably 1 / 10 to 1 / 3, of IL-6. [7] The stem cell population is 1×10 6The stem cell population according to any one of [1] to [6], which produces at least 0.1 ng, 0.13 ng, 0.15 ng, preferably 0.17 ng, more preferably 0.20 ng of SCF per cell in 48 hours. [8] The stem cell population according to any one of [1] to [7], wherein the stem cell population produces ANGPTL4 (angiopoietin-like 4) at a weight ratio of 3 times or more, preferably 5 times or more, of IL-6. [9] The stem cell population according to any one of [1] to [8], wherein the stem cell population produces ANGPTL4 (angiopoietin-like 4) at a weight ratio of 5 to 20 times, preferably 5 to 15 times, that of IL-6.

[10] The stem cell population is 1×10 6 The stem cell population according to any one of [1] to [9], which produces at least 4.0 ng, 4.5 ng, 5.0 ng, preferably 6.9 ng, more preferably 7.0 ng of ANGPTL4 per cell in 48 hours.

[11] A stem cell population described in any of [1] to

[10] , wherein the stem cell population produces BIGH3 (beta-inducing transforming growth factor) at a weight ratio of 400 times or more, preferably 450 times or more, of IL-6.

[12] A stem cell population according to any one of [1] to

[11] , wherein the stem cell population produces BIGH3 (beta-inducing transforming growth factor) at a weight ratio of 450 to 1500 times, preferably 450 to 1000 times, that of IL-6.

[13] The stem cell population is 1×10 6 The stem cell population according to any one of [1] to

[12] , which produces at least 400 ng, 450 ng, 500 ng, preferably 550 ng, more preferably 600 ng of BIGH3 per cell in 48 hours.

[14] A stem cell population according to any of [1] to

[13] , which is obtained by enzymatically isolating cells from human dental pulp (for example, using an enzyme containing collagenase, or an enzyme containing collagenase and a neutral protease (dispase or thermolysin)) and culturing them in a medium that does not contain FBS (fetal bovine serum) in the presence of human platelet lysate (hPL).

[15] The stem cell population according to

[14] , wherein the medium is a serum-free medium.

[16] A culture supernatant of the stem cell population according to any one of [1] to

[15] , which has at least one of the following characteristics 1) to 3): 1) SCF (stem cell factor) is contained in a weight ratio of IL-6 of 1 / 20 or more, preferably 1 / 10 or more, 2) ANGPTL4 (angiopoietin-like 4) is contained in a weight ratio of IL-6 that is 3 times or more, preferably 5 times or more, 3) BIGH3 (beta-inducing transforming growth factor) is contained in a weight ratio of 400 times or more, preferably 450 times or more, of IL-6.

[17] A culture supernatant of the stem cell population according to any one of [1] to

[15] , which has at least one of the following characteristics 1) to 3): 1) SCF (stem cell factor) is contained in a weight ratio of 1 / 10 to 1 / 2, preferably 1 / 10 to 1 / 3 of IL-6, 2) ANGPTL4 (angiopoietin-like 4) is contained in a weight ratio of 5 to 20 times, preferably 5 to 15 times, that of IL-6; 3) BIGH3 (beta-inducing transforming growth factor) is contained in a weight ratio of 450 to 1500 times, preferably 450 to 1000 times, that of IL-6.

[18] A pharmaceutical composition according to the following [18-1] or [18-2]. [18-1] A pharmaceutical composition comprising the stem cell population according to any one of [1] to

[15] , wherein the stem cell population has at least one of the following characteristics 1) to 3): 1) Producing SCF (stem cell factor) at a weight ratio of 1 / 20 or more, preferably 1 / 10 or more, of IL-6; 2) ANGPTL4 (angiopoietin-like 4) is produced at a weight ratio of 3 times or more, preferably 5 times or more, of IL-6; 3) BIGH3 (beta-induced transforming growth factor) is produced at a weight ratio of 400 times or more, preferably 450 times or more, to IL-6. [18-2] A pharmaceutical composition comprising a culture supernatant of the stem cell population according to any one of [1] to

[15] , and having at least one of the following characteristics 1) to 3): 1) SCF (stem cell factor) is contained in a weight ratio of IL-6 of 1 / 20 or more, preferably 1 / 10 or more, 2) ANGPTL4 (angiopoietin-like 4) is contained in a weight ratio of IL-6 that is 3 times or more, preferably 5 times or more, 3) BIGH3 (beta-inducing transforming growth factor) is contained in a weight ratio of 400 times or more, preferably 450 times or more, of IL-6.

[19] A pharmaceutical composition according to the following [19-1] or [19-2]. [19-1] A pharmaceutical composition comprising the stem cell population according to any one of [1] to

[15] , wherein the stem cell population has at least one of the following characteristics 1) to 3): 1) Producing SCF (stem cell factor) at a weight ratio of 1 / 10 to 1 / 2, preferably 1 / 10 to 1 / 3, of IL-6; 2) ANGPTL4 (angiopoietin-like 4) is produced at a weight ratio of 5 to 20 times, preferably 5 to 15 times, that of IL-6. 3) BIGH3 (beta-inducing transforming growth factor) is produced at a weight ratio of 450 to 1500 times, more preferably 450 to 1000 times, that of IL-6. [19-2] A pharmaceutical composition comprising a culture supernatant of the stem cell population according to any one of [1] to

[15] , and having at least one of the following characteristics 1) to 3): 1) SCF (stem cell factor) is contained in a weight ratio of 1 / 10 to 1 / 2, preferably 1 / 10 to 1 / 3 of IL-6, 2) ANGPTL4 (angiopoietin-like 4) is contained in a weight ratio of 5 to 20 times, preferably 5 to 15 times, that of IL-6; 3) BIGH3 (beta-inducing transforming growth factor) is contained in a weight ratio of 450 to 1500 times, preferably 450 to 1000 times, that of IL-6.

[20] The pharmaceutical composition according to

[18] or

[19] , which is for treating or preventing any disease selected from spinal cord injury (including chronic spinal cord injury), ischemic disease (cerebral infarction, lower limb ischemia, etc.), inflammatory disease, autoimmune disease, neurodegenerative disease, peripheral nerve disease, intestinal disease, and bone disease.

[21] A method for producing a stem cell population derived from human dental pulp, comprising: The method includes the steps of enzymatically isolating cells from human dental pulp (for example, using an enzyme containing collagenase, or an enzyme containing collagenase and a neutral protease (dispase or thermolysin)), culturing the cells in a medium containing no FBS (fetal bovine serum) in the presence of human platelet lysate (hPL), and obtaining a colony-forming cell population as the stem cell population. In the method, the cells may be subcultured (expanded) as necessary.

[22] The method according to

[21] , wherein the medium is a serum-free medium.

[23] The method according to

[21] or

[22] , wherein the medium is animal-free.

[24] The method according to any one of

[21] to

[23] , wherein the obtained dental pulp stem cell population is CD117 negative.

[25] The method according to any one of

[21] to

[24] , wherein the obtained dental pulp stem cell population further has the properties of the stem cell population according to any one of [1] to

[13] .

[26] The method according to any one of

[21] to

[24] , wherein the human dental pulp is the dental pulp of a baby tooth extracted within 48 hours.

[27] The stem cell population according to any one of [1] to

[15] , wherein the human dental pulp is the dental pulp of a baby tooth extracted within 48 hours.

[28] The stem cell population according to any one of [1] to

[15] , wherein the stem cell population has the ability to differentiate into adipocytes, osteoblasts, chondrocytes, and nerve cells.

[29] A stem cell population according to any one of [1] to

[15] , a culture supernatant according to

[16] or

[17] , or a pharmaceutical composition according to any one of

[18] to

[20] , which has at least one effect selected from neural progenitor cell proliferation, neurite extension, vascular endothelial cell proliferation, vascular endothelial cell attraction, blood vessel-like structure construction, and immunosuppressive effect.

[30] The method according to

[21] to

[23] , wherein the medium contains 10% human platelet lysate. In the above [1] to

[29] , "positive" and "negative" have the meanings as defined in the specification.

[0011] In a second aspect, the present invention provides the following [1] to

[20] . [1] A method for treating a disease requiring tissue regeneration, comprising administering to a subject in need thereof a (genetically unmodified) stem cell population derived from human dental pulp and / or a culture supernatant of said stem cell population, wherein 90% or more of said stem cell population are characterized as being CD117-negative, CD73-positive, CD90-positive, and CD105-positive, and said human dental pulp is human deciduous dental pulp. [2] The method according to [1], wherein 90% or more of the stem cell population is CD325-negative or CD51-positive; preferably, 90% or more of the stem cell population is CD325-negative and CD49d-positive and / or CD51-positive; more preferably, 90%, 95%, 97%, 98%, or 99% or more of the stem cell population is CD325-negative and CD49d-positive and / or CD51-positive. [3] The method according to [1], wherein the stem cell population is CD31 negative. [4] The method according to [1], wherein the stem cell population is CD34-negative and CD45-negative. [5] The method according to [1], wherein the stem cell population produces SCF (stem cell factor) at a weight ratio of at least one-tenth of IL-6. [6] The method according to [1], wherein the stem cell population produces SCF (stem cell factor) at a weight ratio of 1 / 10 to 1 / 3 of IL-6. [7] The stem cell population is 1×10 6 The method described in [1] produces at least 0.1 ng of SCF per cell in 48 hours. [8] The method according to [1], wherein the stem cell population produces ANGPTL4 (angiopoietin-like 4) at a weight ratio of 5 times or more that of IL-6. [9] The method according to [1], wherein the stem cell population produces ANGPTL4 (angiopoietin-like 4) at a weight ratio of 5 to 20 times that of IL-6.

[10] The stem cell population is 1×10 6 The method described in [1] produces at least 5 ng of ANGPTL4 per cell in 48 hours.

[11] The method according to [1], wherein the stem cell population produces BIGH3 (beta-induced transforming growth factor) at a weight ratio of 450 times or more that of IL-6.

[12] The method according to [1], wherein the stem cell population produces BIGH3 (beta-induced transforming growth factor) at a weight ratio of 450 to 1000 times that of IL-6.

[13] The stem cell population is 1×10 6 The method described in [1] produces at least 500 ng of BIGH3 per cell in 48 hours.

[14] The method described in [1], wherein the stem cell population is obtained by culturing cells enzymatically isolated from human dental pulp in a medium free of FBS (fetal bovine serum) in the presence of human platelet lysate (hPL).

[15] The method according to

[14] , wherein the medium is a serum-free medium.

[16] The method according to

[14] , wherein the medium is animal-free.

[17] The method according to [1], wherein the disease requiring tissue regeneration is any one selected from spinal cord injury (including chronic spinal cord injury), ischemic disease, inflammatory disease, autoimmune disease, and intestinal disease.

[18] The method according to [1], wherein the culture supernatant of the stem cell population has at least one of the following properties 1) to 3): 1) Contains SCF (stem cell factor) at a weight ratio of 1 / 10 or more of IL-6, 2) Contains ANGPTL4 (angiopoietin-like 4) at a weight ratio of 5 times or more that of IL-6, 3) Contains BIGH3 (beta-inducible transforming growth factor) at a weight ratio of 450 times or more that of IL-6.

[19] The method according to [1], wherein the culture supernatant of the stem cell population has at least one of the following properties 1) to 3): 1) Contains SCF (stem cell factor) at a weight ratio of 1 / 10 to 1 / 3 of IL-6, 2) Contains ANGPTL4 (angiopoietin-like 4) at a weight ratio of 5 to 20 times that of IL-6; 3) Contains BIGH3 (beta-inducible transforming growth factor) at a weight ratio of 450 to 1000 times that of IL-6.

[20] The method described in [1], wherein the human dental pulp is the dental pulp of a baby tooth extracted within 48 hours. [Effects of the Invention]

[0012] The present invention provides a highly functional and safe dental pulp stem cell population. Unlike conventional methods, the stem cell population of the present invention is prepared using a serum-free medium that does not contain xenogeneic components such as FBS, and is obtained as a highly homogeneous cell population without any special separation procedures. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 shows the results of surface marker analysis of human dental pulp stem cells according to the present invention. Figure 1A: CD31, Figure 1B: CD34 [Figure 2] Figure 2 shows a microscopic image of fat cells (lipid droplets) differentiated from human dental pulp stem cells stained with oil red. [Figure 3] FIG. 3 shows a microscopic image of mineralization staining of osteoblasts induced to differentiate from human dental pulp stem cells. [Figure 4] FIG. 4 shows a microscopic image of cartilage tissue induced to differentiate from human dental pulp stem cells, stained with Alcian blue. [Figure 5] Figure 5 shows immunostained images of nerve cells induced to differentiate from human dental pulp stem cells (from left: isotype control, anti-Nestin, anti-βIII-tubulin, and anti-neurofilament M stained images). [Figure 6] FIG. 6 shows the neurite outgrowth effect of the culture supernatant of human dental pulp stem cells. [Figure 7] FIG. 7 shows the neural progenitor cell proliferation effect of the culture supernatant of human dental pulp stem cells. [Figure 8] FIG. 8 shows the vascular endothelial cell proliferation effect of the culture supernatant of human dental pulp stem cells. [Figure 9]FIG. 9 shows the action of human dental pulp stem cell culture supernatant in constructing blood vessel-like structures (tube formation). [Figure 10] FIG. 10 shows the vascular endothelial cell attracting effect of the culture supernatant of human dental pulp stem cells. [Figure 11] FIG. 11 shows the immunosuppressive effect of human dental pulp stem cells (the ability to suppress the proliferation of CD4-positive T cells). [Figure 12] FIG. 12 shows the motor function improving effect (A) and nerve regenerating effect (B) of human dental pulp stem cells on chronic spinal cord injury in a rat spinal cord injury model. [Figure 13] FIG. 13 shows the effect of human dental pulp stem cells on improving motor function in perinatal hypoxic-ischemic encephalopathy in a neonatal rat hypoxic-ischemic encephalopathy model. [Figure 14] FIG. 14 shows the effect of improving femoral blood flow (A) and the effect of improving foot necrosis (B) in an immunodeficient rat severe limb ischemia model. [Figure 15] FIG. 15 shows the amount of BDNF (A) and the amount of VEGF (B) produced by human dental pulp stem cells. [Figure 16] FIG. 16 shows the vascular endothelial cell proliferation effect of the culture supernatant of human dental pulp stem cells. [Figure 17] FIG. 17 shows the action of the culture supernatant of human dental pulp stem cells to construct a blood vessel-like structure (tube formation). [Figure 18] FIG. 18 shows the amount of cytokines produced by various stem cells. [Figure 19] FIG. 19 shows the effect of increasing vascular area (A) and the effect of increasing blood vessel number (B) in an immunodeficient rat severe hind limb ischemia model. [Figure 20] FIG. 20 shows the anti-inflammatory effects of various stem cells on THP-1 cells under M2 induction conditions (A) and M1 induction conditions (B). [Figure 21] FIG. 21 shows the proliferation rate of human dental pulp stem cells cultured in various serum-free media and serum-free media containing hPL. [Figure 22]FIG. 22 shows the motor function improving effect of human dental pulp stem cells on severe perinatal hypoxic-ischemic encephalopathy in a neonatal rat model of severe perinatal hypoxic-ischemic encephalopathy. DETAILED DESCRIPTION OF THE INVENTION

[0014] 1. Stem cell population derived from human dental pulp The present invention relates to a population of stem cells derived from non-genetically modified human dental pulp, at least 90% of which are CD117-negative, CD73-positive, CD90-positive, and CD105-positive (hereinafter also referred to as the "stem cell population of the present invention"). The structural and functional characteristics of the stem cell population of the present invention are described below.

[0015] 1.1 Origin The stem cell population of the present invention is a genetically unmodified stem cell population derived from human dental pulp. The "dental pulp" may be the pulp of either a baby tooth or a permanent tooth, but baby teeth and extracted teeth such as wisdom teeth are preferred for ease of acquisition. It is also desirable to use teeth extracted within 72 hours, more preferably within 48 hours. It is particularly desirable to use human baby teeth extracted within 48 hours.

[0016] The stem cell population of the present invention may be autologous cells derived from the dental pulp of the recipient, or allogeneic cells derived from the dental pulp of a donor. In terms of preparation time and cost, and the stability of cell quality, it is preferable that the stem cell population be allogeneic cells.

[0017] 1.2 Markers "CD117" is a transmembrane protein that functions as a tyrosine kinase receptor and is also known as c-kit. Conventionally known dental pulp stem cells are either CD117-positive or a heterogeneous population containing CD117-positive and CD117-negative cells (Hilkens et al., Cell and Tissue Research (2013) 353, 65-78; Deng et al., Frontiers in Cell and Developmental Biology, March 2021, volume 9, Article 661116; Ferro et al., PLoSONE July 2012, volume 7, Issue 7, e41774; Lei et al., Stem Cell International, Volume 2021, Article ID 8886854). However, the stem cell population of the present invention is characterized as CD117-negative. Furthermore, the stem cell population of the present invention is characterized as positive for mesenchymal stem cell markers CD73, CD90, and CD105.

[0018] "CD325," "CD49d," and "CD51" are cell surface proteins that function as adhesion factors and are also known as N-cadherin (cadherin 2), integrin α4, and integrin αV, respectively. Conventionally known dental pulp stem cells are either CD325-positive or a heterogeneous population containing CD325-positive and CD325-negative cells (Deng et al., Frontiers in Cell and Developmental Biology, March 2021, volume 9, Article 661116; Madhoun et al., Frontiers in Cell and Developmental Biology, October 2021, volume 9, Article 717624). However, the stem cell population of the present invention is preferably CD325-negative. Furthermore, while previously known dental pulp stem cells are heterogeneous populations containing both positive and negative cells for CD51 and CD49d (Lei et al., Stem Cell International, Volume 2021, Article ID 8886854; Alvarez et al., International Journal of Oral Science (2015), 7, 205-212), it is preferable that the stem cell population of the present invention exhibits a positivity rate of 90%, 95%, 97%, 98% or 99% or more for CD49d and / or CD51.

[0019] The stem cell population of the present invention is preferably negative for the endothelial cell marker CD31 and also negative for hematopoietic stem cell markers such as CD34 and CD45. Furthermore, it is preferable that the positive rate of CD150, a positive marker for mesenchymal stem cells, is 90% or more, preferably 95% or more, and that the negative markers CD14 and CD19 are negative.

[0020] The stem cell population of the present invention is preferably negative for HLA-DR, HLA-DQ, CD40, CD80, and CD86, which are involved in immunogenicity.

[0021] The stem cell population of the present invention is preferably positive for adhesion factors CD29 (ITGB1), CD44, and CD166 (ALCAM), and negative for CD106 (VCAM).

[0022] The stem cell population of the present invention is CD73-positive, CD90-positive, and CD105-positive, preferably CD73-positive, CD90-positive, and CD105-positive and CD117-negative, and more preferably CD73-positive, CD90-positive, and CD105-positive, and CD117-negative and CD325-negative.

[0023] As used herein, a marker protein or marker gene being "positive" means that, when a stem cell population between passage numbers 2 and 8 is measured using a method known in the art (e.g., analysis of detection data by flow cytometry using an isotype control), the positive rate for the protein or gene in the stem cell population is 30% or more, preferably 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, and more preferably 90% or more. Even more preferably, a marker protein or marker gene being "positive" means that the positive rate for the protein or gene in a stem cell population between passage numbers 2 and 8 is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. Particularly preferably, a marker protein or marker gene being "positive" means that the positive rate of the protein or gene in a stem cell population between passages 2 and 8 is 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or 100%. A marker protein or marker gene being "negative" means that, when detected as described above, the positive rate of the protein or gene in a stem cell population is less than 10%, preferably less than 7%, more preferably less than 6%, less than 5%, and even more preferably less than 3% or less than 2% (however, if a fluorescence intensity of 1% in an isotype control is set as the boundary between positive and negative, the negative value will not be less than 1%). Here, in the case of adherent culture, cells are passaged when they reach 60% confluency.

[0024] The cell population of the present invention is characterized in that, between passage numbers 2 and 8, (1) The CD117 positivity rate is preferably less than 3%, more preferably less than 2%; (2) the CD73 positivity rate is preferably 98% or more, more preferably 99% or more; (3) The CD90 positivity rate is preferably 98% or more, more preferably 99% or more; and (4) The CD105 positivity rate is preferably 98% or more, more preferably 99% or more.

[0025] The cell population of the present invention may also have, between passage numbers 2 and 8, in addition to the characteristics (1) to (4) above, any two or more, three or more, or four or more of the following characteristics (5) to (20): (5) The CD325 positivity rate is preferably less than 7%, more preferably less than 5%. (6) The positive rate of D49d and / or CD51 is preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. (7) The CD31 positivity rate is preferably less than 3%, more preferably less than 2%. (8) The CD34 positivity rate is preferably less than 7%, more preferably less than 5%. (9) The CD45 positivity rate is preferably less than 7%, more preferably less than 5%. (10) The CD150 positivity rate is preferably 90% or more, more preferably 95% or more. (11) The CD14 positivity rate is preferably less than 7%, more preferably less than 5%. (12) The CD19 positivity rate is preferably less than 7%, more preferably less than 5%. (13) The positive rates of HLA-DR and HLA-DQ are preferably less than 7%, more preferably less than 5%, respectively. (14) The CD40 positivity rate is preferably less than 7%, more preferably less than 5%. (15) The CD80 positivity rate is preferably less than 7%, more preferably less than 5%. (16) The CD86 positivity rate is preferably less than 7%, more preferably less than 5%. (17) The CD29 positivity rate is preferably 96% or more, more preferably 98% or more. (18) The CD44 positivity rate is preferably 96% or more, more preferably 98% or more. (19) The CD166 positivity rate is preferably 96% or more, more preferably 98% or more. (20) The CD106 positivity rate is preferably less than 7%, more preferably less than 5%.

[0026] Marker proteins can be detected by antibody-based immunoassays such as ELISA, immunostaining, and flow cytometry. In the case of proteins that are expressed intracellularly but not on the cell surface, the target protein can be detected by expressing a reporter protein together with the protein and detecting the reporter protein. Marker genes can be detected using nucleic acid amplification and / or nucleic acid detection methods such as RT-PCR, microarrays, and biochips.

[0027] 1.3 Cytokine production The stem cell population of the present invention is enriched and produces cytokines useful for tissue regeneration at a higher level than conventional dental pulp stem cells prepared using a medium containing FBS. In particular, the stem cell population of the present invention is characterized by a higher ratio of cytokines useful for tissue regeneration to inflammatory cytokines than conventional dental pulp stem cells.

[0028] Cytokines useful for tissue regeneration include, for example, stem cell factor (SCF), angiopoietin-like 4 (ANGPTL4), beta-induced transforming growth factor (BIGH3), brain-derived trophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), stromal cell-derived factor 1 (SDF-1), monocyte chemotactic protein-1 (MCP-1), and angiopoietin-2. Proinflammatory cytokines include, for example, interleukin-6 (IL-6).

[0029] "SCF" (Stem Cell Factor) is a protein produced and secreted (collectively referred to as "produced" in this specification) by mammalian cells, and is known as a trophic factor that promotes the proliferation of hematopoietic stem cells in cultured cell experiments. SCF also acts on neurons and vascular endothelial cells that express its receptor, CD117, and is known to promote neuroprotection (Dhandapani et al., J Neurochem. 2005 Oct;95(1):9-19.), neurogenesis (Jin et al., J Clin Invest. 2002 Aug;110(3):311-9, Osada et al., J Neurosurg Spine. 2010 Oct;13(4):516-23), neurite outgrowth (Hirata et al., Development. 1993 Sep;119(1):49-56), and angiogenesis (Matsui et al., J Biol Chem. 2004 Apr 30;279(18):18600-7, Sun L., Cancer Cell. 2006 Apr;9(4):287-300, Kimet al., Cardiovasc Res. 2011 Oct 1;92(1):132-40) have been reported in cultured cell and animal experiments. These effects are useful for treating various diseases requiring tissue regeneration, including not only neurological and ischemic diseases, but also intestinal and bone diseases.

[0030] ANGPTL4 (Angiopoietin Like 4) is a protein produced and secreted by mammalian cells. It has been reported in cultured cell and animal experiments to have angiogenic effects (Le Jan et al., Am J Pathol. 2003 May;162(5):1521-8; Hermann et al., Clin Immunol. 2005 Apr;115(1):93-101; Ma et al., Proc Natl Acad Sci US A. 2010 Aug 10;107(32):14363-8) and anti-inflammatory effects (Cho et al., JCI Insight. 2019 Aug 22;4(16):e125437). These effects may be useful in treating ischemic, inflammatory, and autoimmune diseases, as well as various other diseases requiring tissue regeneration.

[0031] Transforming growth factor beta-induced (BIGH3) is a protein produced and secreted by mammalian cells. It is also known as βig-H3, keratoepithelin, or TGFBI. Cultured cell and animal experiments have shown that BIGH3 has angiogenic properties (Aitkenhead et al., MicrovascRes. 2002 Mar;63(2):159-71) and inhibits bone and cartilage degradation (Ruiz et al., Biomaterials. 2020 Jan;226:119544). These properties may be useful in treating ischemic diseases, bone and cartilage disorders, and various other diseases requiring tissue regeneration.

[0032] Brain-derived neurotrophic factor (BDNF) is a protein produced and secreted by mammalian cells and is widely distributed in the mammalian brain. BDNF is a neurotrophic factor known to promote neuronal development and outgrowth and mediate synaptic function (Kowianski et al., Cell Mol Neurobiol (2018) 38:579-593). It has also been reported to have neuroprotective effects (Lau et al., Cell Reports (2015)12:1353-1366; Numakawa et al., Histol Histopathol (2010) 25:237-258). These effects may be useful in the treatment of neurodegenerative diseases as well as various diseases requiring neural tissue regeneration.

[0033] VEGF (vascular endothelial growth factor) is a protein produced and secreted by mammalian cells. VEGF is known to induce angiogenesis by inducing cell division, migration, and differentiation in vascular endothelial cells. VEGF expression is also induced in neural tissue that has become hypoxic due to spinal cord injury, and it is thought to be involved in angiogenesis and neurogenesis at the site of injury (Long et al., Chinese Journal of Traumatology 18 (2015) 293-295). It is also thought to contribute to axon regeneration through angiogenesis at sites of nerve injury in the peripheral nervous system (Saio et al., Int. J. of Mol. Sci. 2021, 22, 11169). These actions may be useful for the treatment of neurodegenerative diseases and various diseases requiring neural tissue regeneration.

[0034] Nerve growth factor (NGF) is a protein produced and secreted by mammalian cells. It is known to have neuroprotective effects in the central nervous system and to be involved in the development and survival of nerve cells in the peripheral nervous system (Keefe et al., Int. J. of Mol. Sci. 2017, 18, 548; Mariga et al., Neurobiol Dis 2017, January; 97 (Pt B): 73-79). These effects may be useful in the treatment of neurodegenerative diseases and various diseases requiring the regeneration of neural tissue.

[0035] "MCP-1" (monocyte chemoattractant protein-1), also known as CC motif chemokine 2 (CCL2), is a protein produced and secreted by mammalian cells. MCP-1 is known to enhance monocyte chemotaxis, enhance T lymphocyte chemotaxis, and induce dendritic cell differentiation. It is also known to be involved in neurodegenerative diseases triggered by chronic inflammation, such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis (Singh et al., International Immunopharmacology 101 (2021) 107598). MCP-1 has also been reported to restore blood flow by improving collateral circulation through arteriogenesis (Ito et al., Circulation Res. (1997), vol.80, Issue 6, 829-837). These findings suggest that MCP-1 may be useful in the treatment of inflammatory diseases, neurodegenerative diseases, and various diseases requiring angiogenesis and neural tissue regeneration.

[0036] Angiopoietin-2 is a protein produced and secreted by mammalian cells and is known to be involved in lymphatic vessel formation in lymphatic endothelial cells and vascular remodeling in tissues (Akwii et al., Cells 2019, 8, 471). Angiopoietin-2 may be useful in treating diseases requiring angiogenesis and tissue regeneration.

[0037] IL-6 is a protein produced and secreted by mammalian cells and is known as an inflammatory cytokine that enhances immune responses and tissue inflammation. IL-6 is also a representative factor of a series of inflammatory cytokines called senescence-associated secretory phenomena (SASP), which are produced by senescent cells and are thought to be a cause of chronic inflammation associated with aging (Rolt et al., Biogerontology. 2019 Jun;20(3):359-371; Di et al., PLoS One. 2014 Nov 24;9(11):e113572). For this reason, it is undesirable for cells administered for the purpose of disease treatment to produce large amounts of IL-6.

[0038] Specifically, the stem cell population of the present invention can produce SCF at a weight ratio of at least 1 / 20 of IL-6, preferably at least 1 / 10, more preferably at a weight ratio of 1 / 10 to 1 / 2, even more preferably at a weight ratio of 1 / 10 to 1 / 3, more preferably at a weight ratio of at least 1 / 5, and even more preferably at a weight ratio of at least 1 / 3 (however, the amount does not exceed the amount of IL-6 produced). Furthermore, the stem cell population of the present invention can produce SCF at a weight ratio of at least 1 / 20 of IL-6, preferably at least 1 / 10 to 1 / 2, even more preferably at a weight ratio of at least 1 / 5, and even more preferably at least 1 / 3, during passages 2 to 8. 6 After 48 hours of culture, the cells can produce at least 0.1 ng, 0.13 ng, or 0.15 ng, preferably at least 0.17 ng, and more preferably at least 0.20 ng of SCF per cell.

[0039] When the cells administered to a subject are themselves CD117-positive, as in the case of conventionally known dental pulp stem cells, the SCF produced by the administered cells is first taken up by the administered cells themselves via CD117 on their own or other administered cells, thereby reducing the efficiency of its action on the endogenous cells of the subject on which it is intended to act.The stem cell population of the present invention has a high SCF-producing ability and is CD117-negative, and therefore can exert excellent tissue regenerative effects when administered to a subject in need of tissue regeneration.

[0040] That is, the stem cell population of the present invention preferably has a CD117 positivity rate of less than 3%, more preferably less than 2%, between passages 2 and 8; and is capable of producing SCF at a weight ratio that is at least 1 / 20 or more of that of IL-6, preferably at least 1 / 10 or more, more preferably at least 1 / 5 or more, and even more preferably at least 1 / 3 or more (however, the amount produced does not exceed that of IL-6).

[0041] The stem cell population of the present invention can produce ANGPTL4 at a weight ratio of at least 3 times, preferably at least 5 times, more preferably 5 to 20 times, even more preferably 5 to 15 times, still more preferably at least 10 times, and particularly preferably at least 20 times, more than IL-6, between passages 2 and 8. Furthermore, the stem cell population of the present invention can produce ANGPTL4 at a weight ratio of at least 3 times, preferably at least 5 times, more preferably 5 to 20 times, even more preferably 5 to 15 times, still more preferably at least 10 times, and particularly preferably at least 20 times. 6 At least 4 ng, 4.5 ng, 5.0 ng, preferably at least 6.9 ng, more preferably at least 7.0 ng of ANGPTL4 can be produced per cell after 48 hours of culture.

[0042] The stem cell population of the present invention can produce BIGH3 at a weight ratio of 400 times or more, preferably 450 times or more, more preferably 450 to 1500 times, and even more preferably 450 to 1000 times, more than IL-6, between passages 2 and 8. Furthermore, the stem cell population of the present invention can produce BIGH3 at a weight ratio of 1 x 10 to IL-6 between passages 2 and 8. 6 At least 400 ng, 450 ng, 500 ng, preferably at least 550 ng, more preferably at least 600 ng of BIGH3 can be produced per cell in 48 hours.

[0043] Here, the weight ratio of each humoral factor in the culture supernatant of the stem cell population of the present invention can be determined by measuring the amount of each humoral factor contained in the culture supernatant after culture.

[0044] The stem cell population of the present invention is characterized in that, between passages 2 and 8, The cell population is capable of producing SCF at a weight ratio of at least 1 / 20 or more of IL-6, preferably at least 1 / 10 or more, more preferably at least 1 / 5 or more, and even more preferably at least 1 / 3 or more (however, not exceeding the amount of IL-6 produced); producing ANGPTL4 at a weight ratio of at least 3 times or more of IL-6, preferably at least 5 times or more, more preferably at least 10 times or more, and even more preferably at least 20 times or more; and producing BIGH3 at a weight ratio of at least 400 times or more of IL-6, preferably at least 450 times or more, more preferably at a weight ratio of 450 to 1500 times, and even more preferably at a weight ratio of 450 to 1000 times.

[0045] When cultured in the presence of human platelet lysate (hPL) in a medium free of other animal-derived components (particularly FBS), the stem cell population of the present invention can produce SCF at a weight ratio of at least one-tenth, preferably at least one-fifth, more preferably at least one-half, and even more preferably at least one-third of that of IL-6 (however, the weight ratio does not exceed that of IL-6) between passages 2 and 8. Furthermore, when cultured in a medium free of other animal-derived components in the presence of hPL, the stem cell population of the present invention can produce SCF at a weight ratio of at least about three-fold, preferably at least about five-fold, and even more preferably at least about seven-fold, compared to when cultured in a medium containing FBS.

[0046] When cultured in the presence of human platelet lysate (hPL) in a medium that does not contain other animal-derived components (particularly FBS), the stem cell population of the present invention can produce ANGPTL4 at a weight ratio of at least about 5 times or more, preferably at least about 10 times or more, and more preferably at least about 20 times or more relative to IL-6, between passages 2 and 8. Furthermore, when cultured in a medium that does not contain other animal-derived components in the presence of hPL, the stem cell population of the present invention can produce ANGPTL4 at a weight ratio of at least about 3 times or more, preferably at least about 5 times or more, and more preferably at least about 7 times or more relative to IL-6, compared to when cultured in a medium containing FBS.

[0047] When cultured in the presence of human platelet lysate (hPL) in a medium that does not contain other animal-derived components (particularly FBS), the stem cell population of the present invention can produce VEGF at a weight ratio of at least about 2-fold or more, preferably at least about 5-fold or more, and more preferably at least about 10-fold or more relative to IL-6, between passages 2 and 8. Furthermore, when cultured in a medium that does not contain other animal-derived components in the presence of hPL, the stem cell population of the present invention can produce VEGF at a weight ratio of at least about 2-fold or more, preferably at least about 3-fold or more, and more preferably at least about 5-fold or more relative to IL-6, compared to when cultured in a medium containing FBS.

[0048] The stem cell population of the present invention can also suppress the amount of IL-6 produced when cultured in the presence of hPL in a medium that does not contain other animal-derived components, compared to when cultured in a medium containing FBS.

[0049] The stem cell population of the present invention has high productivity of SCF, ANGPTL4, and BIGH3, and low IL-6 production rate, and therefore has low risk of inflammatory reactions and can safely exert tissue regeneration effects.

[0050] 1.4 Differentiation potential The stem cell population of the present invention is multipotent and has the ability to differentiate into at least adipocytes, osteoblasts, chondrocytes, and neurons, as shown in the Examples below. Differentiation into target cells can be induced by culturing the stem cell population of the present invention in the presence of a differentiation-inducing factor appropriate for the target cells, according to methods disclosed in the Examples herein or known in the art (e.g., Marion et al., Methods Enzymol. (2006); 420: 339-361; Wang et al., Molecular Medicine Reports (2016); 14: 5551-5555).

[0051] 1.5 Physiological activity The stem cell population of the present invention produces high levels of cytokines useful for tissue regeneration, and has physiological activities (functions) in vivo or ex vivo, such as neural progenitor cell proliferation, neurite extension, vascular endothelial cell proliferation, vascular endothelial cell attraction, blood vessel-like structure construction, and immunosuppressive activity.

[0052] 2. Method for producing stem cell population of the present invention The stem cell population of the present invention can be produced by enzymatically isolating cells from human dental pulp, culturing the cells in a medium that does not contain FBS (fetal bovine serum) in the presence of human platelet lysate (hPL), and obtaining a colony-forming cell population as the stem cell population.

[0053] The "dental pulp" used in the present invention may be the pulp of either a deciduous or permanent tooth; however, for ease of acquisition, the pulp of an extracted tooth, such as a deciduous tooth or a wisdom tooth, is preferred. It is desirable to use an extracted tooth within 72 hours, more preferably within 48 hours, of extraction, and particularly desirable to use a human deciduous tooth within 48 hours of extraction. Examples of enzymatic isolation of a cell population from collected dental pulp include treatment with an enzyme containing collagenase, preferably collagenase and a neutral protease (e.g., dispase or thermolysin), to separate the cells, followed by centrifugation to isolate the cells. When isolating a cell population from collected dental pulp, it is preferable to treat the tissue with a reagent that does not contain mammalian or bacterial components.

[0054] The isolated cells are cultured in the presence of human platelet lysate (hPL) in a medium that does not contain other animal-derived serum components, particularly FBS. "Human platelet lysate (hPL)" is obtained by lysing platelets extracted from human blood using a freeze / thaw cycle, and is rich in various growth factors and cytokines necessary for cell culture. The amount of human platelet lysate (hPL) added to the medium or its content in the medium is not particularly limited, but during primary culture, it is approximately 5-20%, preferably 5-15%, more preferably 7-15%, and most preferably approximately 10%. The same applies to subculture.

[0055] As used herein, the term "about" refers to a value that varies by plus or minus 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1% of the reference value. Preferably, the term "about" refers to a range of plus or minus 10%, 5%, or 1%, of the reference value.

[0056] The "medium" used in the present invention is not particularly limited as long as it does not contain other unpurified animal-derived components, particularly FBS, but it is preferable to use a serum-free medium. As used herein, "serum-free medium" refers to a medium that does not contain unconditioned or unpurified serum, and includes media containing purified blood-derived components or factors (growth factors) derived from animal tissues, as long as they do not contradict the objectives of the present invention. Examples of serum-free media include serum-free basal media such as αMEM medium, DMEM medium, BME medium, Eagle MEM medium, BGJb medium, CMRL1066 medium, Glasgow MEM (GMEM) medium, ImprovedMEM Zinc Option medium, IMDM medium, Medium199 medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof; and commercially available serum-free media for mammalian cells such as MesenCult-ACF (STEMCELL Technologies), STEMPRO MSC SFM (Thermo Fischer Scientific), and UltraCULTURE Serum-free (Lonza).

[0057] For clinical use, the "medium" preferably does not contain any mammalian-derived components, and is particularly preferably animal-free.

[0058] A preferred method for producing the stem cell population of the present invention uses a human deciduous tooth extracted within 48 hours, and comprises the steps of: i) enzymatically isolating a cell population from the collected dental pulp using a reagent that does not contain mammalian- or bacterial-derived components; ii) primary culturing the isolated cell population in a serum-free medium that does not contain other animal-derived unpurified components (particularly FBS) in the presence of human platelet lysate (hPL) to form colonies; and iii) culturing the obtained colonies in a serum-free medium that does not contain other animal-derived unpurified components (particularly FBS) in the presence of hPL.

[0059] The "culture medium" may contain various nutrients necessary for cell maintenance and proliferation and various components necessary for differentiation induction, as appropriate, within the scope of the present invention. For example, nutrient sources may include carbon sources such as glycerol, glucose, fructose, sucrose, lactose, honey, starch, and dextrin; hydrocarbons such as fatty acids, oils and fats, lecithin, and alcohols; nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonium chloride, urea, and sodium nitrate; inorganic salts such as table salt, potassium salts, phosphates, magnesium salts, calcium salts, iron salts, and manganese salts; monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, sodium molybdate, sodium tungstate, and manganese sulfate; various vitamins; and amino acids.

[0060] The pH of the medium obtained by mixing the above components is in the range of 6.0 to 9.0, preferably 6.5 to 8.5, and more preferably 7.0 to 8.0.

[0061] Cells isolated from dental pulp and the stem cell population of the present invention are cultured in adherent culture. The container is not particularly limited as long as it is one used for cell culture, and examples of containers that can be used include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles.

[0062] The seeding density of the cells is not particularly limited, but it is preferable not to seed them too high. For example, cells extracted from the dental pulp of one baby tooth can be seeded at a density of 25 to 225 cm 2 , preferably 75 to 150 cm 2 The cells are cultured at 36°C to 38°C, preferably 36.5°C to 37.5°C, under conditions of 1% to 25% O2 and 1% to 15% CO2, with the medium being changed periodically.

[0063] The primary culture is preferably carried out until colonies are formed, grow, and detached. Once detachment of the colonies is confirmed, the cells are collected using a filter or the like. The collected cells are obtained as a highly homogeneous stem cell population without any special separation procedures.

[0064] The stem cell population may be further subcultured (expansion culture) as needed. Subculture is performed using the same medium as primary culture. Subculture is performed when the cells reach 60% confluency during adherent culture. Typically, primary culture cells are cultured for at least 7 days, preferably 9 days or more, and more preferably 9 to 15 days to confirm colony formation, growth, and detachment, but cells after subculture may be cultured for at least 1 day, preferably 2 days or more, and more preferably 2 to 3 days. The seeding density of cells after subculture is not particularly limited, but it is preferable not to make it too high. For example, 1000 to 2000 cells / cm 2 , preferably 1300 to 1500 cells / cm 2 The seeds are sown at a density of

[0065] For safety reasons, it is desirable to conduct endotoxin tests, mycoplasma tests, sterility tests, etc. again on the collected stem cell population and its culture supernatant.

[0066] The produced stem cell population may be cryopreserved (e.g., stored in a deep freezer at -152°C) until use, if necessary. Cryopreservation is performed, for example, by adding an appropriate cryoprotectant to the medium used for the cell culture. Examples of cryoprotectants that can be used include dextran, DMSO, and commercially available cryopreservation solutions.

[0067] 3. Culture supernatant of the stem cell population of the present invention The culture supernatant of the stem cell population of the present invention is rich in cytokines useful for tissue regeneration, and can be used itself as a pharmaceutical composition for tissue regeneration.

[0068] Specifically, the culture supernatant of the stem cell population of the present invention has at least one of the following properties 1) to 3): 1) The weight ratio of SCF to IL-6 is at least 1 / 20, preferably at least 1 / 10, more preferably 1 / 10 to 1 / 2, and even more preferably 1 / 10 to 1 / 3 (however, the weight ratio does not exceed the amount of IL-6 produced), 2) ANGPTL4 is contained in a weight ratio of 3 times or more, preferably 5 times or more, more preferably 5 to 20 times, and even more preferably 5 to 15 times that of IL-6; 3) BIGH3 is contained at a weight ratio of 400 times or more, preferably 450 times or more, more preferably 450 to 1500 times, and even more preferably 450 to 1000 times that of IL-6.

[0069] The culture supernatant of the stem cell population of the present invention particularly preferably has at least one of the following properties 1') to 3'): 1') SCF is contained in a weight ratio of 1 / 20 or more, preferably 1 / 10 or more, more preferably 1 / 5 or more, and even more preferably 1 / 3 or more of IL-6 (however, the weight ratio does not exceed the amount of IL-6 produced); 2') ANGPTL4 is contained in a weight ratio of IL-6 that is 3 times or more, preferably 5 times or more, more preferably 10 times or more, and even more preferably 20 times or more. 3') BIGH3 is contained in a weight ratio of 400 times or more, preferably 450 times or more, more preferably 450 to 1500 times, and even more preferably 450 to 1000 times that of IL-6.

[0070] The culture supernatant of the present invention can be cryopreserved until clinical use, and can be used to treat various diseases requiring tissue regeneration, similar to the pharmaceutical compositions described below.

[0071] 4. Pharmaceutical compositions containing the stem cell population of the present invention The stem cell population of the present invention abundantly produces cytokines useful for tissue regeneration, such as SCF, has a low production rate of inflammatory cytokines, and is CD117-negative, and therefore has excellent tissue regeneration effects and can be used as a pharmaceutical composition. The stem cell population of the present invention can be provided as a cell medicine together with a culture medium or culture supernatant, as needed.

[0072] The pharmaceutical composition of the present invention has at least one of the following properties 1) to 3): 1) The weight ratio of SCF to IL-6 is at least 1 / 20, preferably at least 1 / 10, more preferably 1 / 10 to 1 / 2, and even more preferably 1 / 10 to 1 / 3 (however, the weight ratio does not exceed the amount of IL-6 produced), 2) ANGPTL4 is contained in a weight ratio of 3 times or more, preferably 5 times or more, more preferably 5 to 20 times, and even more preferably 5 to 15 times that of IL-6; 3) BIGH3 is contained at a weight ratio of 400 times or more, preferably 450 times or more, more preferably 450 to 1500 times, and even more preferably 450 to 1000 times that of IL-6.

[0073] The pharmaceutical composition of the present invention preferably has at least one of the following properties 1') to 3'): 1) SCF is contained in a weight ratio of IL-6 that is 1 / 20 or more, preferably 1 / 10 or more, more preferably 1 / 5 or more, and even more preferably 1 / 3 or more; 2) ANGPTL4 is contained in a weight ratio of IL-6 that is 3 times or more, preferably 5 times or more, more preferably 10 times or more, and even more preferably 20 times or more. 3) BIGH3 is contained at a weight ratio of 400 times or more, preferably 450 times or more, more preferably 450 to 1500 times, and even more preferably 450 to 1000 times that of IL-6.

[0074] The number of stem cells contained in the pharmaceutical composition of the present invention is determined appropriately depending on the subject and the target disease. Taking into consideration the timing of administration to the subject and the time required for culture, it is practical to use the minimum amount that will produce an effect. Generally, the number of cells contained in the pharmaceutical composition is 1 x 10 5 pcs or more, 1x10 6 1x10 or more, preferably 1x10 7 1x10 or more, more preferably 1x10 8 1x10 or more, more preferably 1x10 9 The number of administrations is not limited to one, but may be two or more.

[0075] The pharmaceutical composition of the present invention is preferably a parenteral formulation, more preferably a parenteral systemic formulation, particularly an intravenous formulation. Dosage forms suitable for parenteral administration include injections such as solution injections, suspension injections, emulsion injections, and injections prepared immediately before use, as well as implants. Parenteral formulations are in the form of aqueous or non-aqueous isotonic sterile solutions or suspensions, and are formulated into an appropriate unit dosage form using, for example, an appropriate combination of pharmacologically acceptable carriers or vehicles, specifically, sterile water, physiological saline, culture media (particularly media used for culturing mammalian cells such as RPMI), physiological buffers such as PBS, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, excipients, vehicles, preservatives, binders, etc.

[0076] The pharmaceutical composition of the present invention can be used for any disease requiring tissue regeneration, and is not particularly limited thereto. Examples include spinal cord injury (including traumatic and surgical injuries, and chronic spinal cord injury), ischemic diseases (cerebral infarction, limb ischemia including lower limb ischemia, perinatal hypoxic-ischemic encephalopathy, ischemic heart disease including myocardial infarction, etc.), inflammatory diseases (sepsis, hepatitis, pancreatitis, nephritis, pneumonia, etc.), autoimmune diseases (rheumatism, SLE, type I diabetes, etc.), intestinal diseases (irritable bowel syndrome, ulcerative colitis, Crohn's disease, Hirschsprung's disease and related syndromes, etc.), and treatment and regeneration of bone defects.

[0077] 5. Therapeutic method using the stem cell population of the present invention or its culture supernatant The present invention also provides a therapeutic method comprising administering the stem cell population of the present invention or a culture supernatant thereof to a subject in need thereof. The subject of treatment is not particularly limited as long as it requires tissue regeneration, and examples thereof include the above-mentioned spinal cord injury, ischemic disease, inflammatory disease, autoimmune disease, and intestinal disease, and these diseases can be treated in the acute, subacute, or chronic phase. For example, the therapeutic method of the present invention can treat spinal cord injury in the acute or subacute phase, ischemic disease in the chronic phase, inflammatory disease in the chronic phase, autoimmune disease in the chronic phase, intestinal disease in the chronic phase, bone defect in the acute or subacute phase, or bone defect in the chronic phase.

[0078] 6. Dental pulp stem cell bank According to the method of the present invention, a dental pulp stem cell bank can be created by preparing a population of dental pulp-derived stem cells from multiple donors and cryopreserving them. As described above, the cryopreservation method for cells can be carried out by methods known in the art. [Example]

[0079] The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples.

[0080] Example 1: Preparation of dental pulp stem cells In a sterile environment, dental pulp was excised from extracted human deciduous teeth within 48 hours of extraction, minced, and then treated with Liberase (MNP-S GMP Grade, Roche) at a final concentration of 0.05 mg / ml for 15 minutes at 37°C with agitation. The cell-containing supernatant was collected, and the remaining dental pulp tissue was treated with Liberase at 0.05 mg / ml for 15 minutes at 37°C with agitation. Cells were collected from the treated dental pulp tissue through a 70 mm strainer and centrifuged with the previously collected supernatant. The collected cells were seeded into a CellBind T75 flask using MEMα (Gibco) containing 10% human phospholipids (hPL) (AventaCell BioMedical) and cultured for at least 9 days until colonies formed, grew, and detached. Medium changes were performed 2 and 9 days after seeding. Once colonies detached, cells were harvested using TrypLE Select (Gibco) and expanded in the same medium. Hereinafter, the human dental pulp stem cells prepared by the above method may be referred to as the present human dental pulp stem cells.

[0081] Example 2: Surface marker analysis (1) For the human dental pulp stem cells prepared according to Example 1 for 8 donors, 1 x 10 5 The cells were incubated with phycoerythrin (PE)-conjugated antibodies against various surface markers (CD117 (#313204), CD31 (#303105), CD34 (#343606), CD45 (#368510), CD90 (#328110), CD105 (#323206), CD73 (#344004), CD325 (#350806), CD49d (#304303), CD51 (#327909), CD29 (#303003), CD44 (#338807), CD166 (#343904), and CD106 (#305805), all from Biolegend). Data were collected by flow cytometry. The analysis was performed by gating so that the positive rate of the isotype control antibody was around 1%, and the positive rates of various surface markers were calculated.

[0082] [Table 1]

[0083] The positive rates for CD117, CD325, and CD45 were less than 5% for all donor cells, indicating negative markers. The positive rates for CD73, CD90, CD105, CD49d, and CD51 were greater than 96% for all donor cells, indicating positive markers. The positive rates for CD31 and CD34 were less than 7% for seven of eight donor cells, indicating negative markers. One donor cell showed a positive rate of 15.49% for CD31, and another donor cell showed a positive rate of 19.96% for CD34 (Figure 1). The positive rates for CD29, CD44, and CD166 were greater than 99% for all donors. The positive rate for CD106 was less than 9% for seven of eight donor cells, and 10.75% for one donor cell.

[0084] Example 3: Adipocyte differentiation potential The human dental pulp stem cells obtained in the same manner as in Example 1 were plated in a 6-well plate at 5.76 x 10 5 Cells were seeded at 2.5 mL / well in 10% hPL / MEMα and cultured until 100% confluency was reached. The medium was then replaced with adipogenesis medium (MEMα, 2% FBS, 500 μM IBMX, 50 μM indomethacin, 5 μg / ml insulin, 1 μM dexamethasone). After 28 days of differentiation induction, the cells were stained using a lipid assay kit (Cosmo Bio, #AK09F) and observed under a microscope for lipid droplets.

[0085] As a result, red-stained lipid droplets were observed within the cells (indicated by the arrows in the figure), indicating that these human dental pulp stem cells have the ability to differentiate into adipocytes (Figure 2).

[0086] Example 4: Osteoblast differentiation potential The human dental pulp stem cells obtained in the same manner as in Example 1 were plated on a collagen-coated 6-well plate at 5.76 x 10 5Cells were seeded at 2.5 mL / well in 10% hPL / MEMα and cultured until 100% confluency was reached. The medium was then replaced with osteogenic differentiation medium (MEMα, 5% FBS, 50 μg L-ascorbic acid 2-phosphate, 10 nM dexamethasone, 10 mM β-glycerophosphate). After 28 days of differentiation induction, the cells were stained using a mineralization staining kit (Cosmo Bio, #AK21) and examined for mineralization under a microscope.

[0087] As a result, red-stained mineralization (indicated by the arrow in the figure) was observed, demonstrating that these human dental pulp stem cells have the ability to differentiate into osteoblasts (Figure 3).

[0088] Example 5: Chondrocyte differentiation potential The human dental pulp stem cells (donor 1) obtained in Example 1 were placed on a low-adsorption 96U-bottom plate (Sumitomo Bakelite, SUMS9096U) at 2 x 10 5 Cells were seeded at 200 μL / well in 10% hPL / MEMα. The next day, spheroid formation was confirmed, and the medium was replaced with chondrogenic differentiation medium (PromoCell, #C-28012). After 21 days of differentiation induction, the cells were fixed in 4% paraformaldehyde, embedded in paraffin blocks, and thinly sectioned. The sections were stained with Alcian blue stain and examined under a microscope for cartilage tissue analysis.

[0089] As a result, blue-stained cartilage tissue (indicated by the arrow in the figure) was observed, demonstrating that these human dental pulp stem cells have the ability to differentiate into chondrocytes (Figure 4).

[0090] Example 6: Neuronal differentiation potential The human dental pulp stem cells obtained in the same manner as in Example 1 were placed in a poly-D-lysin / laminin cell ware 8-well culture slid (Corning) at 2.8 x 10 3Cells were seeded at 400 μl / well in 10% hPL / MEMα. After 24 hours, the medium was replaced with N2 medium (Neurobasal-A + N2 supplement, serum-free, 10 ng / ml human EGF, 10 ng / ml human FGF-basic). After 21 days of differentiation, cells were fixed with 4% paraformaldehyde and stained with primary antibodies for neural differentiation markers: anti-BIII-tubulin (clone: ​​TU-20) (Millipore, #MAB1637), anti-Nestin (clone: ​​10C2) (NovusBio, #NB300-266), and anti-neurofilament M (clone: ​​NN18) (Sigma, #5264), and secondary antibodies: Alexa Fluor® 488 anti-mouse IgG (H+L) (Jackson immunoresearch). Cells were then observed under a fluorescent microscope.

[0091] As a result, cells were found that were immunostained with the antibodies for all neural differentiation markers, demonstrating that these human dental pulp stem cells have the ability to differentiate into neural cells (Figure 5).

[0092] Example 7: Cytokine production ability (1) These human dental pulp stem cells were subcultured in 10% hPL / MEMα or 10% FBS / MEMα for 7 days, and then cultured in a 6-well plate at 5.76 x 10 5The cells were seeded in the same medium at a concentration of 2.5 ml / well. The following day, the medium was replaced with 2.5 ml / well of MEMα without hPL, FBS, or phenol red. After 48 hours, the culture supernatant was collected, aliquoted, and stored at -80°C. The frozen samples were thawed, and the concentrations of SCF, ANGPTL4, BIGH3, and IL-6 in the samples were measured using a Multiplex HGF panel (Biolegend, #740180), a Human ANGPTL4 assay kit (IBL, #27749), a Human beta IG-H3 ELISA (abcam, #ab220651), and a Multiplex Neuroinflammatory panel (Biolegend, #740796), respectively. The concentration ratios relative to IL-6 were calculated (Tables 2 and 3). The amount of each factor in the cell culture medium was calculated based on the volume of culture supernatant and the number of cells seeded. 6 The production amount was calculated as the amount produced per cell (Tables 4 and 5).

[0093] As a result, the conditioned medium of dental pulp stem cells subcultured in 10% hPL / MEMα showed a higher concentration ratio of SCF, ANGPTL4, and BIGH3 to IL-6 and a higher concentration of 1x10 compared to the conditioned medium of dental pulp stem cells subcultured in 10% FBS / MEMα. 6 The production amount per cell was high.

[0094] [Table 2]

[0095] [Table 3]

[0096] [Table 4]

[0097] [Table 5]

[0098] Example 8: Neurite outgrowth effect Human dental pulp stem cells (donor 8) were cultured at 1000 cells / cm 2 The cells were seeded at a density of 100 μg / mL in 10% hPL / MEMα and cultured for 6 days, after which the medium was replaced with MEMα. After a further 7 days of culture, the culture supernatant was collected. This culture supernatant was added to a Poly-D lysin (PDL)-coated culture plate (Corning) to coat the plate with the components contained in the culture supernatant. IMR-32 cells were seeded on this plate, and differentiation into neurons was induced by adding all transretinal on days 1-4 and 7-8. On day 9, Tubulin Tracker TM Neurites were stained with Fluorescence Intensity Green (Thermo Fisher Scientific) and nuclei with Hoechst 33342 (Dojindo), and images were taken. Neurite length was then measured by image analysis using AutoneuriteJ.

[0099] As a result, the neurite length extending from neurally differentiated IMR-32 cells was significantly longer on plates coated with culture medium compared to plates coated only with PDL. This suggests that the extracellular matrix secreted by these human dental pulp stem cells promotes neurite extension (Figure 6). This effect is important for nerve regeneration.

[0100] Example 9: Neural progenitor cell proliferation effect Human neural progenitor cells (ENStemA, Millipore) were suspended in ENstem expansion medium containing L-glutamine and FGF (5x10 4The cells were seeded onto Corning BioCoat poly-L-ornithine / laminin-coated multiwell plates (100 μl / well in a 96-well plate). The following day, the medium was replaced with ENstem expansion medium containing L-glutamine supplemented with 20% or 40% of the dental pulp stem cell culture supernatant collected in Example 8 or MEMα (control), and the cells were cultured for an additional 3 days. CCK-8 was added to the neural progenitor cells, and the number of neural progenitor cells was assessed by measuring the absorbance at 450 nm one hour later.

[0101] As a result, the absorbance of neural progenitor cells added with MEMα decreased in a dose-dependent manner, whereas the absorbance of neural progenitor cells added with culture supernatant increased. This suggests that the neural progenitor cells proliferated due to trophic factors secreted by these human dental pulp stem cells (Figure 7). This effect is important for nerve regeneration.

[0102] Example 10: Vascular endothelial cell proliferation effect (1) HUVECs (human umbilical vein endothelial cell line, Kurabou) were suspended in Humedia / 2% FBS and seeded onto a 96-well plate (2.5 x 10 3 The next day, the dental pulp stem cell culture supernatant collected in Example 8 was mixed with MEMα at various ratios, and FBS was added to a final concentration of 2%. This mixture was then added to the Humedia / 2% FBS solution in equal volumes over the wells and cultured for 3 days. CCK-8 was added to the HUVECs, and the mixture was incubated at 37°C for 4 hours. The OD450 was measured to evaluate the number of HUVEC cells.

[0103] As a result, the absorbance increased depending on the proportion of culture supernatant. This suggests that the vascular endothelial cells proliferated due to the trophic factors secreted by these human dental pulp stem cells (Figure 8). This effect is important for angiogenesis.

[0104] Example 11: Vascular structure construction effect (1) Extracellular matrix gel (Angiogenesis Assay Kit, Abcam) was prepared in a 96-well plate, and 1x10 HUVECs were added to the dental pulp stem cell culture supernatant collected in Example 8 or suspended in MEMα. 5 Cells were seeded at 1000 cells / well and cultured for 20 hours. The wells were photographed under bright field light, and the branching interval (distance between branches) was measured by image analysis using Angiogenesis Analyzer for ImageJ to examine tube formation (the formation of blood vessel-like structures).

[0105] As a result, the addition of culture supernatant significantly increased the branching interval (Figure 9). This suggests that the trophic factors secreted by these human dental pulp stem cells promoted tube formation. This effect is important for angiogenesis.

[0106] Example 12: Vascular endothelial cell attraction Seed HUVECs (1x10) in the upper layer of the transwell. 5 After culturing (cells / well), the lower layer was filled with the dental pulp stem cell culture supernatant or MEMα recovered in Example 8 and cultured for 24 hours. HUVECs that had migrated to the back side of the upper layer (lower layer side) were detached with trypsin, and the cell number was measured as luminescence intensity using CellTiterGlo (Promega) to evaluate the attraction of HUVECs to the lower layer.

[0107] As a result, filling the lower layer with culture supernatant significantly increased the luminescence intensity (Figure 10). This suggests that chemokines and other substances secreted by these human dental pulp stem cells strongly attracted vascular endothelial cells. This effect is important for angiogenesis.

[0108] Example 13: Immunosuppressive effect Human dental pulp stem cells (1x10 5CFSE-labeled human PBMCs (peripheral blood mononuclear cells, CTL) were mixed with CFSE (a viability dye, Thermo Fisher Scientific) and stimulated for 6 days with anti-CD3 antibody (Biolegend, #300438) and anti-CD28 antibody (Biolegend, #302934) (final concentration 0.1 μg / ml each). CFSE levels in CFSE-incorporated cells decrease with cell division, so the signal of CD4+ T cells in PBMCs was diluted by proliferation. low The immunosuppressive effect of dental pulp-derived cells was evaluated by quantifying the percentage of CD4-positive T cells as the proliferation rate using flow cytometry using a known method (Killer et al. Stem Cell Research & Therapy (2017) 8:100).

[0109] As a result, the proliferation of CD4+ T cells induced by stimulation with anti-CD3 and anti-CD28 antibodies was significantly suppressed by the human dental pulp stem cells (Figure 11), indicating that the cells have a strong immunosuppressive effect.

[0110] Example 14: Effect on spinal cord injury A spinal cord injury model was created by dropping a weight (2.5 mm diameter, 10 g) from a height of 50 mm onto the spinal cord between the 9th and 10th thoracic vertebrae of anesthetized rats (Jcl: SD, SPF, CLEA Japan) using a MASCIS Impactor (Rutgers University, USA). Seven, 9, 11, and 13 weeks after model creation, vehicle or the human dental pulp stem cells of the present invention were administered intravenously at 1 x 10 doses. 6 pcs, and 5x10 in the spinal canal 5We investigated the motor function recovery and nerve regeneration effects of chronic spinal cord injury by administering 10 mg / kg of cyclosporine intraperitoneally every other day, starting one day before the first administration. Specifically, hindlimb motor function was assessed over time using the Basso-Beattie-Bresnahan (BBB) ​​test. 15 weeks after model creation, rats were perfusion-fixed with 4% paraformaldehyde, and the spinal cord containing the injury site was harvested and coronal frozen sections were prepared using OCT compound. The sections were stained with H&E or Luxol Fast Blue (LFB), and nerve regeneration was assessed by quantifying the stained area using image analysis using Image J. Cyclosporine was administered intraperitoneally at a dose of 10 mg / kg every other day, starting one day before the first administration.

[0111] As a result, a tendency for the BBB score to increase was observed starting four weeks after the initial cell administration, with a statistically significant increase observed two weeks after the final administration (Figure 12A). H&E and LFB staining of spinal cord sections showed a significant increase in the stained area at the epicenter of the injury after cell administration (Figure 12B). Based on these findings, it is believed that these human dental pulp stem cells can regenerate nerve parenchyma and myelin sheaths and restore motor function in the chronic phase of spinal cord injury when administered via a clinically feasible route and at a time that is feasible.

[0112] Example 15: Effect on perinatal hypoxic-ischemic encephalopathy Neonatal rat hypoxic-ischemic encephalopathy models were created by ligating the left carotid artery of 7-day-old Wistar / ST rats under anesthesia for 1-2 hours, followed by placing them in a hypoxic (8% O) environment for 1 hour. The following day, 1x10 human dental pulp stem cells (HPCs) or the vehicle were intravenously administered. 5 The effect of administering 100 mg of acetaminophen to improve motor function in patients with perinatal hypoxic-ischemic encephalopathy was examined. Specifically, a rotarod test was performed 41 days after administration, and the latency to fall from the rod was measured to evaluate motor coordination and endurance of the limbs.

[0113] As a result, compared to the sham treatment group (normal group), the vehicle-administered group showed a significant shortening of the latency to fall, indicating a clear impairment of motor function, but the cell-administered group showed almost no shortening of the latency to fall, indicating a recovery of motor function (Figure 13). Based on these results, it is believed that these human dental pulp stem cells can alleviate the symptoms of cerebral palsy in a relatively short period of time when administered via a route and at a time that is clinically feasible for perinatal hypoxic-ischemic encephalopathy.

[0114] Example 16: Effect on critical limb ischemia The right common iliac artery and femoral artery and vein of immunodeficient rats (F344 / NJcL / -rnu, CLEA Japan) were ligated and separated to create a severe limb ischemia model. The following day, rats were divided into groups based on the degree of reduction in blood flow compared to the normal limb. Then, the human dental pulp stem cells (2 x 10 6 The effect on severe limb ischemia was examined by administering 1000 cells / head (1000 cells / head) into the ischemic limb muscles. The improvement of blood flow due to angiogenesis was evaluated by measuring blood flow volume compared to that of a normal limb. The improvement of foot necrosis was evaluated by visually inspecting each individual for necrosis up to the heel on Day 7 (4 cases per group) and Day 14 (6 cases per group) after model creation.

[0115] The results showed that the vehicle-treated group showed a sustained decrease in blood flow over the 14 days following ischemia, whereas the cell-treated group showed a significant recovery over time (Figure 14A). Furthermore, the cell-treated group showed a high rate of avoidance of foot necrosis over the 14 days, with a statistically significant difference compared to the vehicle-treated group (Figure 14B). Based on these findings, it is believed that the human dental pulp stem cells, when administered via a clinically feasible route and at a time suitable for clinical use, can restore blood flow through angiogenesis in patients with severe limb ischemia, thereby preventing foot amputation due to foot necrosis.

[0116] Example 17: Cytokine production ability (2) The human dental pulp stem cells were subcultured in 10% hPL / MEMα or 10% FBS / MEMα in the same manner as in Example 7, and then cultured in basal medium. The BDNF and VEGF production levels in the culture supernatant were measured using LEGENDplex TMEach was measured using Human Neuroinflammation Panel 1 (biolegend, Cat: 740795).

[0117] As a result, the culture supernatant after subculture in 10% hPL / MEMα had a higher IL-6 concentration ratio and 1x10 6 The production levels of BDNF and VEGF per cell were high (Table 6) (Fig. 15).

[0118] [Table 6]

[0119] Example 18: Vascular endothelial cell proliferation effect (2) As in Example 10, the human dental pulp stem cells were cultured in 10% hPL / MEMα or 10% FBS / MEMα, and the culture supernatant was mixed with MEMα at various ratios. A final concentration of 2% FBS was added, and an equal volume of this mixture was added over the Humedia / 2% FBS in a different well from the one containing the hPL culture supernatant. After adding each culture supernatant, HUVECs were cultured for 3 days. CCK-8 was added, and the cells were incubated at 37°C for 4 hours. The OD450 was measured to evaluate the number of HUVEC cells.

[0120] As a result, HUVECs supplemented with culture supernatant cultured using 10% FBS / MEMα ("FBS") did not exhibit the same proliferation effect as HUVECs supplemented with culture supernatant cultured using 10% hPL / MEMα ("hPL") (Figure 16).

[0121] Example 19: Vascular-like structure construction effect (2) As in Example 11, the tube formation effect was examined using HUVECs suspended in the culture supernatant of human dental pulp stem cells cultured using 10% FBS / MEMα and HUVECs suspended in the dental pulp stem cell culture supernatant recovered in Example 8.

[0122] As a result, it was shown that the dental pulp stem cell culture supernatant collected in Example 8 ("10% hPL / MEM") promoted tube formation more than the dental pulp stem cell culture supernatant cultured using FBS ("10% FBS / MEM") (Figure 17).

[0123] Example 20: Comparison of cytokine production ability in various stem cells Human deciduous tooth-derived dental pulp stem cells (SHED, this human dental pulp stem cells), bone marrow-derived stem cells (BMMSC), and adipose tissue-derived stem cells (ATMSC) were seeded into 6-well plates at 5.76x10^5 cells / well. 24 hours after seeding, the medium was replaced with Minimum Essential Medium α (MEMα). 48 hours later, the culture supernatants were collected and the production levels of brain-derived trophic factor (BDNF), nerve growth factor (NGF), angiopoietin-like 4 (ANGPTL4), vascular endothelial growth factor (VEGF), stromal cell-derived factor 1 (SDF-1), and monocyte chemotactic protein-1 (MCP-1) were compared (Figure 18; each plot in the figure shows the results for cells derived from different donors, and the bars indicate the average values).

[0124] The results showed that dental pulp stem cells derived from human deciduous teeth have a higher production capacity of these humoral factors than other stem cells. BDNF and NGF are known to be closely involved in axonal elongation, synaptic transmission, and neuroprotection in the central nervous system, ANGPTL4 and VEGF in angiogenesis, and SDF1 and MCP-1 in cell migration as chemokines.

[0125] Example 21: Effect on critical limb ischemia (2) In the severe lower limb ischemia model prepared in Example 16, angiogenesis in the dental pulp stem cell administration group and the solvent administration group was quantitatively evaluated using an automated macro in Image J of tissue stained images using an antibody against αSMA (ACTA2), a smooth muscle cell marker.

[0126] The results showed that both the vessel area and vessel number in the ischemic lower limb muscles were increased in the cell-administered group compared to the vehicle-administered group (Figure 19). This indicates that dental pulp stem cells can promote angiogenesis in severely ischemic areas.

[0127] Example 22: Anti-inflammatory effect Human monocytic leukemia cell line (THP-1) cells were cultured in RPMI1640 (Gibco) medium containing 10% FBS and 1x penicillin-streptomycin (10% FBS / RPMI1640 medium) and stimulated with phorbol 12-myristate 13-acetate (PMA) at 10 ng / ml. Five cells were cultured per well. 5 Human deciduous tooth-derived dental pulp stem cells (SHED), bone marrow-derived mesenchymal stem cells (BMMSC), adipose tissue-derived mesenchymal stem cells (ATMSC), or permanent tooth-derived dental pulp stem cells (DPSC) were seeded at 5x10 cells per well in a 24-well plate (Falcon culture insert, 24-well 0.4 μm (HD) PET semi-transparent membrane) with a 0.4 μm pore size mesh. 4 The culture inserts were placed in 10% FBS / RPMI 1640 medium (10% FBS / RPMI 1640 medium) and in control wells (Cont). IL-4 20 ng / ml (M2 macrophage induction conditions) or LPS 1 μg / ml and IFN-γ 20 ng / ml (M1 macrophage induction conditions) were then added. After 24 hours of culture, THP-1 cells were harvested from each well, RNA was extracted, and transglutaminase 2 (TGM2) and IL-12B mRNA levels were measured by RT-qPCR.

[0128] Co-culture with human deciduous tooth-derived dental pulp stem cells significantly enhanced TGM2 transcription in THP-1 cells stimulated to induce M2 macrophages compared with co-culture with other stem cells (Figure 20A; each plot in the figure represents the results of cells from different donors, and the bars represent the average). This indicates that human deciduous tooth-derived dental pulp stem cells can more efficiently promote the polarization of THP-1 cells into M2 macrophages, which have anti-inflammatory effects, than other stem cells. Furthermore, co-culture with human deciduous tooth-derived dental pulp stem cells suppressed the transcription of the pro-inflammatory cytokine IL-12B in THP-1 cells stimulated to induce M1 macrophages to a similar extent as co-culture with other stem cells (Figure 20B; each plot in the figure represents the results of cells from different donors, and the bars represent the average). These results demonstrate that human deciduous tooth-derived dental pulp stem cells have an anti-inflammatory effect.

[0129] Example 23: Comparison with animal-free serum-free medium (1) Cell proliferation rate Human deciduous tooth-derived dental pulp stem cells from four donors were cultured in the same manner as in Example 1. However, after cell collection, the cells were cultured in 10% hPL / MEMα medium (the medium of the present invention), MesenCult TM The seeding density was 1.3-2.6 x 10 cells / well using the ACF Plus Medium Kit (STEMCELL Technologies) (serum-free medium 1) or KBM ADSC-4 (Kohjin Bio) (serum-free medium 2). 3 cells / cm 2 The cells were seeded according to the degree of proliferation so that the passage timing was synchronized between the two.

[0130] The number of cells in each medium at passage numbers 4, 7, 11, 14, and 16 was counted using a cell counter (NucleoCounter NC-202, manufactured by ChemoMetec) and plotted as the proliferation rate (FIG. 21).

[0131] As a result, it was shown that the medium of the present invention had a higher cell proliferation rate than other animal-free serum-free media.

[0132] (2) Surface marker analysis Surface marker analysis was carried out in the same manner as in Example 2 for the human deciduous tooth-derived dental pulp stem cells from four donors that had been cultured up to passage number 8 as in (1).

[0133] [Table 7]

[0134] [Table 8]

[0135] [Table 9]

[0136] [Table 10]

[0137] (3) Cytokine production Human deciduous tooth-derived dental pulp stem cells from four donors that had been cultured up to passage number 8 as in (1) were evaluated for cytokine production ability in the same manner as in Example 7.

[0138] [Table 11]

[0139] When dental pulp stem cells derived from human deciduous teeth were cultured in the medium of the present invention, it was shown that the production of IL-6, SCF, ANGPTL4, BDNF, VEGF, Angiopoietin-2, β-NGF, and MCP-1 was higher than in other serum-free media.

[0140] The ratios of the amounts of SCF, ANGPTL4, BDNF, VEGF, Angiopoietin-2, and β-NGF produced to the amount of IL-6 produced in the medium of the present invention are shown in Table 12.

[0141] [Table 12]

[0142] Example 24: Surface marker analysis (2) Human deciduous tooth-derived dental pulp stem cells from a single donor were minced and treated with Liberase as in Example 1. The tissue was then divided into two halves during cell collection using a strainer. One halve was seeded in a culture vessel containing 10% hPL / αMEM and the other in 20% FBS / αMEM for isolation and culture. Cell populations for which colonies were confirmed to have detached from the 10% hPL / αMEM culture vessel were expanded in 10% hPL / αMEM, while cell populations for which colonies were confirmed to have detached from the 20% FBS / αMEM culture vessel were expanded in either 10% FBS / αMEM or 20% FBS / αMEM. Surface marker analysis was performed by flow cytometry as in Example 2 at passage 2 for each halve. Table 13 shows the positivity rates of surface markers for the cell population expanded in 10% hPL / αMEM medium ("10% hPL"), the cell population expanded in 10% FBS / αMEM medium ("10% FBS"), and the cell population expanded in 20% FBS / αMEM medium ("20% FBS").

[0143] [Table 13]

[0144] 1x10 after 48 hours of culture in the medium of the present invention 6 The amounts of SCF and ANGPTL4 produced per cell were calculated in the same manner as in Example 7 (Table 14).

[0145] [Table 14]

[0146] Example 25: Effect on severe perinatal hypoxic-ischemic encephalopathy A severe hypoxic-ischemic encephalopathy model was created in neonatal SD rats on the 7th day after birth by ligating and cutting the right common carotid artery under anesthesia and placing them in a hypoxic (8% O2) environment for 105 minutes. A sham-treated group was also created at the same time. At the age of 35 days, the rats were divided into three groups using the rotarod method: a sham-treated group, a vehicle-administered group, and a cell-administered group. At the ages of 5, 7, 9, and 11 weeks (four times at 2-week intervals), the human dental pulp stem cells of the present invention (1 x 10 7 The mice were administered a dose of 1000 mg / kg of the drug via the tail vein. At 13 weeks of age, a cylinder test was performed in a blinded manner. After euthanasia at 14 weeks of age, the whole brains of half of the animals in each group (5 in the sham group, 6 in each of the vehicle-treated and cell-treated groups) were removed and fixed in 4% paraformaldehyde in phosphate buffer. The cerebral tissue specimens were embedded in paraffin and sliced ​​according to standard methods. All animals underwent HE staining and myelin basic protein (MBP) immunostaining. Because most of the cerebrum on the injured side (right side) was missing in almost all animals in both the vehicle-treated and cell-treated groups, image analysis of the uninjured left cerebral tissue was performed in a blinded manner to measure the total length of MBP.

[0147] The results of the cylinder test showed that the cell-administered group ("Cells" in the figure) showed a significantly higher percentage of use of the left forelimb, which is innervated by the affected side of the brain, compared to the vehicle-administered group ("Vehicle" in the figure), demonstrating the improvement of motor dysfunction through cell administration (Figure 22A). To examine the relationship between cell administration and histological changes in cerebral tissue, MBP immunostaining was performed, and nerve fiber length was calculated from the stained image. A significant increase in total nerve fiber length was observed in the cell-administered group compared to the vehicle-administered group (Figure 22B). Based on these findings, it is possible that under these test conditions, these human dental pulp stem cells contribute to the functional recovery of the injured left forelimb by extending (or increasing) nerve fibers in the unaffected side of the brain.

[0148] These results suggest that repeated administration of this drug to patients with severe perinatal hypoxic-ischemic encephalopathy using a clinically feasible route and timing improves motor dysfunction in cerebral palsy, and that nerve elongation on the unaffected side contributes to part of the mechanism of action.

[0149] As described above, compared to other mesenchymal stem cells, human deciduous tooth-derived dental pulp stem cells have a higher ability to produce cytokines necessary for tissue regeneration in the cranial nervous system, such as axonal elongation, synaptic transmission, angiogenesis, and cell migration, while suppressing the induction of inflammatory macrophages and having a high ability to induce anti-inflammatory macrophages, thereby suppressing inflammation. Therefore, human deciduous tooth-derived dental pulp stem cells have the potential to be a safer and more highly functional cell medicine.

[0150] Furthermore, by culturing dental pulp stem cells derived from human deciduous teeth in serum-free medium without FBS in the presence of hPL, it is possible to suppress the production of inflammatory cytokines (IL-6) and produce high levels of useful cytokines such as SCF, ANGPTL4, BDNF, VEGF, Angiopoietin-2, β-NGF, and MCP-1 compared to culturing them in medium containing FBS. [Industrial Applicability]

[0151] The dental pulp stem cells of the present invention have multipotency, neural progenitor cell proliferation activity, neurite extension activity, vascular endothelial cell proliferation activity, vascular endothelial cell attraction activity, vascular structure construction activity, and immunosuppressive activity, and are useful for treating spinal cord injury, ischemic disease, inflammatory disease, neurodegenerative disease, etc.

[0152] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A stem cell population derived from human dental pulp, 90% or more of which are characterized as CD117 negative, CD73 positive, CD90 positive, and CD105 positive; More than 90% of them are CD325 negative or CD51 positive, and The stem cell population, wherein the human dental pulp is human deciduous dental pulp.

2. The stem cell population according to claim 1, wherein the stem cell population has at least one of the following characteristics 1) to 3): 1) Produces stem cell factor (SCF) at a weight ratio of 1 / 10 or more of IL-6, 2) ANGPTL4 (angiopoietin-like 4) is produced at a weight ratio of 5 times or more to IL-6. 3) Produces BIGH3 (beta-induced transforming growth factor) at a weight ratio of more than 450 times that of IL-6.

3. The stem cell population according to claim 1, wherein the stem cell population has at least one of the following characteristics 1) to 3): 1) 1 x 10 6 Produce at least 0.1 ng of SCF (stem cell factor) per cell in 48 hours. 2) 1 x 10 6 Produce at least 500 ng of BIGH3 (beta-inducing transforming growth factor) per cell in 48 hours, 3) 1 x 10 6 Produce at least 5 ng of ANGPTL4 per cell in 48 hours.

4. The stem cell population of claim 1, wherein the stem cell population is obtained by culturing cells enzymatically isolated from human dental pulp in a medium free of FBS (fetal bovine serum) in the presence of human platelet lysate.

5. The stem cell population of claim 4 , wherein the medium is a serum-free medium.

6. A culture supernatant of the stem cell population according to any one of claims 1 to 5, which has at least one of the following properties 1) to 3): 1) Contains stem cell factor (SCF) at a weight ratio of at least 1 / 10 of IL-6, 2) Contains ANGPTL4 (angiopoietin-like 4) at a weight ratio of 5 times or more that of IL-6, 3) Contains BIGH3 (beta-inducible transforming growth factor) at a weight ratio of 450 times or more that of IL-6.

7. A pharmaceutical composition comprising the stem cell population according to any one of claims 1 to 5, wherein the stem cell population has at least one of the following characteristics 1) to 3): 1) Produces stem cell factor (SCF) at a weight ratio of 1 / 10 or more of IL-6, 2) ANGPTL4 (angiopoietin-like 4) is produced at a weight ratio of 5 times or more to IL-6. 3) Produces BIGH3 (beta-induced transforming growth factor) at a weight ratio of more than 450 times that of IL-6.

8. A pharmaceutical composition comprising a culture supernatant of the stem cell population according to any one of claims 1 to 5, wherein the culture supernatant has at least one of the following properties 1) to 3): 1) Contains stem cell factor (SCF) at a weight ratio of at least 1 / 10 of IL-6, 2) Contains ANGPTL4 (angiopoietin-like 4) at a weight ratio of 5 times or more that of IL-6, 3) Contains BIGH3 (beta-inducible transforming growth factor) at a weight ratio of 450 times or more that of IL-6.

9. 9. The pharmaceutical composition according to claim 7 or 8, which is for treating or preventing any disease selected from spinal cord injury, ischemic disease, inflammatory disease, autoimmune disease, and intestinal disease.

10. A method for producing a stem cell population derived from human dental pulp, comprising: The method comprises the steps of enzymatically isolating cells from human dental pulp, culturing the cells in a medium containing no FBS (fetal bovine serum) in the presence of human platelet lysate, and obtaining a colony-forming cell population as the stem cell population, wherein the human dental pulp is human deciduous dental pulp.

11. The method of claim 10, wherein the medium is a serum-free medium.

12. The method of claim 10, wherein the medium is animal-free.

13. The method according to any one of claims 10 to 12, wherein 90% or more of the obtained dental pulp stem cell population is CD117 negative.

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