Novel dental pulp stem cell population

Abstract

The present invention relates to: a stem cell population derived from human deciduous tooth pulp characterized by 90% or more of which being CD117-negative, CD73-positive, CD90-positive, and CD105-positive; a method for producing a stem cell population derived from human deciduous tooth pulp; and the like. The method for producing a stem cell population derived from human deciduous tooth pulp comprises a step for culturing cells isolated from human deciduous tooth pulp in a culture medium that does not contain FBS (fetal bovine serum) in the presence of human platelet lysate (hPL).

Description

Technical field:

[0004] The present invention relates to a stem cell population derived from human deciduous tooth pulp and a culture supernatant thereof, a pharmaceutical composition containing the stem cell population, and a method for producing the pharmaceutical composition. Background Art

[0005] It has been reported that stem cells derived from human dental pulp (dental pulp stem cells) have therapeutic effects on spinal cord injury, cerebral palsy (perinatal hypoxic-ischemic encephalopathy), lower limb ischemia, etc. in experiments using animal models. However, in previous reports, cells were directly applied to the spinal cord immediately after spinal cord injury (non-patent document 1), or in cerebral palsy models, cells were directly applied to the brain several hours after brain injury (non-patent document 2), and these were unrealistic methods of administration considering clinical applications. In other cerebral palsy models, cells were administered intravenously 24 hours after brain injury, and motor function was improved after more than 5 months (non-patent document 3), hoping to find a method of treatment that can be effective faster. For lower limb ischemia, cells were administered to model animals with moderate symptoms immediately after vascular ligation (non-patent document 4) or several hours later (non-patent document 5), which is inconsistent with the actual treatment of severe lower limb ischemia as the main target of stem cell therapy.

[0006] In the past, fetal bovine serum (FBS) was used as a method for isolating dental pulp stem cells from dental pulp tissue (Non-patent Documents 1 to 6). However, the possibility of residual xenogeneic serum components in the administered preparation cannot be completely ruled out, so from the perspective of clinical application, the use of FBS is not preferred. When using cells isolated from the patient's own dental pulp tissue, although the patient's own serum can be used to prepare the cells, in addition to increasing the burden on the patient, there is also the problem of increased time and cost due to product customization. In order to allow more patients to be treated with dental pulp stem cells, it is necessary to produce a large number of homologous cells for treatment.

[0007] For dental pulp stem cells isolated by the previous method using FBS, there are reports on the analysis of their cell characteristics, such as containing more than 50% or 96.2% CD117 (c-kit) positive cells (Patent Document 1, Non-Patent Document 7). There are also previous studies that have isolated CD117 positive cells and reported that their characteristics are multi-differentiation ability (Non-Patent Document 8). Regarding dental pulp stem cells, there are other reports indicating that CD325 (N-cadherin) is positive (Non-Patent Document 9), and for CD51 (integrin αV) and CD49d (integrin α4), there are two situations where they are positive and negative (Non-Patent Documents 10, 11).

[0008] There are also reports indicating that pluripotent stem cells can be obtained by treating human dental pulp tissue with trypsin and then culturing the entire tissue in a serum-free medium containing human platelet lysate (hPL), but the obtained cell population is positive for the hematopoietic stem cell markers CD34 and CD45 and is therefore considered to be a heterogeneous cell population (Non-Patent Literature 12).

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: WO2014 / 141210A2 (JP6491606B)

[0012] Non-patent literature

[0013] Non-patent literature 1: Sakai et al., J Clin Invest. 2012 Jan; 122(1): 80-90. doi: 10.1172 / JCI59251.

[0014] Non-patent document 2: Yamagata et al., Stroke. 2013 Feb; 44(2): 551-4. doi: 10.1161 / STROKEAHA.112.676759.

[0015] Non-patent literature 3: Kitase et al., Stem Cells Dev. 2020 Jan 15; 29(2): 63-74. doi: 10.1089 / scd.2019.0221. PMID: 31801412.

[0016] Non-patent literature 4: Yong et al., Tissue Eng Regen Med. 2022Aug; 19(4): 861-870. doi: 10.1007 / s13770-022-00452-6.

[0017] Non-patent literature 5: Li et al., Stem Cells Int. 2021Aug 31; 2021: 5585255. doi: 10.1155 / 2021 / 5585255.

[0018] Non-patent document 6: Miura et al., Proc Natl Acad Sci US A. 2003 May 13; 100(10): 5807-12. doi: 10.1073 / pnas.0937635100.

[0019] Non-patent document 7: Suchanek et al. Acta Medica (Hradec Kralove). 2010; 53(2): 93-9. doi: 10.14712 / 18059694.2016.66.

[0020] Non-patent literature 8: Carnevale et al., J Tissue Eng Regen Med. 2018 Feb; 12(2): e774-e785. doi: 10.1002 / term.2378.

[0021] Non-patent document 9: Takahashi et al., Arch Oral Biol. 2012 Jan; 57(1): 44-51. doi: 10.1016 / j.archoralbio.2011.07.013.

[0022] Non-patent literature 10: Lee et al., Stem Cell Res Ther. 2020 Jun 3; 11(1): 210. doi: 10.1186 / s13287-020-01714-7.

[0023] Non-patent literature 11: Hata et al., Stem Cell Res Ther. 2015 Sep 7; 6(1): 162. doi: 10.1186 / s13287-015-0156-4.

[0024] Non-patent literature 12: Pilbauerova et al., Biomolecules. 2022Aug 8; 12(8): 1091. doi: 10.3390 / biom12081091. Summary of the invention

[0025] Problem that the invention aims to solve

[0026] The technical problem of the present invention is to provide safe and highly functional stem cells suitable for clinical application and a method for producing the same.

[0027] Solutions for solving problems

[0028] The present 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 those of dental pulp stem cells reported previously. In addition, it has been found that the stem cell population obtained by this method has a higher production of cytokines useful for tissue regeneration than dental pulp stem cells derived from human deciduous teeth obtained by a conventional method using FBS, and can exert excellent tissue regeneration ability.

[0029] The present invention was accomplished based on the above findings, and in a first aspect, provides the following [1] to

[30] .

[0030] [1] A stem cell population, which is a (non-genetically modified) stem cell population derived from human dental pulp, characterized by more than 90% of them being CD117 negative, CD73 positive, CD90 positive and CD105 positive, wherein the human dental pulp is human deciduous tooth pulp.

[0031] [2] The stem cell population according to [1], wherein more than 90% of the stem cell population is CD325 negative or CD51 positive; preferably, more than 90% of the stem cell population is CD325 negative and CD49d positive and / or CD51 positive; more preferably, more than 90%, 95%, 97%, 98% or 99% of the stem cell population is CD325 negative and CD49d positive and / or CD51 positive.

[0032] [3] The stem cell population according to [1] or [2], wherein the stem cell population is CD31 negative.

[0033] [4] The stem cell population according to any one of [1] to [3], wherein the stem cell population is CD34-negative and CD45-negative.

[0034] [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 1 / 20 or more, preferably 1 / 10 or more, of IL-6.

[0035] [6] The 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 one-tenth to one-half, preferably one-tenth to one-third, of IL-6.

[0036] [7] The stem cell population according to any one of [1] to [6], wherein the stem cell population has a growth rate of 1×10 6 Each cell produces at least 0.1 ng, 0.13 ng, 0.15 ng of SCF, preferably 0.17 ng of SCF, and more preferably 0.20 ng of SCF.

[0037] [8] The stem cell population according to any one of [1] to [7], which produces ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 3 times or more, preferably 5 times or more, that of IL-6.

[0038] [9] The stem cell population according to any one of [1] to [8], which produces ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 5 to 20 times that of IL-6, preferably at a weight ratio of 5 to 15 times.

[0039]

[10] The stem cell population according to any one of [1] to [9], wherein the stem cell population has a growth rate of 1×10 6 Each cell produces at least 4.0 ng, 4.5 ng, 5.0 ng of ANGPTL4, preferably 6.9 ng of ANGPTL4, and more preferably 7.0 ng of ANGPTL4.

[0040]

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

[10] , which produces BIGH3 (transforming growth factor-β-induced protein) at a weight ratio of 400 times or more, preferably 450 times or more, to IL-6.

[0041]

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

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

[0042]

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

[12] , wherein the stem cell population has a growth rate of 1×10 6 Each cell produces at least 400 ng, 450 ng, 500 ng of BIGH3, preferably 550 ng of BIGH3, and more preferably 600 ng of BIGH3.

[0043]

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

[13] , wherein the stem cell population is obtained by culturing cells isolated from human dental pulp using an enzyme (e.g., an enzyme containing collagenase, or an enzyme containing collagenase and a neutral protease (dispase or thermolysin)) in a culture medium without FBS (fetal bovine serum) in the presence of human platelet lysate (hPL).

[0044]

[15] The stem cell population according to

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

[0045]

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

[15] , having at least one of the following characteristics 1) to 3),

[0046] 1) containing SCF (stem cell factor) at a weight ratio of 1 / 20 or more, preferably 1 / 10 or more of IL-6;

[0047] 2) containing ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 3 times or more, preferably 5 times or more, that of IL-6;

[0048] 3) BIGH3 (transforming growth factor-β-induced protein) is contained in a weight ratio of 400 times or more, preferably 450 times or more, that of IL-6.

[0049]

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

[15] , having at least one of the following characteristics 1) to 3),

[0050] 1) SCF (stem cell factor) is contained in an amount of one-tenth to one-half, preferably one-tenth to one-third, of IL-6 by weight;

[0051] 2) containing ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 5 to 20 times, preferably 5 to 15 times, that of IL-6;

[0052] 3) BIGH3 (transforming growth factor-β-induced protein) is contained in a weight ratio of 450 to 1500 times, preferably 450 to 1000 times, that of IL-6.

[0053]

[18] The pharmaceutical composition of the following [18-1] or [18-2].

[0054] [18-1] A pharmaceutical composition comprising the stem cell population described in any one of [1] to

[15] , wherein the stem cell population has at least one of the following characteristics 1) to 3),

[0055] 1) producing SCF (stem cell factor) at a weight ratio of 1 / 20 or more, preferably 1 / 10 or more, of IL-6;

[0056] 2) producing ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 3 times or more, preferably 5 times or more, that of IL-6;

[0057] 3) BIGH3 (transforming growth factor-β-induced protein) is produced at a weight ratio of 400 times or more, preferably 450 times or more, that of IL-6.

[0058] [18-2] A pharmaceutical composition comprising a culture supernatant of the stem cell population described in any one of [1] to

[15] , wherein the pharmaceutical composition has at least one of the following characteristics 1) to 3):

[0059] 1) containing SCF (stem cell factor) at a weight ratio of 1 / 20 or more, preferably 1 / 10 or more of IL-6;

[0060] 2) containing ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 3 times or more, preferably 5 times or more, that of IL-6;

[0061] 3) BIGH3 (transforming growth factor-β-induced protein) is contained in a weight ratio of 400 times or more, preferably 450 times or more, that of IL-6.

[0062]

[19] The pharmaceutical composition of the following [19-1] or [19-2].

[0063] [19-1] A pharmaceutical composition comprising the stem cell population described in any one of [1] to

[15] , wherein the stem cell population has at least one of the following characteristics 1) to 3),

[0064] 1) Producing SCF (stem cell factor) at a weight ratio of one-tenth to one-half, preferably one-tenth to one-third of that of IL-6;

[0065] 2) producing ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 5 to 20 times, preferably 5 to 15 times, that of IL-6;

[0066] 3) BIGH3 (transforming growth factor-β-induced protein) is produced at a weight ratio of 450 to 1500 times, more preferably 450 to 1000 times, that of IL-6.

[0067] [19-2] A pharmaceutical composition comprising a culture supernatant of the stem cell population described in any one of [1] to

[15] , wherein the pharmaceutical composition has at least one of the following characteristics 1) to 3),

[0068] 1) SCF (stem cell factor) is contained in an amount of one-tenth to one-half, preferably one-tenth to one-third, of IL-6 by weight;

[0069] 2) containing ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 5 to 20 times, preferably 5 to 15 times, that of IL-6;

[0070] 3) BIGH3 (transforming growth factor-β-induced protein) is contained in a weight ratio of 450 to 1500 times, preferably 450 to 1000 times, that of IL-6.

[0071]

[20] A pharmaceutical composition according to

[18] or

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

[0072]

[21] A method for producing a stem cell population derived from human dental pulp, the method comprising:

[0073] A step of isolating cells from human dental pulp using an enzyme (e.g., an enzyme containing collagenase, an enzyme containing collagenase and a neutral protease (dispase or thermolysin)); a step of culturing the cells in a medium not containing FBS (fetal bovine serum) in the presence of human platelet lysate (hPL); and a step of obtaining a colony-forming cell population as the stem cell population. In the method, the cells may be subcultured (expanded) as needed.

[0074]

[22] The method according to

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

[0075]

[23] The method according to

[21] or

[22] , wherein the culture medium does not contain animal components.

[0076]

[24] The method according to any one of

[21] to

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

[0077]

[25] The method according to any one of

[21] to

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

[13] .

[0078]

[26] The method according to

[21] to

[24] , wherein the human dental pulp is the pulp of a deciduous tooth within 48 hours after tooth extraction.

[0079]

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

[15] , wherein the human dental pulp is the pulp of a deciduous tooth within 48 hours after tooth extraction.

[0080]

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

[15] , which has the ability to differentiate into adipocytes, osteoblasts, chondrocytes and neurons.

[0081]

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

[15] , the culture supernatant according to

[16] or

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

[18] to

[20] has at least one action selected from the group consisting of an action of promoting the proliferation of neural progenitor cells, an action of promoting neurite outgrowth, an action of promoting the proliferation of vascular endothelial cells, an action of chemotaxis of vascular endothelial cells, an action of constructing vascular-like structures, and an action of immunosuppression.

[0082]

[30] In the method according to

[21] to

[23] , the culture medium contains 10% human platelet lysate.

[0083] As used in the above [1] to

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

[0084] The second aspect of the present invention provides the following [1] to

[20] .

[0085] [1] A method for treating a disease requiring tissue regeneration, the method comprising administering to a subject in need thereof a (non-genetically modified) stem cell population derived from human dental pulp and / or the culture supernatant of the stem cell population, the stem cell population being characterized in that more than 90% thereof is negative for CD117, positive for CD73, positive for CD90, and positive for CD105, and the human dental pulp being human deciduous dental pulp.

[0086] [2] The method according to [1], wherein more than 90% of the stem cell population is negative for CD325 or positive for CD51, preferably more than 90% of the stem cell population is negative for CD325, and positive for CD49d and / or CD51, more preferably 90%, 95%, 97%, 98% or more than 99% of the stem cell population is negative for CD325, and positive for CD49d and / or CD51.

[0087] [3] The method according to [1], wherein the stem cell population is negative for CD31.

[0088] [4] The method according to [1], wherein the stem cell population is negative for CD34 and negative for CD45.

[0089] [5] The method according to [1], wherein the stem cell population produces SCF (stem cell factor) at a weight ratio of more than one-tenth of IL-6.

[0090] [6] The method according to [1], wherein the stem cell population produces SCF (stem cell factor) at a weight ratio of one-tenth to one-third of IL-6.

[0091] [7] The method according to [1], wherein the stem cell population produces per 1×10 within 48 hours 6Each cell produced at least 0.1 ng of SCF.

[0092] [8] The method according to [1], wherein the stem cell population produces ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 5 times or more to IL-6.

[0093] [9] The method according to [1], wherein the stem cell population produces ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 5 to 20 times that of IL-6.

[0094]

[10] The method according to [1], wherein the stem cell population is 1×10 6 Each cell produced at least 5 ng of ANGPTL4.

[0095]

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

[0096]

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

[0097]

[13] The method according to [1], wherein the stem cell population is 1×10 6 Each cell produced at least 500 ng of BIGH3.

[0098]

[14] The method according to [1], wherein the stem cell population is obtained by culturing cells isolated using an enzyme from human dental pulp in a medium without FBS (fetal bovine serum) in the presence of human platelet lysate (hPL).

[0099]

[15] The method according to

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

[0100]

[16] The method according to

[14] , wherein the culture medium does not contain animal components.

[0101]

[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.

[0102]

[18] The method according to [1], wherein the culture supernatant of the stem cell population has at least one of the following characteristics 1) to 3),

[0103] 1) containing SCF (stem cell factor) at a weight ratio of one-tenth or more of IL-6;

[0104] 2) containing ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 5 times or more to IL-6;

[0105] 3) BIGH3 (transforming growth factor-β-induced protein) is contained at a weight ratio of 450 times or more that of IL-6.

[0106]

[19] The method according to [1], wherein the culture supernatant of the stem cell population has at least one of the following characteristics 1) to 3),

[0107] 1) SCF (stem cell factor) is included at a weight ratio of one-tenth to one-third of IL-6;

[0108] 2) containing ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 5 to 20 times that of IL-6;

[0109] 3) BIGH3 (transforming growth factor-β-induced protein) is contained at a weight ratio of 450 to 1000 times that of IL-6.

[0110]

[20] The method according to [1], wherein the human dental pulp is the pulp of a deciduous tooth within 48 hours after tooth extraction.

[0111] Effects of the Invention

[0112] According to the present invention, a highly functional and safe dental pulp stem cell population is provided. Unlike conventional methods, the stem cell population of the present invention is prepared using a serum-free medium that does not contain foreign components such as FBS, and a highly homogeneous cell population can be obtained without requiring special separation means. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] FIG. 1 shows the analysis results of surface markers of human dental pulp stem cells of the present invention. Figure 1A : CD31, Figure 1B :CD34

[0114] Figure 2 Microscopic images showing oil red staining of adipocytes (lipid droplets) induced by differentiation of human dental pulp stem cells.

[0115] Figure 3 Microscopic images showing mineralization staining of osteoblasts induced by differentiation of human dental pulp stem cells.

[0116] Figure 4 Microscopic images showing Alcian blue staining of cartilage tissue induced by differentiation of human dental pulp stem cells.

[0117] Figure 5The immunostaining images of neural cells induced by differentiation of human dental pulp stem cells are shown (from left to right, the images are stained with isotype control, anti-nestin antibody, anti-tubulin β3 antibody, and anti-middle molecular weight neurofilament antibody).

[0118] Figure 6 The results show the neurite outgrowth effect induced by the culture supernatant of human dental pulp stem cells.

[0119] Figure 7 The results show that the culture supernatant of human dental pulp stem cells induces the proliferation of neural progenitor cells.

[0120] Figure 8 The results show that the culture supernatant of human dental pulp stem cells induces proliferation of vascular endothelial cells.

[0121] Fig. 9 The results show that the culture supernatant of human dental pulp stem cells induces the formation of blood vessel-like structures (tube formation).

[0122] Fig.10 The figure shows the chemotactic effect of the culture supernatant of human dental pulp stem cells on vascular endothelial cells.

[0123] Fig.11 The immunosuppressive effect (CD4-positive T cell proliferation inhibitory ability) of human dental pulp stem cells was shown.

[0124] FIG. 12 shows the motor function improvement effect (A) and the nerve regeneration effect (B) of human dental pulp stem cells on chronic spinal cord injury in a rat spinal cord injury model.

[0125] Fig.13 The results show that human dental pulp stem cells improve the motor function of perinatal hypoxic-ischemic encephalopathy in the neonatal rat hypoxic-ischemic encephalopathy model.

[0126] Fig.14 The diagram shows the improving effect on thigh blood flow (A) and the improving effect on foot necrosis (B) in the severe hindlimb ischemia model of immunodeficient rats.

[0127] Fig.15 The amounts of BDNF (A) and VEGF (B) produced by human dental pulp stem cells are shown.

[0128] Fig.16 The results show that the culture supernatant of human dental pulp stem cells induces proliferation of vascular endothelial cells.

[0129] Fig.17 The results show that the culture supernatant of human dental pulp stem cells induces the formation of blood vessel-like structures (tube formation).

[0130] Fig.18 The amounts of cytokine production by various stem cells are shown.

[0131] Fig.19 Shows the effect of increasing the vascular area (A) and the effect of increasing the number of blood vessels (B) in a severe hindlimb ischemia model of immunodeficient rats.

[0132] Fig. 20 Shows the anti-inflammatory effects of various stem cells under M2 induction conditions (A) and M1 induction conditions (B) of THP-1 cells.

[0133] Fig.21 Shows the proliferation rate of human dental pulp stem cells cultured in various serum-free media and serum-free media containing hPL.

[0134] Fig. 22 Shows the effect of human dental pulp stem cells on improving the motor function of severe perinatal hypoxic-ischemic encephalopathy in a severe perinatal hypoxic-ischemic encephalopathy model of neonatal rats. Detailed implementation mode

[0135] 1. Stem cell population derived from human dental pulp

[0136] The present invention relates to a population of stem cells derived from human dental pulp without genetic modification (hereinafter, also referred to as "the stem cell population of the present invention"), at least 90% or more of which are negative for CD117, positive for CD73, positive for CD90, and positive for CD105. The structural or functional characteristics of the stem cell population of the present invention are described as follows.

[0137] 1.1 Source

[0138] The stem cell population of the present invention is a population of stem cells derived from human dental pulp without genetic modification. The "dental pulp" can be the dental pulp of either primary teeth or permanent teeth, but from the perspective of easy acquisition, etc., the dental pulp of primary teeth, wisdom teeth, etc. that have been extracted is preferred. In addition, teeth within 72 hours after tooth extraction are used, and more preferably teeth within 48 hours after tooth extraction. Human primary teeth within 48 hours after tooth extraction are particularly preferred.

[0139] The stem cell population of the present invention can be autologous cells from the dental pulp of the subject to be administered, or allogeneic cells from the dental pulp of others. From the perspective of preparation time, cost, and stability of cell quality, it is preferred that the stem cell population is allogeneic cells.

[0140] 1.2 Markers

[0141] "CD117" is a transmembrane protein that functions as a tyrosine kinase receptor, also known as c-kit. Existing known dental pulp stem cells are CD117 positive, or a heterogeneous population containing CD117 positive cells 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., PLoS ONE July 2012, volume 7, Issue 7, e41774; Lei et al., Stem Cell International, Volume 2021, Article ID 8886854), and the stem cell population of the present invention is characterized by CD117 negativity. In addition, the stem cell population of the present invention is characterized by being positive for mesenchymal stem cell markers CD73, CD90 and CD105.

[0142] "CD325", "CD49d" and "CD51" are cell surface proteins that function as adhesion factors, and are also called N-cadherin (cadherin 2), integrin α4 and integrin αV, respectively. Existing known dental pulp stem cells are CD325 positive, or a heterogeneous population containing CD325 positive cells 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), but the stem cell population of the present invention is preferably CD325 negative. In addition, the currently known dental pulp stem cells are a heterogeneous population containing both positive cells and negative cells of 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), but the stem cell population of the present invention preferably shows a positivity rate of 90%, 95%, 97%, 98% or 99% or more for CD49d and / or CD51.

[0143] In the stem cell population of the present invention, it is preferred that the endothelial cell marker CD31 is negative, and hematopoietic stem cell markers such as CD34 and CD45 are also negative. In addition, CD150, which is a positive marker for mesenchymal stem cells, has a positive rate of 90% or more, preferably a positive rate of 95% or more, and CD14 and CD19, which are negative markers for mesenchymal stem cells, are preferably negative.

[0144] In the stem cell population of the present invention, HLA-DR, HLA-DQ, CD40, CD80, and CD86, which are associated with immunogenicity, are preferably negative.

[0145] In the stem cell population of the present invention, it is preferred that the adhesion factors CD29 (ITGB1), CD44, and CD166 (ALCAM) are positive and CD106 (VCAM) is negative.

[0146] 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.

[0147] It should be noted that, in the present specification, "positive" for a marker protein or marker gene means that when a stem cell population with passage numbers 2 to 8 is measured by a method known in the art (for example, using an isotype control to analyze the detection data obtained by flow cytometry), the positive rate of 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. More preferably, "positive" for a marker protein or marker gene means that the positive rate of the protein or gene in a stem cell population with passage numbers 2 to 8 is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Particularly preferably, a marker protein or marker gene being "positive" means that the positivity of the protein or gene in a stem cell population of passage numbers 2 to 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 positivity 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%, further preferably less than 3%, less than 2% (wherein, when the fluorescence intensity of 1% is set as the boundary value between positive and negative in the isotype control, the negative value is not less than 1%). Here, in the case of adherent culture, cell passage is performed when the confluence reaches 60%.

[0148] The cell population of the present invention is between passage 2 and 8.

[0149] (1) The positive rate of CD117 is preferably less than 3%, more preferably less than 2%;

[0150] (2) The CD73 positivity rate is preferably 98% or more, more preferably 99% or more;

[0151] (3) the CD90 positivity rate is preferably 98% or more, more preferably 99% or more; and

[0152] (4) The CD105 positive rate is preferably 98% or more, more preferably 99% or more.

[0153] In addition, the cell population of the present invention may have, in addition to the above characteristics (1)-(4), any two or more, three or more, or four or more of the following characteristics (5)-(20) at passage number 2 to 8.

[0154] (5) The positive rate of CD325 is preferably less than 7%, more preferably less than 5%

[0155] (6) The positive rates of D49d and / or CD51 are preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more.

[0156] (7) The positive rate of CD31 is preferably less than 3%, more preferably less than 2%

[0157] (8) The positive rate of CD34 is preferably less than 7%, more preferably less than 5%

[0158] (9) The positive rate of CD45 is preferably less than 7%, more preferably less than 5%

[0159] (10) The positive rate of CD150 is preferably 90% or more, more preferably 95% or more

[0160] (11) The positive rate of CD14 is preferably less than 7%, more preferably less than 5%

[0161] (12) The positive rate of CD19 is preferably less than 7%, more preferably less than 5%

[0162] (13) The positive rates of HLA-DR and HLA-DQ are preferably less than 7%, more preferably less than 5% respectively.

[0163] (14) The positive rate of CD40 is preferably less than 7%, more preferably less than 5%

[0164] (15) The positive rate of CD80 is preferably less than 7%, more preferably less than 5%

[0165] (16) The positive rate of CD86 is preferably less than 7%, more preferably less than 5%

[0166] (17) The CD29 positive rate is preferably 96% or more, more preferably 98% or more

[0167] (18) The positive rate of CD44 is preferably 96% or more, more preferably 98% or more

[0168] (19) The positive rate of CD166 is preferably 96% or more, more preferably 98% or more

[0169] (20) The positive rate of CD106 is preferably less than 7%, more preferably less than 5%

[0170] The marker protein can be detected by immunological analysis using antibodies such as ELISA, immunostaining, and flow cytometry. For proteins that are expressed in cells and do not appear on the cell surface, the target protein can be detected by expressing a reporter protein together with the protein and detecting the reporter protein. The marker gene can be detected using nucleic acid amplification methods such as RT-PCR, microarrays, and biochips and / or nucleic acid detection methods.

[0171] 1.3 Cytokine production

[0172] Compared to conventional dental pulp stem cells prepared using a culture medium containing FBS, the stem cell population of the present invention produces a high amount of cytokines useful for tissue regeneration and is more abundant. In particular, compared to conventional dental pulp stem cells, the stem cell population of the present invention is characterized in that the ratio of the amount of cytokines produced useful for tissue regeneration to inflammatory cytokines is high.

[0173] Examples of cytokines useful for tissue regeneration include SCF (stem cell factor), ANGPTL4 (angiopoietin-like protein 4), BIGH3 (transforming growth factor-β-induced protein), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), stromal cell-derived factor 1 (SDF-1), monocyte chemoattractant protein-1 (MCP-1), and angiopoietin-2. Examples of inflammatory cytokines include IL-6 (interleukin-6).

[0174] "SCF" (Stem Cell Factor) is a protein produced and secreted (collectively referred to as "produced" in this specification) in mammalian cells, and is a trophic factor known in cultured cell experiments to promote the proliferation of hematopoietic stem cells. In addition, it has been reported in cultured cell experiments and animal experiments that SCF promotes 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, Kim et al., Cancer Cell. 2006 Apr; 9(4): 287-300). al., Cardiovasc Res. 2011 Oct 1; 92(1): 132-40). In addition to ischemic diseases, these effects are considered to be useful for the treatment of various diseases requiring tissue regeneration, such as bone and cartilage diseases.

[0175] "ANGPTL4" (angiopoietin-like protein 4) is a protein produced and secreted in mammalian cells. It has been reported in cultured cell experiments and animal experiments that it has angiogenic effects (Le Jan et al., Am J Pathol. 2003 May; 162 (5): 1521-8, Hermann et al., Clin Immunol. 2005 April; 115 (1): 93-101, Ma et al., Proc Natl Acad Sci US A. 2010 August 10; 107 (32): 14363-8) and anti-inflammatory effects (Cho et al., JCI Insight. 2019 August 22; 4 (16): e125437). In addition to ischemic diseases, inflammatory diseases and autoimmune diseases, these effects are also useful for the treatment of various diseases that require tissue regeneration.

[0176] BIGH3 (transforming growth factor-β-induced protein) is a protein produced and secreted in mammalian cells, also known as βig-H3, corneal epithelial protein, or TGFBI. In cultured cell experiments and animal experiments, it was reported that BIGH3 has angiogenic effects (Aitkenhead et al., Microvasc Res. 2002 Mar; 63 (2): 159-71) and inhibits the decomposition of bone and cartilage (Ruiz et al., Biomaterials. 2020 Jan; 226: 119544). In addition to ischemic diseases, bone and cartilage diseases, these effects are also considered to be useful for the treatment of various diseases that require tissue regeneration.

[0177] "BDNF" (brain-derived neurotrophic factor) is a protein produced and secreted in mammalian cells and is widely distributed in the brain of mammals. BDNF is a type of neurotrophic factor, which is known to promote the generation and growth of nerve cells and mediate the regulation of synaptic function (Kowianski et al., Cell Mol Neurobiol (2018) 38: 579-593), and is reported to have neuroprotective effects (Lau et al., Cell Reports (2015) 12: 1353-1366, Numakawa et al., Histol Histopathol (2010) 25: 237-258). In addition to neurodegenerative diseases, these effects are also considered to be useful for the treatment of various diseases that require neural tissue regeneration.

[0178] "VEGF" (vascular endothelial growth factor) is a protein produced and secreted in mammalian cells. It is known that VEGF causes angiogenesis by inducing cell division, migration and differentiation of vascular endothelial cells. In addition, it is believed that VEGF is also induced to be expressed in neural tissue that is trapped in a hypoxic state due to spinal cord injury, and participates in angiogenesis and neurogenesis at the site of injury (Long et al., Chinese Journal of Traumatology 18 (2015) 293-295). In addition, it is also believed that angiogenesis at the site of nerve injury in the peripheral nervous system contributes to axon regeneration (Saio et al., Int. J. of Mol. Sci. 2021, 22, 11169). These effects are considered to be useful for the treatment of various diseases such as neurodegenerative diseases and the need for neural tissue regeneration.

[0179] NGF (nerve growth factor) is a protein produced and secreted in mammalian cells. It is known to have a neuroprotective effect in the central nervous system and is 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 are considered to be useful for the treatment of neurodegenerative diseases and various diseases requiring nerve tissue regeneration.

[0180] "MCP-1" (monocyte chemoattractant protein-1) is a protein generated and secreted in mammalian cells, also known as CC motif chemokine 2 (CCL2). MCP-1 is known to have the effect of enhancing monocyte chemotaxis, enhancing the effect of T lymphocyte chemotaxis, inducing the ability of dendritic cell differentiation, and is involved in neurodegenerative diseases (Parkinson's disease, Alzheimer's disease, multiple sclerosis, etc.) (Singh et al., International Immunopharmacology 101 (2021) 107598) such as those caused by chronic inflammation. In addition, it is also reported that MCP-1 improves collateral circulation through arterial generation, thereby restoring blood flow (Ito et al., Circulation Res. (1997), vol.80, Issue 6, 829-837). Based on these findings, MCP-1 is considered to be useful for the treatment of various diseases such as inflammatory diseases, neurodegenerative diseases, angiogenesis, and the need for neural tissue regeneration.

[0181] "Angiopoietin-2" is a protein produced and secreted in mammalian cells, and is known to be involved in the formation of lymphatic vessels in lymphatic endothelial cells and vascular remodeling in tissues (Akwii et al., Cells 2019, 8, 471). Angiopoietin-2 is believed to be useful for the treatment of diseases requiring angiogenesis and tissue regeneration.

[0182] IL-6 is a protein generated and secreted in mammalian cells, and it is known to be a pro-inflammatory cytokine that enhances immune response and tissue inflammation. In addition, IL-6 is a representative factor of a series of pro-inflammatory cytokines called SASP (aging-related secretory phenotype) produced by senescent cells, and is considered to be the 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). Therefore, it is not preferred that cells administered for the purpose of disease treatment produce a large amount of IL-6.

[0183] Specifically, the stem cell population of the present invention can produce SCF (but not exceeding the production amount of IL-6) at a weight ratio of at least 1 / 20 of IL-6, preferably at least 1 / 10 of IL-6, more preferably at a weight ratio of 1 / 10 to 1 / 2 of IL-6, further preferably at a weight ratio of 1 / 10 to 1 / 3 of IL-6, more preferably at least 1 / 5 of IL-6, and further preferably at least 1 / 3 of IL-6. 6 Each cell cultured for 48 hours can produce at least 0.1 ng, 0.13 ng, 0.15 ng of SCF, preferably at least 0.17 ng of SCF, and more preferably at least 0.20 ng of SCF.

[0184] As with conventionally known dental pulp stem cells, when the cells administered to the subject are CD117 positive, the SCF produced by the administered cells is first absorbed by the administered cells themselves through their own or other administered cells' CD117, thereby reducing the efficiency of the action on the endogenous cells of the administered subject that are supposed to act. The stem cell population of the present invention has a high SCF production capacity and is CD117 negative, so when administered to a subject requiring tissue regeneration, it can exert an excellent tissue regeneration effect.

[0185] That is to say, the CD117 positivity rate of the stem cell population of the present invention is preferably less than 3%, more preferably less than 2% between passage numbers 2 and 8; and it is capable of producing SCF at a weight ratio of at least one twentieth of IL-6, preferably at least one tenth, more preferably at least one fifth, and further preferably at least one third of the weight ratio.

[0186] 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, further preferably 5 to 15 times, more preferably at least 10 times, and particularly preferably at least 20 times as much as IL-6 at passages 2 to 8. In addition, 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, more preferably at least 10 times, and particularly preferably at least 20 times as much as IL-6 at passages 2 to 8. 6 The cells can produce at least 4 ng, 4.5 ng, 5.0 ng of ANGPTL4 after culturing for 48 hours, preferably at least 6.9 ng of ANGPTL4, and more preferably at least 7.0 ng of ANGPTL4.

[0187] 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 times to 1500 times, and even more preferably 450 times to 1000 times that of IL-6 between passages 2 to 8. 6 The cells can produce at least 400 ng, 450 ng, 500 ng of BIGH3 after culturing for 48 hours, preferably at least 550 ng of BIGH3, and more preferably at least 600 ng of BIGH3.

[0188] 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.

[0189] The stem cell population of the present invention is a cell population that is capable of producing SCF at a weight ratio of at least one twentieth of IL-6, preferably at least one tenth of IL-6, more preferably at least one fifth of IL-6, and further preferably at least one third of IL-6 (but not exceeding the production amount of IL-6) between passage numbers 2 and 8; capable of producing ANGPTL4 at a weight ratio of at least three times, preferably at least five times, more preferably at least ten times, and further preferably at least twenty times of IL-6; and capable of producing BIGH3 at a weight ratio of at least 400 times, preferably at a weight ratio of 450 times, more preferably at a weight ratio of 450 to 1500 times, and further preferably at a weight ratio of 450 to 1000 times that of IL-6.

[0190] When the stem cell population of the present invention is cultured in a medium containing no other animal-derived components (particularly FBS) and in the presence of human platelet lysate (hPL), it is capable of producing 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 the weight ratio of IL-6 between passage numbers 2 and 8 (but not exceeding the amount of IL-6 produced). In addition, when the stem cell population of the present invention is cultured in a medium containing no other animal-derived components and in the presence of hPL, it is capable of producing SCF at a weight ratio of at least about 3 times, preferably at least about 5 times, and more preferably at least about 7 times, compared to when cultured in a medium containing FBS.

[0191] When the stem cell population of the present invention is cultured in a medium containing no other animal-derived components (particularly FBS) and in the presence of human platelet lysate (hPL), ANGPTL4 can be produced at a weight ratio of at least about 5 times, preferably at least about 10 times, and more preferably at least about 20 times that of IL-6 between passage numbers 2 and 8. In addition, when the stem cell population of the present invention is cultured in a medium containing no other animal-derived components and in the presence of hPL, ANGPTL4 can be produced at a weight ratio of at least about 3 times, preferably at least about 5 times, and more preferably at least about 7 times as much as when cultured in a medium containing FBS.

[0192] When the stem cell population of the present invention is cultured in a medium containing no other animal-derived components (particularly FBS) and in the presence of human platelet lysate (hPL), VEGF can be produced at a weight ratio of at least about 2 times, preferably at least about 5 times, and more preferably at least about 10 times that of IL-6 between passage numbers 2 and 8. In addition, when the stem cell population of the present invention is cultured in a medium containing no other animal-derived components and in the presence of hPL, VEGF can be produced at a weight ratio of at least about 2 times, preferably at least about 3 times, and more preferably at least about 5 times as much as when cultured in a medium containing FBS.

[0193] Furthermore, when the stem cell population of the present invention is cultured in a medium containing no other animal-derived components and in the presence of hPL, the amount of IL-6 produced can be suppressed compared to when cultured in a medium containing FBS.

[0194] The stem cell population of the present invention has high production capacity of SCF, ANGPTL4, and BIGH3 and low production rate of IL-6, and therefore has a low risk of inflammatory response and can safely exert tissue regeneration effects.

[0195] 1.4 Differentiation ability

[0196] The stem cell population of the present invention is multipotent, and as shown in the following examples, it has at least the ability to differentiate into adipocytes, osteoblasts, chondrocytes, and neurons. For differentiation induction into target cells, the stem cell population of the present invention can be cultured in the presence of differentiation induction factors corresponding to the target cells according to the methods disclosed in the examples of this specification or methods 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).

[0197] 1.5 Physiological activity

[0198] The stem cell population of the present invention produces high levels of cytokines useful for tissue regeneration, and has physiological activities (functions) such as neural progenitor cell proliferation, neurite outgrowth, endothelial cell proliferation, endothelial cell chemotaxis, vascular-like structure construction, and immunosuppression in vivo or in vitro.

[0199] 2. Method for producing stem cell population of the present invention

[0200] The stem cell population of the present invention can be produced by isolating cells from human dental pulp using an enzyme, culturing the cells in the presence of human platelet lysate (hPL) in a medium free of FBS (fetal bovine serum), and obtaining a colony-forming cell population as the stem cell population.

[0201] The "dental pulp" used in the present invention may be the pulp of either a deciduous tooth or a permanent tooth, but from the perspective of easy access, the pulp of extracted teeth such as deciduous teeth and wisdom teeth is preferred. In addition, for extracted teeth, it is desirable to use teeth within 72 hours after extraction, more preferably within 48 hours after extraction, and it is particularly preferred to use human deciduous teeth within 48 hours after extraction. As a method for isolating a cell population from the collected dental pulp using an enzyme, for example, a method of isolating cells by treating with an enzyme containing collagenase, preferably collagenase and a neutral protease (such as dispase or thermolysin), and isolating by centrifugation can be cited. When isolating a cell population from the collected dental pulp, it is preferably treated with a reagent that does not contain mammalian and bacterial-derived components.

[0202] The isolated cells are cultured in the presence of human platelet lysate (hPL) in a culture medium that does not contain other animal-derived serum components, especially FBS. "Human platelet lysate (hPL)" refers to a substance obtained by dissolving platelets extracted from human blood using a freeze / thaw cycle, and is rich in various growth factors and cytokines required for cell culture. There is no particular restriction on the amount of human platelet lysate (hPL) added to the culture medium or its content in the culture medium, but in primary culture it is about 5 to 20%, preferably 5 to 15%, more preferably 7 to 15%, and most preferably about 10%. The same applies to subculture.

[0203] It should be noted that, in this specification, "about" means a value that fluctuates by 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference value. Preferably, the term "about" means a range of plus or minus 10%, 5% or 1% relative to a reference value.

[0204] The "culture medium" used in the present invention is not particularly limited as long as it does not contain other unpurified components of animal origin, especially FBS, but it is preferred to use a serum-free culture medium. In this specification, "serum-free culture medium" refers to a culture medium that does not contain unregulated or unpurified serum, and a culture medium mixed with purified blood-derived components and animal tissue-derived factors (growth factors) is also included in the serum-free culture medium as long as it does not conflict with the purpose of the present invention. Examples of serum-free culture media include basal culture media to which serum is not added, such as αMEM medium, DMEM medium, BME medium, EagleMEM medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEMZinc Option medium, IMDM medium, Medium 199 medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, or mixtures thereof; and commercially available serum-free culture media for mammalian cells, such as MesenCult-ACF (STEMCELL Technologies), STEMPROMSC SFM (Thermo Fischer Scientific), UltraCULTURE Serum-free (Lonza), and the like.

[0205] For clinical use, the "culture medium" preferably does not contain components derived from mammals, and particularly preferably does not contain animal components.

[0206] As a method for producing a stem cell population of the present invention, it is preferred to use human deciduous teeth within 48 hours after tooth extraction, and the method includes the following steps: i) a step of isolating a cell population from the collected dental pulp using an enzyme using a reagent that does not contain components derived from mammals or bacteria; ii) a step of primary culturing the isolated cell population in a serum-free medium that does not contain unpurified components from other animal sources (especially FBS) in the presence of human platelet lysate (hPL) to form colonies; and iii) a step of culturing the obtained colonies in a serum-free medium that does not contain unpurified components from other animal sources (especially FBS) in the presence of hPL.

[0207] As long as the purpose of the present invention is not impaired, the "culture medium" may be appropriately supplemented with various nutrient sources required for the maintenance and proliferation of cells and various components required for differentiation induction. For example, as nutrient sources, it may include carbon sources such as glycerol, glucose, fructose, sucrose, lactose, honey, starch, and dextrin; in addition, it may include hydrocarbons such as fatty acids, oils, lecithin, and alcohols; nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonium chloride, urea, and sodium nitrate; inorganic salts such as salt, potassium salt, phosphate, magnesium salt, calcium salt, iron salt, and manganese salt, potassium dihydrogen phosphate, potassium monohydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, sodium molybdate, sodium tungstate, and manganese sulfate, various vitamins, amino acids, and the like.

[0208] 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.

[0209] Cells isolated from dental pulp and the stem cell population of the present invention are cultured by adherence. The container is not particularly limited as long as it is a container for cell culture, and flasks, tissue culture bottles, culture dishes, petri dishes, tissue culture dishes, multi-purpose culture dishes, microplates, microplates, multiplates, multi-well plates, microslides, chamber slides, culture plates, tubes, trays, culture bags, and roller bottles can be used.

[0210] There is no particular limit on the cell seeding density, but it is preferably not too high. For example, cells extracted from the pulp of a deciduous tooth are seeded at 25 to 225 cm 2 The area of ​​​​inoculation is preferably 75 to 150cm 2 At 36℃~38℃, preferably 36.5℃~37.5℃, 1%~25%O 2 1%~15%CO 2 The culture was carried out while replacing the culture medium under the conditions of .

[0211] The primary culture is preferably carried out until colony formation, growth, and detachment are confirmed. Once detachment of the colony is confirmed, the cells are recovered using a filter, etc. The recovered cells are obtained as a highly homogeneous stem cell population without the need for special separation means.

[0212] If necessary, the stem cell population can be further subcultured (expanded culture). Subculture is carried out using the same culture medium as the primary culture. Subculture is performed when the confluence reaches 60% in adherent culture. Typically, primary culture cells are cultured for at least 7 days, preferably for more than 9 days, more preferably for 9 to 15 days, in order to confirm the formation, growth and detachment of colonies, and the subcultured cells are cultured for at least 1 day, preferably for more than 2 days, more preferably for 2 to 3 days. There is no particular limitation on the seeding density of the subcultured cells, but it is preferably not too high. For example, 1000 to 2000 cells / cm2 , preferably 1300 to 1500 cells / cm 2 The density of inoculation.

[0213] For safety reasons, it is best to re-submit endotoxin tests, mycoplasma tests, sterility tests, etc. to the recovered stem cell population and its culture supernatant.

[0214] If necessary, the produced stem cell population can be cryopreserved (for example, stored in a deep freezer at -152°C) until use. For example, cryopreservation is performed by adding an appropriate cryoprotectant to the culture medium used for the above-mentioned cell culture. As a cryoprotectant, dextran, DMSO, a commercially available cryopreservation solution, etc. can be used.

[0215] 3. Culture supernatant of the stem cell population of the present invention

[0216] The culture supernatant of the stem cell population of the present invention is rich in cytokines useful for tissue regeneration, and can be used as a pharmaceutical composition for tissue regeneration.

[0217] Specifically, the culture supernatant of the stem cell population of the present invention has at least one of the following characteristics 1) to 3),

[0218] 1) SCF is contained in a weight ratio of 1 / 20 or more, preferably 1 / 10 or more, more preferably 1 / 10 to 1 / 2, and further preferably 1 / 10 to 1 / 3 of IL-6 (but not exceeding the amount of IL-6 produced);

[0219] 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;

[0220] 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.

[0221] The culture supernatant of the stem cell population of the present invention particularly preferably has at least one of the following characteristics 1') to 3'):

[0222] 1') containing SCF in a weight ratio of 1 / 20 or more, preferably 1 / 10 or more, more preferably 1 / 5 or more, and further preferably 1 / 3 or more of IL-6 (but not exceeding the production amount of IL-6);

[0223] 2') containing ANGPTL4 at a weight ratio of 3 times or more, preferably 5 times or more, more preferably 10 times or more, and even more preferably 20 times or more than IL-6;

[0224] 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.

[0225] The culture supernatant of the present invention can be frozen and stored until clinical use, and can be used to treat various diseases requiring tissue regeneration, as can the pharmaceutical composition described below.

[0226] 4. Pharmaceutical compositions comprising the stem cell population of the present invention

[0227] The stem cell population of the present invention produces a large amount of cytokines such as SCF that are useful for tissue regeneration, has a low pro-inflammatory cytokine production ratio, and is CD117 negative, so it 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 drug together with a culture medium or a culture supernatant as needed.

[0228] The pharmaceutical composition of the present invention has at least one of the following characteristics 1) to 3),

[0229] 1) SCF is contained in a weight ratio of 1 / 20 or more, preferably 1 / 10 or more, more preferably 1 / 10 to 1 / 2, and further preferably 1 / 10 to 1 / 3 of IL-6 (but not exceeding the amount of IL-6 produced);

[0230] 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;

[0231] 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.

[0232] The pharmaceutical composition of the present invention preferably has at least one of the following characteristics 1') to 3'),

[0233] 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;

[0234] 2) ANGPTL4 is contained in a weight ratio of 3 times or more, preferably 5 times or more, more preferably 10 times or more, and even more preferably 20 times or more than IL-6;

[0235] 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.

[0236] The number of stem cells contained in the pharmaceutical composition of the present invention is appropriately determined according to the subject and the target disease. Considering the timing of administration to the subject and the time required for culture, it is practical to use the minimum number of cells that show the effect. Generally, the number of cells contained in the pharmaceutical composition is 1×10 5 More than 1×10 6 More than 1×10 7 More than 1×10 8 More than 1×10 9 The number of administrations is not limited to once, and may be administered twice or more.

[0237] The pharmaceutical composition of the present invention is preferably an oral administration preparation, more preferably an oral systemic administration preparation, particularly an intravenous administration preparation. As dosage forms suitable for oral administration, there can be listed: injections such as solution-type injections, suspension-type injections, emulsion-type injections, injections prepared before use, implants, etc. The preparation for oral administration is in the form of an aqueous or non-aqueous isotonic sterile solution or suspension, appropriately combined with, for example, a pharmacologically acceptable carrier or medium, specifically sterile water, physiological saline, culture medium (particularly a culture medium for the culture of mammalian cells such as RPMI), physiological buffers such as PBS, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, excipients, vehicles, preservatives, adhesives, etc., formulated into a suitable unit administration form.

[0238] The diseases to which the pharmaceutical composition of the present invention can be applied are not particularly limited as long as they are diseases requiring tissue regeneration. For example, spinal cord injury (including traumatic and surgical injuries, 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, congenital megacolon and related syndromes, etc.), and treatment and regeneration of bone defects, etc. can be listed.

[0239] 5. Therapeutic methods using the stem cell population of the present invention or their culture supernatant

[0240] The present invention also provides a treatment method, the method comprising administering the stem cell population of the present invention or its culture supernatant to a subject in need. The treatment object is not particularly limited as long as it is a subject requiring tissue regeneration, and the above-mentioned spinal cord injury, ischemic disease, inflammatory disease, autoimmune disease, intestinal disease, etc. can be listed, and these diseases in the acute phase, subacute phase, or chronic phase can be used as treatment objects. For example, the treatment object that can be used as the treatment method of the present invention includes acute or subacute spinal cord injury, chronic ischemic disease, chronic inflammatory disease, chronic autoimmune disease, chronic intestinal disease, acute or subacute bone defect, chronic bone defect.

[0241] 6. Dental Pulp Stem Cell Bank

[0242] According to the method of the present invention, a dental pulp stem cell bank can be created by preparing a dental pulp-derived stem cell population from multiple donors and cryopreserving the population. As described above, the cryopreservation method of the cells can be performed by methods known in the art.

[0243] Example

[0244] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

[0245] Example 1: Preparation of dental pulp stem cells

[0246] Under a sterile environment, the pulp was scraped from the extracted tooth (human deciduous tooth) within 48 hours after extraction, cut, and added with a final concentration of 0.05 mg / ml release enzyme (MNP-S GMP Grade, Roche), and treated at 37°C with stirring for 15 minutes. The reaction supernatant containing cells was recovered, and 0.05 mg / ml of release enzyme was further added to the remaining pulp tissue, and treated at 37°C with stirring for 15 minutes. Cells were recovered from the treated pulp tissue through a 70mm filter and centrifuged together with the previously recovered supernatant. The recovered cells were inoculated in a culture container (CellBind T75 bottle) using MEMα (Gibco) containing 10% hPL (AventaCell BioMedical) and isolated cultured for more than 9 days until colonies were formed, grown, and detached. The culture medium was changed 2 days after inoculation and 9 days after inoculation. Once colony detachment was confirmed, TrypLE Select (Gibco) was used to recover the cells and the same culture medium was used for expansion culture. Hereinafter, the human dental pulp stem cells prepared by the above method may be referred to as human dental pulp stem cells of the present invention.

[0247] Example 2: Surface marker analysis (1)

[0248] For the human dental pulp stem cells from 8 donors prepared according to Example 1, 1×10 5 Each cell was reacted with antibodies to various surface markers labeled with phycoerythrin (PE) (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), CD106 (#305805), all manufactured by Biolegend), and data were acquired by flow cytometry. For the analysis, a gate was set so that the positive rate of the isotype control antibody was approximately 1%, and the positive rate of each surface marker was calculated based on this.

[0249] Table 1

[0250] Positive rate of surface markers (%)

[0251]

[0252] As a result, for CD117, CD325, and CD45, the positive rates in all donor cells were less than 5%, which were judged to be negative. For CD73, CD90, CD105, CD49d, and CD51, the positive rates in all donor cells were all above 96%, which were judged to be positive. For CD31 and CD34, the positive rates in 7 of 8 donor cells were less than 7%, which were judged to be negative. In one donor cell, CD31 showed a positive rate of 15.49%, and in another donor cell, CD34 showed a positive rate of 19.96% (Figure 1). In addition, for CD29, CD44, and CD166, the positive rates in all donors were all above 99%. For CD106, the positive rates in 7 of 8 donor cells were less than 9%, and the positive rate in 1 donor cell was 10.75%.

[0253] Example 3: Adipocyte differentiation ability

[0254] The human dental pulp stem cells of the present invention obtained in the same manner as in Example 1 were added to 5.76×10 5Cells were seeded in 6-well plates at 100% confluence per well and then replaced with adipogenic differentiation 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 (CosmoBio, #AK09F) and lipid droplet images were observed under a microscope.

[0255] As a result, red lipid droplets were confirmed in the cells (indicated by arrows in the figure), indicating that the human dental pulp stem cells of the present invention have the ability to differentiate into adipocytes ( Figure 2 ).

[0256] Example 4: Osteoblast differentiation ability

[0257] The human dental pulp stem cells of the present invention obtained in the same manner as in Example 1 were added to 5.76×10 5 Cells / 2.5 mL / well were seeded in a collagen-coated 6-well plate and cultured until 100% confluence, then replaced with bone 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 (CosmoBio, #AK21) and the mineralization images were observed under a microscope.

[0258] As a result, a mineralized image stained red was observed (indicated by the arrow in the figure), indicating that the human dental pulp stem cells of the present invention have the ability to differentiate into osteoblasts. Figure 3 ).

[0259] Example 5: Chondrocyte differentiation ability

[0260] The human dental pulp stem cells (donor 1) obtained in Example 1 were cultured at 2×10 5 The cells were inoculated in a low-adsorption 96U bottom plate (Sumitomo Bakelite, SUMS9096U) at 100 cells / 200 μL / well. After confirming the formation of spheroids the next day, the medium for cartilage differentiation (PromoCell, #C-28012) was replaced. After 21 days of differentiation induction, the cells were fixed with 4% paraformaldehyde, embedded in paraffin blocks, and then cut into thin slices. The slices were stained with Alcian blue staining solution, and the cartilage tissue images were observed under a microscope.

[0261] As a result, the cartilage tissue image stained blue was confirmed (indicated by the arrow in the figure), indicating that the human dental pulp stem cells of the present invention have the ability to differentiate into chondrocytes ( Figure 4 ).

[0262] Example 6: Neuronal cell differentiation ability

[0263] The human dental pulp stem cells of the present invention obtained in the same manner as in Example 1 were cultured at 2.8×10 3 Cells / 400 μl / well were seeded on poly-D-lysine / laminin coated 8-well culture slides (Corning). After 24 hours, the medium was replaced with N2 medium (Neurobasal-A + N2 serum-free supplement, human EGF 10 ng / ml, human basic fibroblast growth factor 10 ng / ml). After 21 days of differentiation induction, the cells were fixed with 4% paraformaldehyde, and anti-tubulin β3 antibody (clone: ​​TU-20) (Millipore, #MAB1637), anti-nestin antibody (clone: ​​10C2) (Novus Bio, #NB300-266), and anti-middle molecular weight neurofilament antibody (clone: ​​NN18) (Sigma, #5264) were used as primary antibodies for neural differentiation markers, and Alexa Fluor (registered trademark) 488 anti-mouse IgG (H+L) (Jackson immuno research) was used as secondary antibody for staining and observation was performed under a fluorescence microscope.

[0264] The results showed that cells immunostained with antibodies against any neural differentiation marker were confirmed, indicating that the human dental pulp stem cells of the present invention have the ability to differentiate into neural cells. Figure 5 ).

[0265] Example 7: Cytokine production capacity (1)

[0266] The human dental pulp stem cells of the present invention were subcultured in 10% hPL / MEMα or 10% FBS / MEMα for 7 days and then cultured in the same medium at 5.76×10 5Cells / 2.5 ml / well were inoculated in a 6-well plate. The next day, the culture medium was replaced with 2.5 ml / well of MEMα without hPL, FBS and phenol red. After 48 hours, the culture supernatant was recovered, distributed 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 Multiplex HGF panel (Biolegend, #740180), Human ANGPTL4 assay kit (IBL, #27749), Human beta IG-H3 ELISA (abcam, #ab220651) and Multiplex Neuroinflammatory panel (Biolegend, #740796), and the concentration ratios relative to IL-6 were calculated (Tables 2 and 3). In addition, based on the amount of culture supernatant and the number of cells inoculated, the amount of various factors contained in the cell culture medium was calculated as per 1×10 6 The production of cells was 1.34 wt % (Tables 4 and 5).

[0267] The results showed that the concentration ratio of SCF, ANGPTL4, and BIGH3 to IL-6 and the concentration of 1×10 6 The production of each cell is high.

[0268] Table 2 Concentrations and ratios of SCF, ANGPTL4, BIGH3 and IL-6 produced by human dental pulp stem cells cultured in 10% hPL / MEMα

[0269]

[0270] Table 3

[0271] Concentrations and ratios of SCF, ANGPTL4, BIGH3, and IL-6 produced by human dental pulp stem cells cultured in 10% FBS / MEMα

[0272]

[0273] Table 4

[0274] Amounts of SCF, ANGPTL4, BIGH3, and IL-6 produced by human dental pulp-derived cells cultured in 10% hPL / MEMα

[0275]

[0276] Table 5

[0277] The amounts of SCF, ANGPTL4, BIGH3, and IL-6 produced by cells derived from human dental pulp cultured in 10% FBS / MEMα

[0278]

[0279] Example 8: Neurite outgrowth effect

[0280] The human dental pulp stem cells of the present invention (donor 8) were seeded at a density of 1000 cells / cm² in 10% hPL / MEMα. After culturing for 6 days, the medium was replaced with MEMα. After further culturing for 7 days, the culture supernatant was recovered. The culture supernatant was added to a poly-D-lysine (PDL)-coated culture plate (Corning), thereby coating the culture plate with the components contained in the culture supernatant. IMR-32 cells were seeded on this culture plate and induced to differentiate into nerve cells by adding all-trans retinoic acid on days 1-4 and days 7-8. On day 9, the neurites were stained with Tubulin Tracker 2 Green (Thermo Fisher Scientific), the cell nuclei were stained with Hoechst 33342 (Dojindo), and images were taken, and then the neurite length was measured by image analysis using Autoneurite J TM As a result, compared with the culture plate coated only with PDL, the length of the neurites extended from the IMR-32 cells after induction of nerve differentiation was significantly longer in the culture plate coated with the culture supernatant. That is, it is considered that the extracellular matrix secreted by the human dental pulp stem cells of the present invention promoted neurite outgrowth(

[0281] )). This effect is important for nerve regeneration Figure 6 )

[0282] Example 9: Neural progenitor cell proliferation effect

[0283] Human neural progenitor cells (ENStemA, Millipore) were suspended in ENstem expansion medium containing L-glutamine and FGF (5×10⁴ cells / well in a 96-well plate) 4Cells / 100 μl / well) were plated in Corning BioCoat poly-L-ornithine / laminin coated multiwell plates. The next day, the culture medium was replaced with a culture medium containing ENstem expansion medium containing L-glutamine, to which the dental pulp stem cell culture supernatant recovered in Example 8 or MEMα (control) was added at a ratio of 20% or 40%, and cultured for a further 3 days. CCK-8 was added to the neural progenitor cells, and the number of neural progenitor cells was evaluated by measuring the absorbance at 450 nm after 1 hour.

[0284] As a result, the absorbance of the neural progenitor cells to which MEMα was added decreased in a manner dependent on the amount of MEMα added, while the absorbance of the neural progenitor cells to which the culture supernatant was added increased. That is, it is believed that the trophic factors secreted by the human dental pulp stem cells of the present invention promote the proliferation of neural progenitor cells ( Figure 7 ). This effect is important for nerve regeneration.

[0285] Example 10: Vascular endothelial cell proliferation (1)

[0286] HUVEC (human umbilical vein endothelial cell line, Kurabou) were suspended in Humedia / 2% FBS and seeded in 96-well plates (2.5×10 3 The next day, the culture supernatant of dental pulp stem cells recovered in Example 8 was mixed with MEMα in various ratios, and FBS was further added at a final concentration of 2%. The resulting mixture was added in equal amounts from above the Humania / 2% FBS in the wells and cultured for 3 days. CCK-8 was added to HUVEC, and after reacting at 37°C for 4 hours, OD450 was measured to evaluate the cell number of HUVEC.

[0287] As a result, the absorbance increased in a manner dependent on the ratio of the culture supernatant. That is, it is believed that the trophic factors secreted by the human dental pulp stem cells of the present invention cause the proliferation of vascular endothelial cells ( Figure 8 ). This effect is important for angiogenesis.

[0288] Example 11: Construction of blood vessel-like structures (1)

[0289] HUVEC suspended in the culture supernatant of dental pulp stem cells recovered in Example 8 or in MEMα were cultured at 1×10 5Cells / well were seeded in an extracellular matrix gel (Angiogenesis Assay Kit, abcam) prepared in a 96-well plate and cultured for 20 hours. The wells were photographed in bright field, and the branching interval was measured by image analysis using Angiogenesis Analyzer for ImageJ to examine the effect of tube formation (construction of blood vessel-like structures).

[0290] As a result, the branch spacing was significantly increased by adding the culture supernatant ( Fig. 9 ). That is, it is believed that the trophic factors secreted by the human dental pulp stem cells of the present invention promote tube formation. This effect is important for angiogenesis.

[0291] Example 12: Chemotaxis of vascular endothelial cells

[0292] HUVEC were seeded on the upper layer of the chamber (1×10 5 After that, 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 migrated to the back side of the upper layer (lower layer side) were peeled off using trypsin, and the number of cells was measured in the form of luminescence intensity using CellTiterGlo (Promega) to evaluate the effect of HUVECs being attracted to the lower layer.

[0293] As a result, the luminescence intensity increased significantly by filling the lower layer with the culture supernatant ( Fig.10 ). That is, it is believed that vascular endothelial cells are strongly attracted by chemokines and the like secreted by the human dental pulp stem cells of the present invention. This action is important for angiogenesis.

[0294] Example 13: Immunosuppressive Effects

[0295] Human dental pulp stem cells (1×10 5 After mixing with human PBMC (peripheral blood mononuclear cells, CTL Corporation) labeled with CFSE (stain for live cell staining, Thermo Fisher Scientific), PBMC were stimulated for 6 days by adding anti-CD3 antibody (Biolegend, #300438) and anti-CD28 antibody (Biolegend, #302934) (final concentration of each was 0.1 μg / ml). Since the amount of CFSE in cells that have absorbed CFSE decreases according to the number of divisions, the CFSE in CD4-positive T cells in PBMC whose signals are diluted due to proliferation was analyzed by flow cytometry using a known method. lowThe ratio of was quantified (Killer et al. Stem Cell Research & Therapy (2017) 8: 100) and used as the proliferation rate of CD4-positive T cells to evaluate the immunosuppressive effect of cells derived from dental pulp.

[0296] As a result, the proliferation of CD4-positive T cells stimulated by anti-CD3 antibody and anti-CD28 antibody was significantly suppressed by the human dental pulp stem cells of the present invention ( Fig.11 ). That is, the cells have a strong immunosuppressive effect.

[0297] Example 14: Effect on spinal cord injury

[0298] A spinal cord injury model was prepared by dropping a weight (2.5 mm in 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, Japan CLEA) using a MASCIS Impactor (Rutgers University, USA). After 7, 9, 11, and 13 weeks of model preparation, the solvent or 1×10 human dental pulp stem cells of the present invention were added to the spinal cord. 6 5×10 5 The rats were administered into the spinal cavity to investigate the recovery of motor function and the effect of nerve regeneration on chronic spinal cord injury. Specifically, the motor function of the hind limbs was evaluated over time using the BBB (behavioral score of spinal cord injury) test. 15 weeks after the model was prepared, the rats were perfused and fixed with 4% paraformaldehyde, the spinal cord including the injured part was collected, and coronal frozen sections were prepared using OCT compound. The sections were stained with H&E or fast blue (LFB), and the stained areas were quantified by image analysis using Image J to evaluate nerve regeneration. It should be noted that cyclosporine was administered intraperitoneally at a dosage of 10 mg / kg every other day starting from the day before the first administration.

[0299] As a result, an upward trend in the BBB score was observed 4 weeks after the first cell administration, and a statistically significant increase was observed 2 weeks after the final administration ( Fig. 12A ). In both H&E staining and LFB staining of spinal cord sections, it was confirmed that the administration of cells significantly increased the staining area in the epicenter of injury (Epicenter). Fig. 12B Based on the above, it is believed that in the chronic stage of spinal cord injury, the human dental pulp stem cells of the present invention can be administered in a clinically feasible way and at a suitable time to regenerate nerve parenchyma and myelin sheath and restore motor function.

[0300] Example 15: Effect on perinatal hypoxic-ischemic encephalopathy

[0301] The left common carotid artery of 7-day-old Wistar / ST rats was ligated under anesthesia for 1-2 hours, and then placed in hypoxia (8% O 2 ) environment for 1 hour to prepare the neonatal rat hypoxic-ischemic encephalopathy model. The next day, the solvent or 1×10 human dental pulp stem cells of the present invention were intravenously administered 5 Specifically, the rotarod test was performed 41 days after administration to measure the latency to fall from the rod, thereby evaluating the motor coordination and endurance of the limbs.

[0302] As a result, compared with the sham treatment group (normal group), the fall latency of the solvent administration group was significantly shortened, that is, motor function was significantly impaired, while the fall latency of the cell administration group was almost not shortened, and motor function was confirmed to be recovered ( Fig.13 ). It is therefore believed that by administering the human dental pulp stem cells of the present invention to perinatal hypoxic-ischemic encephalopathy in a clinically feasible manner and at a time, the symptoms of cerebral palsy can be restored at a relatively early stage.

[0303] Example 16: Effect on severe lower limb ischemia

[0304] The right iliac artery and femoral artery and vein of immunodeficient rats (F344 / NJcL / -rnu, Japan CLEA) were ligated and isolated to prepare a severe lower limb ischemia model. The next day, the rats were divided into groups based on the degree of blood flow reduction compared with normal limbs, and then human dental pulp stem cells (2×10 6 The effect of severe lower limb ischemia was investigated by administering 100 cells / mouse) to ischemic lower limb muscles. The blood flow improvement effect brought about by angiogenesis was evaluated by comparing the blood flow with that of normal limbs. On the 7th day (4 cases in each group) and 14th day (6 cases in each group) after model preparation, the presence or absence of necrosis in the heel of each individual was visually observed to evaluate the improvement effect on foot necrosis.

[0305] As a result, in the solvent administration group, a continuous decrease in blood flow was observed within 14 days after ischemia, whereas in the cell administration group, a significant recovery of blood flow was observed day by day ( Fig.14 A). In addition, the cell administration group showed a higher rate of avoiding foot necrosis within 14 days, which was statistically significantly different from the solvent administration group ( Fig.14 B) Based on the above, it is believed that by administering the human dental pulp stem cells of the present invention to severe lower limb ischemia in a clinically feasible way and at a time, blood flow can be restored through angiogenesis, thereby avoiding the need for amputation due to foot necrosis.

[0306] Example 17: Cytokine production capacity (2)

[0307] Using LEGENDplex TMHuman Neuroinflammation panel 1 (biolegend, Cat: 740795) measures the BDNF production and VEGF production in the culture supernatant obtained by subculturing the human dental pulp stem cells of the present invention in 10% hPL / MEMα or 10% FBS / MEMα in the same manner as in Example 7 and then culturing in basal medium.

[0308] The results showed that the concentration ratio of IL-6 in the culture supernatant after subculture in 10% hPL / MEMα and the concentration of IL-6 per 1×10 6 The production of BDNF and VEGF in each cell was high (Table 6) Fig.15 ).

[0309] Table 6

[0310]

[0311] Example 18: Vascular endothelial cell proliferation (2)

[0312] The culture supernatant obtained by culturing the human dental pulp stem cells of the present invention using 10% hPL / MEMα or 10% FBS / MEMα in the same manner as in Example 10 was mixed with MEMα at various ratios, and FBS was further added to obtain a mixture at a final concentration of 2%, and the mixture was added in equal amounts from above the Humana / 2% FBS in a well different from the well to which the hPL culture supernatant was added. After adding each culture supernatant and culturing for 3 days, CCK-8 was added to the obtained HUVECs, and after reacting at 37°C for 4 hours, the OD450 was measured to evaluate the cell number of HUVECs.

[0313] As a result, the proliferation effect shown by HUVECs supplemented with the culture supernatant obtained by culturing with 10% FBS / MEMα ("FBS") was not observed as that by HUVECs supplemented with the culture supernatant obtained by culturing with 10% hPL / MEMα ("hPL"). Fig.16 ).

[0314] Example 19: Construction of blood vessel-like structures (2)

[0315] The tube formation effect was examined using HUVECs suspended in the culture supernatant obtained by culturing the human dental pulp stem cells of the present invention with 10% FBS / MEMα in the same manner as in Example 11 and HUVECs suspended in the culture supernatant of the dental pulp stem cells collected in Example 8.

[0316] The results showed that the dental pulp stem cell culture supernatant ("10% hPL / MEM") recovered in Example 8 promoted tube formation more than the dental pulp stem cell culture supernatant ("10% FBS / MEM") obtained by culturing with FBS. Fig.17 ).

[0317] Example 20: Comparison of cytokine production capacity in various stem cells

[0318] Dental pulp stem cells derived from human deciduous teeth (SHED, human dental pulp stem cells of the present invention), stem cells derived from bone marrow (BMMSC), and stem cells derived from adipose tissue (ATMSC) were seeded in a 6-well plate at 5.76×10^5 cells / well, and the medium was replaced with Minimum Essential Medium α (MEMα) 24 hours after seeding. Then, the production amounts of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), angiopoietin-like protein 4 (ANGPTL4), vascular endothelial growth factor (VEGF), stromal cell-derived factor 1 (SDF-1), and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatant recovered after 48 hours were compared ( Fig.18 , each graph in the figure shows the results of cells derived from different donors, and the bars represent the average value).

[0319] The results showed that dental pulp stem cells derived from human deciduous teeth had a higher ability to produce these humoral factors compared to other stem cells. BDNF and NGF are known to be deeply involved in axon growth, synaptic transmission and neuroprotection in the brain nervous system, ANGPTL4 and VEGF are deeply involved in angiogenesis, and SDF1 and MCP-1 are deeply involved in cell migration as chemokines.

[0320] Example 21: Effect on severe lower limb ischemia (2)

[0321] In the severe lower limb ischemia model prepared in Example 16, the angiogenesis in the dental pulp stem cell administration group and the solvent administration group was quantitatively evaluated using an automated macro in Image J using tissue staining images using an antibody against the smooth muscle cell marker αSMA (ACTA2).

[0322] The results showed that in the ischemic lower limb muscle, the vessel area ("vessel area") and vessel number ("vessel number") of the cell administration group increased compared with the solvent administration group ( Fig.19 ). This suggests that dental pulp stem cells can promote angiogenesis in severe ischemic areas.

[0323] Example 22: Inflammation inhibition effect

[0324] Human monocytic leukemia cell line (THP-1) was stimulated with 10 ng / ml of phorbol 12-myristate 13-acetate (DMA) in RPMI1640 (Gibco) medium containing 10% FBS and 1× penicillin-streptomycin (10% FBS / RPMI1640 medium) and then 5×10 5 5×10 dental pulp stem cells (SHED) derived from human deciduous teeth, mesenchymal stem cells (BMMSC) derived from bone marrow, mesenchymal stem cells (ATMSC) derived from adipose tissue, or dental pulp stem cells (DPSC) derived from permanent teeth were placed on the plates at 5×10 4 The cells were added to a small chamber (Falcon culture insert, 0.4μm (HD) PET translucent membrane for 24 wells) with a grid of 0.4μm pore size (the culture medium was 10% FBS / RPMI1640 culture medium), and a culture insert (Cont) was placed on it in a well as a control in which only 10% FBS / RPMI1640 culture medium was added. Then, IL-4 20ng / ml (M2 macrophage induction condition) or LPS1μg / ml, IFN-γ20ng / ml (M1 macrophage induction condition) was added. After culturing for 24 hours, only THP-1 cells were recovered from each well and RNA was extracted, and the mRNA levels of transglutaminase 2 (TGM2) and IL-12B were measured by RT-qPCR.

[0325] The results showed that co-culture with dental pulp stem cells derived from human deciduous teeth significantly promoted TGM2 transcription in THP-1 cells after M2 macrophage induction stimulation compared with co-culture with other stem cells ( Fig. 20 A, each graph in the figure shows the results of cells from different donors, and the bars represent the average value). This suggests that dental pulp stem cells derived from human deciduous teeth can promote polarization into M2 macrophages that have anti-inflammatory effects on THP-1 cells more effectively than other stem cells. In addition, co-culture with dental pulp stem cells derived from human deciduous teeth inhibited the transcription of the proinflammatory cytokine IL-12B in THP-1 cells after M1 macrophage induction stimulation to the same extent as co-culture with other stem cells ( Fig. 20 B, each graph in the figure shows the results of cells from different donors, and the bars represent the average value.) These results indicate that dental pulp stem cells derived from human deciduous teeth have an inhibitory effect on inflammation.

[0326] Example 23: Comparison with serum-free medium without animal components

[0327] (1) Cell proliferation rate

[0328] The dental pulp stem cells derived from human deciduous teeth of 4 donors were cultured in the same manner as in Example 1. However, after cell recovery, they were cultured using a 10% hPL / MEMα medium (the medium of the present invention), MesenCult TM ACF Plus Medium Kit (manufactured by STEMCELL Technologies) (serum-free medium 1), or KBM ADSC-4 (manufactured by KOHJIN BIO) (serum-free medium 2). The seeding density was 1.3 - 2.6×10 3 cells / cm 2 , and inoculation was performed according to the proliferation degree to make the passage time consistent.

[0329] The cell counter (NucleoCounter NC-202, manufactured by ChemoMetec) was used to measure the cell numbers in each medium at passage numbers 4, 7, 11, 14, and 16, and they were plotted as the proliferation rate ( Fig.21 ).

[0330] The results showed that the medium of the present invention had a higher cell proliferation rate than other serum-free media without animal components.

[0331] (2) Surface marker analysis

[0332] For the dental pulp stem cells derived from human deciduous teeth of 4 donors cultured to passage 8 in the manner of (1), surface marker analysis was performed in the same manner as in Example 2.

[0333] Table 7 Donor 9 (Positive rate of surface markers (%))

[0334] Serum-free medium 1 Serum-free medium 2 Culture medium of the present invention CD73 99.93 99.95 99.86 CD105 99.80 99.55 99.41 CD90 99.98 99.92 99.92 CD49d 97.85 99.32 99.67 CD51 99.92 99.90 99.94 CD325 1.18 1.07 1.32 CD117 1.26 0.71 1.29 CD31 2.66 12.21 1.79 CD34 2.53 1.49 0.81 CD45 1.24 1.32 0.87

[0335] Table 8 Donor 10 (Positive rate of surface markers (%))

[0336] Serum-free medium 1 Serum-free medium 2 Culture medium of the present invention CD73 99.95 99.94 99.92 CD105 99.90 98.82 98.82 CD90 99.90 99.97 99.93 CD49d 96.38 98.94 99.90 CD51 99.92 99.91 99.93 CD325 1.34 1.10 0.87 CD11 1.41 0.87 0.80 CD31 8. 5 14.03 2.70 CD34 5.47 1.82 1.31 CD45 1.69 0.65 0.61

[0337] Table 9 Donor 11 (Positive rate of surface markers (%))

[0338] Serum-free medium 1 Serum-free medium 2 Culture medium of the present invention CD73 99.93 99.91 99.93 CD105 99.52 99.59 99.84 CD90 99.97 99.96 99.97 CD49d 78.77 99.49 99.88 CD51 99.94 99.93 99.95 CD325 1.80 1.74 3.09 CD11 7 1.32 1.24 0.58 CD31 10.83 1.18 1.96 CD34 3.77 2.34 3.39 CD45 0.55 0.08 1.39

[0339] Table 10

[0340] Donor 12 (Positive rate of surface markers (%))

[0341] Serum-free medium 1 Serum-free medium 2 Culture medium of the present invention CD73 99.88 99.94 99.96 CD105 99.67 97.96 99.39 CD90 99.86 99.94 99.92 CD49d 59.38 97.94 99.71 CD51 99.97 99.92 99.92 CD325 1.34 0.74 1.81 CD117 1.56 0.94 0.88 CD31 3.13 6.03 2.28 CD34 4.34 1.84 1.02 CD45 1.21 0.63 0.69

[0342] (3) Cytokine production ability

[0343] The cytokine production ability was evaluated in the same manner as in Example 7 for four donors of human deciduous tooth-derived dental pulp stem cells cultured to the eighth passage according to the method (1).

[0344] Table 11

[0345] Cytokine production (pg / ml)

[0346]

[0347] When dental pulp stem cells derived from human deciduous teeth are cultured in the culture medium of the present invention, the production amounts of IL-6, SCF, ANGPTL4, BDNF, VEGF, Angiopoietin-2, β-NGF, and MCP-1 are higher than those in other serum-free culture media.

[0348] Table 12 shows the ratios of the production amounts of SCF, ANGPTL4, BDNF, VEGF, Angiopoietin-2, and p-NGF relative to IL-6 in the culture medium of the present invention.

[0349] Table 12

[0350] The ratio of the production of SCF, ANGPTL4, BDNF, VEGF, Angiopoietin-2, and β-NGF to IL-6

[0351] Donor 9 Donor 10 Donor 11 Donor 12 SCF 0.84 0.17 0.20 0.42 ANGPTL4 134.78 8.84 6.27 46.98 BDNF 2.20 0.34 0.36 1.67 VEGF 41.74 6.07 2.04 13.48 Angiopoietin-2 1.44 0.29 0.41 0.40 β-NGF 0.11 0.05 0.03 0.07

[0352] Example 24: Surface marker analysis (2)

[0353] Dental pulp stem cells derived from human deciduous teeth from a single donor were cut in the same manner as in Example 1, treated with a releasing enzyme, and then divided into two parts at the cell recovery stage using a filter, one of which was inoculated in a culture vessel containing 10% hPL / αMEM as a medium, and the other was inoculated in a culture vessel containing 20% ​​FBS / αMEM as a medium for isolation culture. The cell population in which colonies were confirmed to be detached from the culture vessel containing 10% hPL / αMEM medium was expanded in 10% hPL / αMEM medium, and the cell population in which colonies were confirmed to be detached from the culture vessel containing 20% ​​FBS / αMEM medium was expanded in 10% FBS / αMEM medium or 20% FBS / αMEM medium, and surface marker analysis was performed by flow cytometry in the same manner as in Example 2 at the second passage number. The positive rates of surface markers of 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") are shown in Table 13.

[0354] Table 13

[0355] Positive rate of surface markers (%)

[0356]

[0357] The amount of 1×10 6 The production levels of SCF and ANGPTL4 per cell were measured (Table 14).

[0358] Table 14 SCF and ANGPTL4 produced by human dental pulp-derived cells cultured in 10% hPL / MEMα (ng / dL)

[0359] Donor 9 Donor 10 Donor 11 Donor 12 SCF 0.11 0.13 0.21 0.11 ANGPTL4 17.49 6.68 6.56 11.68

[0360] Example 25: Effect on severe perinatal hypoxic-ischemic encephalopathy

[0361] The right common carotid artery of SD rats at day 7 after birth was ligated and then cut under anesthesia, and the rats were placed in hypoxia (8% O 2 ) environment for 105 minutes to prepare a severe hypoxic-ischemic encephalopathy model of neonatal rats. A sham treatment group was also prepared at the same time. At the 35th day of age, the rats were divided into three groups according to the rotating rod method: a sham treatment group, a solvent administration group, and a cell administration group. At 5 weeks of age, 7 weeks of age, 9 weeks of age, and 11 weeks of age (four times every two weeks), human dental pulp stem cells (1×107 / kg) was administered into the tail vein. At 13 weeks of age, the cylinder test was performed in a blinded manner. After the animals were euthanized at 14 weeks of age, the whole brain was removed from half of each group (5 cases in the sham treatment group, 6 cases in the solvent administration group and the cell administration group), and the tissue was fixed with 4% paraformaldehyde phosphate buffer. The brain tissue specimens were embedded in paraffin and sectioned according to conventional methods, and all samples were stained with HE and immunostained with myelin basic protein (MBP). In the solvent administration group and the cell administration group, most of the brain on the damaged side (right side) of almost all individuals was missing, so the total length of MBP was imaged and analyzed in a blinded manner on the left brain tissue on the undamaged side.

[0362] The results of the cylinder test showed that the use rate of the left forelimb controlled by the damaged side of the brain was significantly higher in the cell administration group ("cell" in the figure) compared with the solvent administration group ("solvent" in the figure), confirming the improvement effect of cell administration on motor dysfunction ( Fig. 22 A). In order to investigate the relationship between cell administration and histological changes in brain tissue, MBP immunostaining was performed and nerve fiber length was calculated from the staining images. It was confirmed that the total nerve fiber length in the cell administration group was significantly increased compared with the solvent administration group ( Fig. 22 B) Therefore, it is believed that under the conditions of this experiment, the human dental pulp stem cells of the present invention may cause the growth (or increase) of nerve fibers in the brain of the uninjured side, thereby contributing to the functional recovery of the left forelimb on the injured side.

[0363] The above indicates that repeated administration of the drug to severe perinatal hypoxic-ischemic encephalopathy using a clinically feasible route and timing of administration can improve the motor dysfunction of cerebral palsy, and part of its mechanism of action is to help nerve growth on the undamaged side.

[0364] As mentioned above, compared with other mesenchymal stem cells, dental pulp stem cells derived from human deciduous teeth have a stronger ability to produce cytokines required for tissue regeneration such as axon growth, synaptic transmission, angiogenesis, and cell migration in the brain nervous system. On the other hand, they inhibit the induction of inflammatory macrophages and have a high ability to induce anti-inflammatory macrophages, so they have an inhibitory effect on inflammation. Therefore, dental pulp stem cells derived from human deciduous teeth can become safer and more functional cell drugs.

[0365] In addition, by culturing dental pulp stem cells derived from human deciduous teeth in a serum-free medium without FBS in the presence of hPL, the production of proinflammatory cytokines (IL-6) can be suppressed compared to culturing in a medium containing FBS, while useful cytokines such as SCF, ANGPTL4, BDNF, VEGF, Angiopoietin-2, β-NGF, MCP-1, etc. can be produced in large quantities.

[0366] Industrial Applicability

[0367] The dental pulp stem cells of the present invention have pluripotency of differentiation, neuroprogenitor cell proliferation effect, neurite growth effect, vascular endothelial cell proliferation effect, vascular endothelial cell chemotaxis effect, vascular-like structure construction effect, and immunosuppressive effect, and are useful for the treatment of spinal cord injury, ischemic diseases, inflammatory diseases, neurodegenerative diseases, etc.

[0368] All publications, patents, and patent applications cited in this specification are incorporated herein by reference.

Claims

1. A stem cell population derived from human dental pulp, characterized by more than 90% of the stem cells being CD117 negative, CD73 positive, CD90 positive, and CD105 positive, wherein the human dental pulp is human deciduous tooth pulp. 2 . The stem cell population according to claim 1 , wherein more than 90% of the stem cell population are CD325 negative or CD51 positive.

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) Produces SCF (stem cell factor) at a weight ratio of more than one-tenth that of IL-6; 2) produces ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 5 times greater than IL-6; 3) Produces BIGH3 (transforming growth factor-β-induced protein) at a weight ratio 450 times greater than that of IL-6.

4. 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) Every 1×10 6 Each cell produces at least 0.1 ng of SCF (stem cell factor); 2) Every 1×10 6 Each cell produces at least 500 ng of BIGH3 (transforming growth factor-β-induced protein); 3) Every 1×10 6 Each cell produced at least 5 ng of ANGPTL4.

5. The stem cell population according to claim 1, which is obtained by culturing cells isolated from human dental pulp using an enzyme in the presence of human platelet lysate in a medium free of FBS (fetal bovine serum).

6. The stem cell population according to claim 5, in, The culture medium is a serum-free culture medium.

7. A culture supernatant of a stem cell population according to any one of claims 1 to 6, having at least one of the following characteristics 1) to 3): 1) containing SCF (stem cell factor) at a weight ratio of one-tenth or more of IL-6; 2) containing ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 5 times or more to IL-6; 3) BIGH3 (transforming growth factor-β-induced protein) is contained at a weight ratio of 450 times or more that of IL-6.

8. A pharmaceutical composition comprising the stem cell population according to any one of claims 1 to 6, wherein the stem cell population has at least one of the following characteristics 1) to 3), 1) Produces SCF (stem cell factor) at a weight ratio of more than one-tenth that of IL-6; 2) produces ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 5 times greater than IL-6; 3) Produces BIGH3 (transforming growth factor-β-induced protein) at a weight ratio 450 times greater than that of IL-6.

9. A pharmaceutical composition comprising the culture supernatant of the stem cell population according to any one of claims 1 to 6, wherein the pharmaceutical composition has at least one of the following characteristics 1) to 3): 1) containing SCF (stem cell factor) at a weight ratio of one-tenth or more of IL-6; 2) containing ANGPTL4 (angiopoietin-like protein 4) at a weight ratio of 5 times or more to IL-6; 3) BIGH3 (transforming growth factor-β-induced protein) is contained at a weight ratio of 450 times or more that of IL-6.

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

11. A method for producing a stem cell population derived from human dental pulp, the method comprising: include: The steps of isolating cells from human dental pulp using enzymes; culturing the cells in a medium free of 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 tooth pulp.

12. The method according to claim 11, in, The culture medium is a serum-free culture medium.

13. The method according to claim 11, in, The culture medium contains no animal components.

14. The method according to any one of claims 11 to 13, in, More than 90% of the dental pulp stem cells obtained were CD117 negative.

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