Agent for treating or preventing cerebral palsy

Abstract

The present invention pertains to an agent that is for treating or preventing cerebral palsy, that contains dental pulp stem cells, and that is to be administered to a subject having a symptom of cerebral palsy.

Description

Preventive or therapeutic agent for cerebral palsy

[0001] Related Applications: This specification includes the contents described in the specifications of Japanese Patent Application No. 2022-159434 (filed October 3, 2022), PCT / JP2022 / 036892 (filed October 3, 2022), and Japanese Patent Application No. 2023-071438 (filed April 25, 2023), which are priority documents of this application. Technical Field: The present invention relates to a preventive or therapeutic agent for cerebral palsy, etc.

[0002] Cerebral palsy is caused by various perinatal brain disorders, including neonatal hypoxic-ischemic encephalopathy (HIE), and neurological symptoms such as motor disorders and epilepsy appear in the chronic stage. It is necessary to develop treatments that are effective even after symptoms of cerebral palsy are confirmed in the chronic stage.

[0003] Non-Patent Document 1 reports that administering mesenchymal stem cells derived from umbilical cord blood into the subarachnoid space of cerebral palsy patients aged 4 to 14 improved the symptoms of cerebral palsy. However, there are still few cases of preserving umbilical cord blood at birth, and it is difficult to use mesenchymal stem cells derived from one's own umbilical cord blood (autologous transplantation), especially in the distant phase. Furthermore, although there are various stem cells other than those derived from umbilical cord blood, stem cells differ greatly in their differentiation ability, secretion factors, etc. depending on their origin, and therefore it is not possible to predict which stem cells will be effective in improving cerebral palsy symptoms.

[0004] Amanat et al. Stem Cell Research & Therapy (2021) 12:439

[0005] An objective of the present invention is to provide an agent for preventing or treating cerebral palsy.

[0006] The present inventors have conducted extensive research in light of the above-mentioned problems and have surprisingly found that dental pulp stem cells have the effect of preventing or treating cerebral palsy and promoting neurogenesis in the brain. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0007] In a first aspect, the present invention relates to the following [1] to

[20] . [1] A preventive or therapeutic agent for cerebral palsy, comprising dental pulp stem cells and administered to a subject with symptoms of cerebral palsy. [2] The preventive or therapeutic agent according to [1], wherein the cerebral palsy is long-term cerebral palsy. [3] The preventive or therapeutic agent according to [2], wherein the long-term cerebral palsy is in infancy or later. [4] The preventive or therapeutic agent according to any of [1] to [3], wherein the preventive or therapeutic agent is administered multiple times (2 to 20 times, preferably 2 to 10 times, more preferably 2 to 5 times). [5] The preventive or therapeutic agent according to any of [1] to [4], wherein the dental pulp stem cells are human deciduous dental pulp stem cells (stem cells derived from human deciduous dental pulp). [6] The preventive or therapeutic agent according to any of [1] to [5], wherein the cerebral palsy is cerebral palsy caused by hypoxia and / or ischemia. [7] The preventive or therapeutic agent according to any of [1] to [6], wherein the cerebral palsy is caused by perinatal brain damage. [8] The preventive or therapeutic agent according to any one of [1] to [7], which is used to improve motor function and / or memory and learning function in a subject with symptoms of cerebral palsy. [9] The preventive or therapeutic agent according to any one of [1] to [8], wherein the subject is a human.

[10] The preventive or therapeutic agent according to any one of [1] to [9], which induces brain neurogenesis in a subject.

[11] The preventive or therapeutic agent according to any one of [1] to

[10] , wherein the dental pulp stem cells consist of a stem cell population derived from human deciduous dental pulp, characterized by 90% or more being CD117-negative, CD73-positive, CD90-positive, and CD105-positive.

[12] The preventive or therapeutic agent according to

[11] , wherein 90% or more of the stem cell population is CD325-negative or CD51-positive.

[13] The prophylactic or therapeutic agent according to

[11] or

[12] , wherein the stem cell population has at least one of the following characteristics 1) to 3): 1) producing SCF (stem cell factor) at a weight ratio of at least 1 / 10 of IL-6, 2) producing ANGPTL4 (angiopoietin-like 4) at a weight ratio of at least 5 times IL-6, 3) producing BIGH3 (beta-inducible transforming growth factor) at a weight ratio of at least 450 times IL-6.

[14] The prophylactic or therapeutic agent according to any of

[11] to

[13] , wherein the stem cell population has at least one of the following characteristics 1) to 3): 1) 1 x 10 62) 1 x 10 cells producing at least 0.1 ng of SCF (stem cell factor) per 48 hours 6 3) 1 x 10 cells producing at least 500 ng of BIGH3 (beta-inducing transforming growth factor) per cell in 48 hours 6 The prophylactic or therapeutic agent according to any one of

[11] to

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

[16] The prophylactic or therapeutic agent according to any one of [1] to

[15] , wherein the dental pulp stem cells are human deciduous dental pulp stem cells that have not been induced to differentiate and / or have not been genetically modified.

[17] The prophylactic or therapeutic agent according to any one of [1] to

[16] , wherein the dental pulp stem cells are human deciduous dental pulp stem cells that have not been induced to differentiate into neurons.

[18] The prophylactic or therapeutic agent according to any one of [1] to

[17] , wherein the dental pulp stem cells are human deciduous dental pulp stem cells that have not been induced to differentiate into neurons.

[19] The prophylactic or therapeutic agent according to any one of [1] to

[18] , wherein the subject is a human who has suffered neonatal asphyxia and / or has been diagnosed with periventricular leukomalacia.

[20] The therapeutic agent according to any one of [1] to

[19] , wherein the subject is a human aged 6 months or older.

[0008] In a second aspect, the present invention relates to the following [1] to

[15] . [1] A composition comprising dental pulp stem cells for use in the prevention or treatment of cerebral palsy, the composition being administered to a subject with symptoms of cerebral palsy. [2] The composition for use according to [1], wherein the cerebral palsy is long-term cerebral palsy. [3] The composition for use according to [2], wherein the long-term cerebral palsy is in infancy or later. [4] The composition for use according to any of [1] to [3], wherein the dental pulp stem cells are administered to the subject multiple times (2 to 20 times, preferably 2 to 10 times, more preferably 2 to 5 times). [5] The composition for use according to any of [1] to [4], wherein the dental pulp stem cells are human deciduous dental pulp stem cells (stem cells derived from human deciduous dental pulp), preferably human deciduous dental pulp stem cells that have not been induced to differentiate (particularly, have not been induced to differentiate into neurons) and / or have not been genetically modified. [6] The composition for use according to any of [1] to [5], wherein the cerebral palsy is cerebral palsy caused by hypoxia and / or ischemia. [7] The composition for use according to any one of [1] to [6], wherein the cerebral palsy is caused by perinatal brain damage. [8] The composition for use according to any one of [1] to [7], wherein the composition is used to improve motor function and / or memory and learning function in a subject with symptoms of cerebral palsy. [9] The composition for use according to any one of [1] to [8], wherein the subject is a human, for example, a human aged 6 months or older, or a human who suffered neonatal asphyxia and / or has been diagnosed with periventricular leukomalacia.

[10] The composition for use according to any one of [1] to [9], wherein the subject is a human, for example, a human aged 6 months or older, or a human who has suffered neonatal asphyxia and / or has been diagnosed with periventricular leukomalacia.

[11] The composition for use according to any one of [1] to

[10] , wherein the dental pulp stem cells consist of a stem cell population derived from human deciduous dental pulp, 90% or more of which are characterized as CD117-negative, CD73-positive, CD90-positive, and CD105-positive.

[12] The composition for use according to

[11] , wherein 90% or more of the stem cell population is CD325 negative or CD51 positive.

[13] The composition for use according to

[11] or

[12] , wherein the stem cell population has at least one of the following characteristics 1) to 3): 1) producing SCF (stem cell factor) at a weight ratio of at least 1 / 10 of IL-6, 2) producing ANGPTL4 (angiopoietin-like 4) at a weight ratio of at least 5 times IL-6, 3) producing BIGH3 (beta-inducible transforming growth factor) at a weight ratio of at least 450 times IL-6.

[14] The composition for use according to any of

[11] to

[13] , wherein the stem cell population has at least one of the following characteristics 1) to 3): 1) 1 x 10 6 2) 1 x 10 cells producing at least 0.1 ng of SCF (stem cell factor) per 48 hours 6 3) 1 x 10 cells producing at least 500 ng of BIGH3 (beta-inducing transforming growth factor) per cell in 48 hours 6 The composition for use according to any one of

[11] to

[14] , wherein the stem cell population is obtained by culturing cells enzymatically isolated from human dental pulp in a medium free of FBS (fetal bovine serum) in the presence of human platelet lysate, and the composition produces at least 5 ng of ANGPTL4 per cell in 48 hours.

[0009] In a third aspect, the present invention relates to the following [1] to

[20] . [1] A method for preventing or treating cerebral palsy, comprising administering dental pulp stem cells to a subject, wherein the subject has symptoms of cerebral palsy. [2] The method described in [1], wherein the cerebral palsy is long-term cerebral palsy. [3] The method described in [2], wherein the long-term cerebral palsy is in infancy or later. [4] The method described in [1], wherein the cerebral palsy is administered multiple times (2 to 20 times, preferably 2 to 10 times, more preferably 2 to 5 times). [5] The method described in [1], wherein the dental pulp stem cells are human deciduous dental pulp stem cells (stem cells derived from human deciduous dental pulp). [6] The method described in [1], wherein the cerebral palsy is caused by hypoxia and / or ischemia. [7] The method described in [1], wherein the cerebral palsy is caused by perinatal brain damage. [8] The method described in [1], wherein the motor function and / or memory and learning function of a subject with symptoms of cerebral palsy is improved. [9] The method according to [1], wherein the subject is a human.

[10] The method according to [1], wherein brain nerves are regenerated in a subject.

[11] The method according to [1], wherein the dental pulp stem cells consist of a stem cell population derived from human deciduous dental pulp, characterized by 90% or more being CD117-negative, CD73-positive, CD90-positive, and CD105-positive.

[12] The method according to

[11] , wherein 90% or more of the stem cell population are CD325-negative or CD51-positive.

[13] The method according to

[11] , wherein the stem cell population has at least one of the following properties 1) to 3): 1) producing SCF (stem cell factor) at a weight ratio of at least 1 / 10 of IL-6, 2) producing ANGPTL4 (angiopoietin-like 4) at a weight ratio of at least 5 times that of IL-6, or 3) producing BIGH3 (beta-inducing transforming growth factor) at a weight ratio of at least 450 times that of IL-6.

[14] The method according to

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

[15] The method according to

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

[16] The method according to [1], wherein the dental pulp stem cells are administered as a single active ingredient.

[17] The method according to [1], wherein the dental pulp stem cells are human deciduous dental pulp stem cells that have not been induced to differentiate and / or have not been genetically modified.

[18] The method according to [1], wherein the dental pulp stem cells are human deciduous dental pulp stem cells that have not been induced to differentiate into neurons.

[19] The method according to [1], wherein the subject is a human who has suffered neonatal asphyxia and / or has been diagnosed with periventricular leukomalacia.

[20] The method according to [1], wherein the subject is a human who is 6 months or older after birth.

[0010] In a fourth aspect, the present invention relates to the following [1] to

[13] . [1] Use of dental pulp stem cells in the manufacture of a preventive or therapeutic agent for cerebral palsy, the preventive or therapeutic agent being for a subject with symptoms of cerebral palsy. [2] The use according to [1], wherein the cerebral palsy is long-term cerebral palsy. [3] The use according to [2], wherein the long-term cerebral palsy is in infancy or later. [4] The use according to any of [1] to [3], wherein the dental pulp stem cells are human deciduous dental pulp stem cells (stem cells derived from human deciduous dental pulp). [5] The use according to any of [1] to [4], wherein the cerebral palsy is cerebral palsy caused by hypoxia and / or ischemia. [6] The use according to any of [1] to [5], wherein the cerebral palsy is caused by perinatal brain damage. [7] The use according to any of [1] to [6], wherein the preventive or therapeutic agent is for improving motor function and / or memory and learning function in a subject with symptoms of cerebral palsy. [8] The use according to any one of [1] to [7], wherein the subject is a human. [9] The use according to any one of [1] to [8], wherein the dental pulp stem cells consist of a stem cell population derived from human deciduous dental pulp, characterized by 90% or more being CD117-negative, CD73-positive, CD90-positive, and CD105-positive.

[10] The use according to [9], wherein 90% or more of the stem cell population are CD325-negative or CD51-positive.

[11] The use according to [9] or

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

[12] The use according to any one of [9] to

[11] , wherein the stem cell population has at least one of the following characteristics 1) to 3): 1) 1 x 10 6 2) 1 x 10 cells producing at least 0.1 ng of SCF (stem cell factor) per 48 hours 6 3) 1 x 10 cells producing at least 500 ng of BIGH3 (beta-inducing transforming growth factor) per cell in 48 hours 6The stem cell population according to any one of [9] to

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

[0011] According to the present invention, it is possible to provide a preventive or therapeutic agent for cerebral palsy that utilizes dental pulp stem cells.

[0012] 1 shows the results of the Horizontal Ladder test when cells were administered once (Test Example 2). 2 shows the results of the Horizontal Ladder test when cells were administered multiple times (Test Example 2). 3 shows a diagram comparing the results of the Horizontal Ladder test (Test Example 2) when cells were administered once and multiple times. 4 shows the results of the Cylinder test when cells were administered multiple times (Test Example 2). 5 shows the results of the Shuttle avoidance test when cells were administered multiple times (Test Example 2). 6 shows the results of body weight measurement (Test Example 3). 7 shows the results of brain weight ratio measurement (Test Example 3). 8 shows the fluorescence intensity ratio of the cells in the brain after administration of deciduous dental pulp stem cells (representing the amount of the cells present). 9 shows the proportion of BrdU / DCX double staining positive cells in the injured area of ​​the hippocampal dentate gyrus after cell administration. 10 shows the number of BrdU positive cells in the injured area of ​​the hippocampal dentate gyrus after cell administration. 11 shows the number of NeuN positive cells in the injured area of ​​the hippocampal dentate gyrus after cell administration. 1 shows the results of a rotarod test in a perinatal hypoxic-ischemic encephalopathy model when cells are administered once (Test Example 7). 1 shows the results of a cylinder test in a severe perinatal hypoxic-ischemic encephalopathy model when cells are administered multiple times (Test Example 8). 1 shows the proliferation rate of neural stem cells when co-cultured with deciduous dental pulp stem cells, bone marrow-derived mesenchymal stem cells, and fibroblasts (Test Example 11). 1 shows the amount of BDNF in the culture supernatant when co-cultured with neural stem cells (Test Example 11).

[0013] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0014] In one aspect, the present invention relates to a preventive or therapeutic agent for cerebral palsy, which contains dental pulp stem cells. In another aspect, the present invention relates to a neurogenesis promoter in the brain of a subject with cerebral palsy symptoms, which contains dental pulp stem cells (also collectively referred to as the "preventive or therapeutic agent of the present invention" in this specification). These will be explained below.

[0015] 1. Dental Pulp Stem Cells 1.1 Origin Dental pulp stem cells are not particularly limited as long as they are obtained from dental pulp (dental pulp-derived). Examples of dental pulp stem cells include deciduous tooth pulp stem cells (dental pulp stem cells derived from deciduous teeth) and permanent tooth pulp stem cells (dental pulp stem cells derived from permanent teeth). Dental pulp stem cells are neural crest-derived stem cells that can be easily collected with minimal invasive procedures and are a cell population that co-express various undifferentiated neural markers and mesenchymal stem cell markers (Miura M. et al.; Proc Natl Acad Sci USA. 2003 May 13;100(10):5807-123). From the perspective of preventing or treating cerebral palsy, deciduous tooth pulp stem cells are preferred, and deciduous tooth pulp stem cells collected from deciduous teeth within 48 hours after tooth extraction or detachment are particularly preferred.

[0016] The organism from which dental pulp stem cells are derived is not particularly limited, and examples include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, deer, etc. Human dental pulp stem cells are preferred, more preferably human deciduous tooth pulp stem cells, and particularly preferably deciduous tooth pulp stem cells obtained from deciduous teeth within 48 hours after shed or extraction.

[0017] In relation to the target (e.g., patient) of the prophylactic or therapeutic agent of the present invention, the dental pulp stem cells are preferably dental pulp stem cells of the same biological species (human-derived if the target is human) in order to suppress or avoid rejection reactions. Furthermore, the dental pulp stem cells can be autologous or allogeneic. "Prevention" includes the suppression or slowing of the onset of symptoms and the decline in physical function, and "treatment" includes the alleviation of symptoms and the improvement of declined physical function. Examples of such symptoms and functions in the case of cerebral palsy include motor function, posture, memory, and learning.

[0018] Dental pulp stem cells can be selected as adhesive cells among dental pulp cells. Adhesive cells among dental pulp cells collected from deciduous or permanent teeth or their subcultured cells can be used as "dental pulp stem cells." For example, dental pulp stem cells can be prepared by the method described below in Japanese Patent Laid-Open No. 2011-219432.

[0019] (1) Pulp collection: After disinfecting naturally fallen baby teeth (or extracted baby teeth or permanent teeth) with chlorhexidine or povidone-iodine solution, the crown is divided and the pulp tissue is collected using a dental reamer.

[0020] (2) Enzyme treatment: The collected dental pulp tissue is suspended in a basal medium (e.g., MEMα) and treated with 0.02-2 mg / ml of a tissue-degrading enzyme (e.g., collagenase, dispase, etc.). After the enzyme treatment, dental pulp cells are collected by centrifugation.

[0021] (3) Cell culture (selection of adherent cells) The cells are resuspended in basal medium and seeded on a dish for adherent cell culture. After culturing in an incubator adjusted to 5% CO2 and 37°C, the cells are washed as necessary, and the adherent cells that have formed colonies are treated with 0.05% trypsin-EDTA for 5 minutes at 37°C. The dental pulp cells detached from the dish are washed as necessary and seeded on a dish for adherent cell culture for expansion. For example, when the cells reach subconfluence (a state in which the cells occupy approximately 70% of the surface of the culture vessel) or confluence as observed with the naked eye, they are detached and collected from the culture vessel and seeded again on a culture vessel filled with culture medium. Subculture may be repeated. For example, subculture is performed 1 to 8 times to obtain the required number of cells (for example, approximately 1 x 10 7 The cells are grown to a density of 1000 cells / ml. Cells can be detached from the culture vessel by conventional methods such as trypsin treatment. After the above culture, the cells may be collected and stored (storage conditions may be, for example, -150°C).

[0022] The culture medium can be a basal medium or a basal medium supplemented with serum or the like. Examples of basal media that can be used include DMEM, Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO, etc.), Ham's F12 Medium (HamF12) (Sigma, GIBCO, etc.), and RPMI 1640 medium. Two or more basal media can also be used in combination. An example of a mixed medium is a medium containing equal parts of IMDM and HamF12 (e.g., commercially available under the trade name IMDM / HamF12 (GIBCO)). Examples of components that can be added to the medium include serum (e.g., fetal bovine serum, human serum, sheep serum), serum substitutes (e.g., knockout serum replacement (KSR)), bovine serum albumin (BSA), antibiotics, various vitamins, and various minerals.

[0023] (4) Cell Recovery Cells can be recovered by detaching them from the culture vessel using trypsin or other methods, followed by centrifugation. Dental pulp stem cells recovered in this manner can be used to prepare the preventive or therapeutic agent of the present invention.

[0024] In one embodiment, the dental pulp stem cells are a population of stem cells derived from non-genetically modified human deciduous dental pulp, at least 90% of which are CD117-negative, CD73-positive, CD90-positive, and CD105-positive (hereinafter also referred to as the "stem cell population of the present invention"). The structural and functional characteristics of the stem cell population of the present invention are described below.

[0025] 1.2 Marker "CD117" is a transmembrane protein that functions as a tyrosine kinase receptor and is also known as c-kit. Conventionally known dental pulp stem cells are either CD117-positive or a heterogeneous population containing CD117-positive and CD117-negative cells (Hilkens et al., Cell and Tissue Research (2013) 353, 65-78; Deng et al., Frontiers in Cell and Developmental Biology, March 2021, volume 9, Article 661116; Ferro et al., PLoS ONE, July 2012, volume 7, Issue 7, e41774; Lei et al., Stem Cell International, Volume 2021, Article ID 8886854). However, the stem cell population of the present invention is characterized as CD117-negative. The stem cell population of the present invention is also characterized by being positive for the mesenchymal stem cell markers CD73, CD90, and CD105.

[0026] "CD325," "CD49d," and "CD51" are cell surface proteins that function as adhesion factors and are also known as N-cadherin (cadherin 2), integrin α4, and integrin αV, respectively. Conventionally known dental pulp stem cells are either CD325-positive or a heterogeneous population containing CD325-positive and CD325-negative cells (Deng et al., Frontiers in Cell and Developmental Biology, March 2021, volume 9, Article 661116; Madhoun et al., Frontiers in Cell and Developmental Biology, October 2021, volume 9, Article 717624). However, the stem cell population of the present invention is preferably CD325-negative. Furthermore, while conventionally known dental pulp stem cells are heterogeneous populations containing both positive and negative cells for CD51 and CD49d (Lei et al., Stem Cell International, Volume 2021, Article ID 8886854; Alvarez et al., International Journal of Oral Science (2015), 7, 205-212), it is preferable that the stem cell population of the present invention exhibits a positivity rate of 90%, 95%, 97%, 98% or 99% or more for CD49d and / or CD51.

[0027] The stem cell population of the present invention is preferably negative for the endothelial cell marker CD31 and negative for hematopoietic stem cell markers such as CD34 and CD45. Furthermore, it is preferable that the positive rate of CD150, a positive marker for mesenchymal stem cells, is 90% or more, preferably 95% or more, and that the negative markers CD14 and CD19 are negative.

[0028] The stem cell population of the present invention is preferably negative for HLA-DR, HLA-DQ, CD40, CD80, and CD86, which are involved in immunogenicity.

[0029] The stem cell population of the present invention is preferably positive for adhesion factors CD29 (ITGB1), CD44, and CD166 (ALCAM), and negative for CD106 (VCAM).

[0030] 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.

[0031] As used herein, a marker protein or marker gene being "positive" means that, when a stem cell population between passages 2 and 8 is measured using a method known in the art (e.g., analyzing detection data by flow cytometry using an isotype control), the positive rate for the protein or gene in the stem cell population is 30% or more, preferably 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, and more preferably 90% or more. Even more preferably, a marker protein or marker gene being "positive" means that the positive rate for the protein or gene in a stem cell population between passages 2 and 8 is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. Particularly preferably, a marker protein or marker gene being "positive" means that the positive rate of the protein or gene in a stem cell population between passages 2 and 8 is 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or 100%. A marker protein or marker gene being "negative" means that, when detected as described above, the positive rate of the protein or gene in a stem cell population is less than 10%, preferably less than 7%, more preferably less than 6%, less than 5%, and even more preferably less than 3% or less than 2% (however, when a fluorescence intensity of 1% in an isotype control is set as the boundary between positive and negative, the negative value is not less than 1%). Here, in the case of adherent culture, cells are passaged when they reach 60% confluency.

[0032] The cell population of the present invention has, between passages 2 and 8, (1) a CD117 positivity rate of preferably less than 3%, more preferably less than 2%; (2) a CD73 positivity rate of preferably 98% or more, more preferably 99% or more; (3) a CD90 positivity rate of preferably 98% or more, more preferably 99% or more; and (4) a CD105 positivity rate of preferably 98% or more, more preferably 99% or more.

[0033] The cell population of the present invention may further have, between passage numbers 2 and 8, any two or more, three or more, or four or more of the following characteristics (5) to (20), in addition to the characteristics (1) to (4) described above: (5) The positive rate of CD325 is preferably less than 7%, more preferably less than 5%. (6) The positive rates of D49d and / or CD51 are each preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. (7) The positive rate of CD31 is preferably less than 3%, more preferably less than 2%. (8) The positive rate of CD34 is preferably less than 7%, more preferably less than 5%. (9) The positive rate of CD45 is preferably less than 7%, more preferably less than 5%. (10) The positive rate of CD150 is preferably 90% or more, more preferably 95% or more. (11) The positive rate of CD14 is preferably less than 7%, more preferably less than 5%. (12) The positive rate of CD19 is preferably less than 7%, more preferably less than 5%. (13) The positive rates of HLA-DR and HLA-DQ are each preferably less than 7%, more preferably less than 5%. (14) The positive rate of CD40 is preferably less than 7%, more preferably less than 5%. (15) The positive rate of CD80 is preferably less than 7%, more preferably less than 5%. (16) The positive rate of CD86 is preferably less than 7%, more preferably less than 5%. (17) The positive rate of CD29 is preferably 96% or more, more preferably 98% or more. (18) The positive rate of CD44 is preferably 96% or more, more preferably 98% or more. (19) The positive rate of CD166 is preferably 96% or more, more preferably 98% or more. (20) The positive rate of CD106 is preferably less than 7%, more preferably less than 5%.

[0034] Marker proteins can be detected by antibody-based immunoassays such as ELISA, immunostaining, and flow cytometry. In the case of proteins that are expressed intracellularly but not on the cell surface, the target protein can be detected by expressing a reporter protein together with the protein and detecting the reporter protein. Marker genes can be detected using nucleic acid amplification and / or nucleic acid detection methods such as RT-PCR, microarrays, and biochips.

[0035] 1.3 Cytokine Production The stem cell population of the present invention produces cytokines useful for tissue regeneration at a higher level and is enriched compared to conventional dental pulp stem cells prepared using a medium containing FBS. In particular, the stem cell population of the present invention is characterized by a higher ratio of cytokines useful for tissue regeneration to inflammatory cytokines compared to conventional dental pulp stem cells.

[0036] Cytokines useful for tissue regeneration include, for example, stem cell factor (SCF), angiopoietin-like 4 (ANGPTL4), beta-induced transforming growth factor (BIGH3), brain-derived trophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), stromal cell-derived factor 1 (SDF-1), monocyte chemotactic protein-1 (MCP-1), and angiopoietin-2. Proinflammatory cytokines include, for example, interleukin-6 (IL-6).

[0037] "SCF" (Stem Cell Factor) is a protein produced and secreted (collectively referred to as "produced" in this specification) by mammalian cells, and is known as a trophic factor that promotes the proliferation of hematopoietic stem cells in cultured cell experiments. SCF also acts on neurons and vascular endothelial cells that express its receptor, CD117, and is known to promote neuroprotection (Dhandapani et al., J Neurochem. 2005 Oct;95(1):9-19), neurogenesis (Jin et al., J Clin Invest. 2002 Aug;110(3):311-9, Osada et al., J Neurosurg Spine. 2010 Oct;13(4):516-23), neurite outgrowth (Hirata et al., Development. 1993 Sep;119(1):49-56), and angiogenesis (Matsui et al., J Biol Chem. 2004 Apr 30;279(18):18600-7, Sun L., Cancer Cell. 2006 Apr;9(4):287-300, Kim et al., Cardiovasc Res. 2011 Oct). 1;92(1):132-40) in cultured cell and animal experiments. These effects are useful for treating various diseases requiring tissue regeneration, including not only neurological and ischemic diseases, but also intestinal and bone diseases.

[0038] ANGPTL4 (Angiopoietin Like 4) is a protein produced and secreted by mammalian cells. It has been reported in cultured cell and animal experiments to have angiogenic effects (Le Jan et al., Am J Pathol. 2003 May;162(5):1521-8; Hermann et al., Clin Immunol. 2005 Apr;115(1):93-101; Ma et al., Proc Natl Acad Sci US A. 2010 Aug 10;107(32):14363-8) and anti-inflammatory effects (Cho et al., JCI Insight. 2019 Aug 22;4(16):e125437). These effects may be useful in treating ischemic, inflammatory, and autoimmune diseases, as well as various other diseases requiring tissue regeneration.

[0039] Transforming growth factor beta-induced (BIGH3) is a protein produced and secreted by mammalian cells. It is also known as βig-H3, keratoepithelin, or TGFBI. Cultured cell and animal experiments have shown that BIGH3 has angiogenic properties (Aitkenhead et al., Microvasc Res. 2002 Mar;63(2):159-71) and inhibits bone and cartilage degradation (Ruiz et al., Biomaterials. 2020 Jan;226:119544). These properties may be useful in treating ischemic diseases, bone and cartilage disorders, and various other diseases requiring tissue regeneration.

[0040] Angiopoietin-2 is a protein produced and secreted by mammalian cells and is known to be involved in lymphatic vessel formation in lymphatic endothelial cells and vascular remodeling within tissues (Akwii et al., Cells 2019, 8, 471). Angiopoietin-2 may be useful in treating diseases requiring angiogenesis and tissue regeneration.

[0041] IL-6 is a protein produced and secreted by mammalian cells and is known as a pro-inflammatory cytokine that enhances immune responses and tissue inflammation. IL-6 is also a representative factor of a group of pro-inflammatory cytokines called senescence-associated secretory phenomena (SASP), which are produced by senescent cells and are thought to be a cause of chronic inflammation associated with aging (Rolt et al., Biogerontology. 2019 Jun;20(3):359-371; Di et al., PLoS One. 2014 Nov 24;9(11):e113572). For this reason, it is undesirable for cells administered for the purpose of disease treatment to produce large amounts of IL-6.

[0042] Specifically, the stem cell population of the present invention can produce SCF at a weight ratio of at least 1 / 20 or more of IL-6, preferably at least 1 / 10 or more, more preferably 1 / 10 to 1 / 2, even more preferably 1 / 10 to 1 / 3, more preferably at least 1 / 5 or more, and even more preferably at least 1 / 3 (however, the amount does not exceed the amount of IL-6 produced). Furthermore, the stem cell population of the present invention can produce SCF at a weight ratio of at least 1 / 20 or more of IL-6, preferably at least 1 / 3 or more, more preferably at least 1 / 4 or more, more preferably at least 1 / 4 or more, and even more preferably at least 1 / 3 or more, between passages 2 and 8. 6 After 48 hours of culture, the cells can produce at least 0.1 ng, 0.13 ng, or 0.15 ng, preferably at least 0.17 ng, and more preferably at least 0.20 ng of SCF.

[0043] When the cells administered to a subject are themselves CD117-positive, as in the case of conventionally known dental pulp stem cells, the SCF produced by the administered cells is first taken up by the administered cells themselves via CD117 on their own or other administered cells, thereby reducing the efficiency of its action on the endogenous cells of the subject on which it is intended to act.The stem cell population of the present invention has a high SCF-producing ability and is CD117-negative, and therefore can exert excellent tissue regenerative effects when administered to a subject in need of tissue regeneration.

[0044] That is, the stem cell population of the present invention preferably has a CD117 positivity rate of less than 3%, more preferably less than 2%, between passages 2 and 8; and is capable of producing SCF at a weight ratio that is at least 1 / 20 or more of that of IL-6, preferably at least 1 / 10 or more, more preferably at least 1 / 5 or more, and even more preferably at least 1 / 3 or more (however, the amount does not exceed the amount of IL-6 produced).

[0045] The stem cell population of the present invention can produce ANGPTL4 at a weight ratio of at least 3 times, preferably at least 5 times, more preferably 5 to 20 times, even more preferably 5 to 15 times, still more preferably at least 10 times, and particularly preferably at least 20 times, more than IL-6, between passages 2 and 8. Furthermore, the stem cell population of the present invention can produce ANGPTL4 at a weight ratio of at least 1 x 10 6 At least 4 ng, 4.5 ng, 5.0 ng, preferably at least 6.9 ng, more preferably at least 7.0 ng of ANGPTL4 can be produced per cell after 48 hours of culture.

[0046] The stem cell population of the present invention can produce BIGH3 at a weight ratio of 400 times or more, preferably 450 times or more, more preferably 450 to 1500 times, and even more preferably 450 to 1000 times, more than IL-6, between passages 2 and 8. Furthermore, the stem cell population of the present invention can produce BIGH3 at a weight ratio of 1 x 10 to IL-6 between passages 2 and 8. 6 At least 400 ng, 450 ng, 500 ng, preferably at least 550 ng, more preferably at least 600 ng of BIGH3 can be produced per cell in 48 hours.

[0047] 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.

[0048] The stem cell population of the present invention is a cell population that, between passages 2 and 8, is capable of producing SCF at a weight ratio of at least 1 / 20 or more of IL-6, preferably at least 1 / 10 or more, more preferably at least 1 / 5 or more, and even more preferably at least 1 / 3 or more (however, not exceeding the amount of IL-6 produced); producing ANGPTL4 at a weight ratio of at least 3 times or more of IL-6, preferably at least 5 times or more, more preferably at least 10 times or more, and even more preferably at least 20 times or more; and producing BIGH3 at a weight ratio of at least 400 times or more of IL-6, preferably at least 450 times or more, more preferably at a weight ratio of 450 to 1500 times, and even more preferably at a weight ratio of 450 to 1000 times.

[0049] When cultured in the presence of human platelet lysate (hPL) in a medium that does not contain other animal-derived components (particularly FBS), the stem cell population of the present invention can produce SCF at a weight ratio that is at least one-tenth or more of IL-6, preferably at least one-fifth or more, more preferably at least one-half or more, and even more preferably at least one-third or more (however, the weight ratio does not exceed that of IL-6) between passages 2 and 8. Furthermore, when cultured in a medium that does not contain other animal-derived components in the presence of hPL, the stem cell population of the present invention can produce SCF at a weight ratio that is at least about three-fold or more, preferably at least about five-fold or more, and even more preferably at least about seven-fold or more compared to when cultured in a medium containing FBS.

[0050] When cultured in the presence of human platelet lysate (hPL) in a medium free of other animal-derived components (particularly FBS), the stem cell population of the present invention can produce ANGPTL4 at a weight ratio of at least about 5-fold or more, preferably at least about 10-fold or more, and more preferably at least about 20-fold or more relative to IL-6, between passages 2 and 8. Furthermore, when cultured in a medium free of other animal-derived components in the presence of hPL, the stem cell population of the present invention can produce ANGPTL4 at a weight ratio of at least about 3-fold or more, preferably at least about 5-fold or more, and more preferably at least about 7-fold or more relative to IL-6, compared to when cultured in a medium containing FBS.

[0051] When cultured in the presence of human platelet lysate (hPL) in a medium that does not contain other animal-derived components (particularly FBS), the stem cell population of the present invention can produce VEGF at a weight ratio of at least about 2-fold or more, preferably at least about 5-fold or more, and more preferably at least about 10-fold or more relative to IL-6, between passages 2 and 8. Furthermore, when cultured in a medium that does not contain other animal-derived components in the presence of hPL, the stem cell population of the present invention can produce VEGF at a weight ratio of at least about 2-fold or more, preferably at least about 3-fold or more, and more preferably at least about 5-fold or more relative to IL-6, compared to when cultured in a medium containing FBS.

[0052] The stem cell population of the present invention can also suppress the amount of IL-6 produced when cultured in the presence of hPL in a medium that does not contain other animal-derived components, compared to when cultured in a medium containing FBS.

[0053] The stem cell population of the present invention has high productivity of SCF, ANGPTL4, and BIGH3, and low IL-6 production rate, and therefore has low risk of inflammatory reactions and can safely exert tissue regeneration effects.

[0054] 1.4 Differentiation Potential The stem cell population of the present invention is multipotent and has the potential to differentiate into at least adipocytes, osteoblasts, chondrocytes, and neurons, as shown in the Examples below. Differentiation into target cells can be induced by culturing the stem cell population of the present invention in the presence of a differentiation-inducing factor appropriate for the target cells, according to methods disclosed in the Examples herein or known in the art (e.g., Marion et al., Methods Enzymol. (2006); 420: 339-361; Wang et al., Molecular Medicine Reports (2016); 14: 5551-5555).

[0055] 1.5 Physiological Activity The stem cell population of the present invention produces high levels of cytokines useful for tissue regeneration, and has physiological activities (functions) in vivo or ex vivo, such as neural progenitor cell proliferation, neurite extension, vascular endothelial cell proliferation, vascular endothelial cell attraction, blood vessel-like structure construction, and immunosuppressive activity.

[0056] 2. Method for Producing the Stem Cell Population of the Present Invention The stem cell population of the present invention can be produced by enzymatically isolating cells from human dental pulp, culturing the cells in a medium free of FBS (fetal bovine serum) in the presence of human platelet lysate (hPL), and obtaining a colony-forming cell population as the stem cell population.

[0057] The "dental pulp" used in the present invention may be the pulp of either a deciduous or permanent tooth; however, for ease of acquisition, the pulp of an extracted tooth, such as a deciduous tooth or a wisdom tooth, is preferred. It is desirable to use an extracted tooth within 72 hours, more preferably within 48 hours, of extraction, and particularly desirable to use a human deciduous tooth within 48 hours of extraction. Enzymatic isolation of a cell population from the collected dental pulp includes, for example, treating the pulp with an enzyme containing collagenase, preferably collagenase and a neutral protease (e.g., dispase or thermolysin), to separate the cells, followed by centrifugation. When isolating a cell population from the collected dental pulp, it is preferable to treat it with a reagent that does not contain mammalian or bacterial components.

[0058] The isolated cells are cultured in the presence of human platelet lysate (hPL) in a medium that does not contain other animal-derived serum components, particularly FBS. "Human platelet lysate (hPL)" is obtained by lysing platelets extracted from human blood using a freeze / thaw cycle and is rich in various growth factors and cytokines necessary for cell culture. The amount of human platelet lysate (hPL) added to the medium or its content in the medium is not particularly limited, but for primary culture, it is approximately 5-20%, preferably 5-15%, more preferably 7-15%, and most preferably about 10%. The same applies for subculture.

[0059] As used herein, the term "about" refers to a value that varies by plus or minus 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1% of the reference value. Preferably, the term "about" refers to a range of plus or minus 10%, 5%, or 1%, of the reference value.

[0060] The "medium" used in the present invention is not particularly limited as long as it does not contain other unpurified animal-derived components, particularly FBS, but it is preferable to use a serum-free medium. As used herein, "serum-free medium" refers to a medium that does not contain unconditioned or unpurified serum, and includes media containing purified blood-derived components or factors (growth factors) derived from animal tissues, as long as they do not contradict the objectives of the present invention. Examples of serum-free media include serum-free basal media such as αMEM medium, DMEM medium, BME medium, Eagle MEM medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc 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, and mixtures thereof; and commercially available serum-free media for mammalian cells such as MesenCult-ACF (STEMCELL Technologies), STEMPRO MSC SFM (Thermo Fischer Scientific), and UltraCULTURE Serum-free (Lonza).

[0061] For clinical use, the "medium" preferably does not contain any mammalian-derived components, and is particularly preferably animal-free.

[0062] A preferred method for producing the stem cell population of the present invention uses a human deciduous tooth extracted within 48 hours, and comprises the steps of: i) enzymatically isolating a cell population from the collected dental pulp using a reagent that does not contain mammalian- or bacterial-derived components; ii) primary culturing the isolated cell population in a serum-free medium that does not contain other animal-derived unpurified components (particularly FBS) in the presence of human platelet lysate (hPL) to form colonies; and iii) culturing the obtained colonies in a serum-free medium that does not contain other animal-derived unpurified components (particularly FBS) in the presence of hPL.

[0063] The "culture medium" may contain various nutrients necessary for cell maintenance and proliferation and various components necessary for differentiation induction, as appropriate, within the scope of the present invention. For example, nutrient sources may include carbon sources such as glycerol, glucose, fructose, sucrose, lactose, honey, starch, and dextrin; hydrocarbons such as fatty acids, oils and fats, lecithin, and alcohols; nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonium chloride, urea, and sodium nitrate; inorganic salts such as table salt, potassium salts, phosphates, magnesium salts, calcium salts, iron salts, and manganese salts; monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, sodium molybdate, sodium tungstate, and manganese sulfate; various vitamins; and amino acids.

[0064] 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.

[0065] Cells isolated from dental pulp and the stem cell population of the present invention are cultured in adherent culture. The container is not particularly limited as long as it is one used for cell culture, and examples of containers that can be used include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles.

[0066] The seeding density of cells is not particularly limited, but it is preferable not to seed them too high. For example, cells extracted from the dental pulp of one baby tooth should be seeded at a density of 25 to 225 cm 2 , preferably 75 to 150 cm 2 The cells are cultured at 36°C to 38°C, preferably 36.5°C to 37.5°C, under conditions of 1% to 25% O2 and 1% to 15% CO2, with medium changes being performed.

[0067] The primary culture is preferably carried out until colonies are formed, grow, and detached. Once detachment of the colonies is confirmed, the cells are collected using a filter or the like. The collected cells are obtained as a highly homogeneous stem cell population without any special separation procedures.

[0068] The stem cell population may be further subcultured (expansion culture) as needed. Subculture is performed using the same medium as the primary culture. Subculture is performed when the cells reach 60% confluency during adherent culture. Typically, primary culture cells are cultured for at least 7 days, preferably 9 days or more, and more preferably 9 to 15 days to confirm colony formation, growth, and detachment, while cells after subculture may be cultured for at least 1 day, preferably 2 days or more, and more preferably 2 to 3 days. The seeding density of cells after subculture is not particularly limited, but it is preferable not to make it too high. For example, 1000 to 2000 cells / cm 2 , preferably 1300 to 1500 cells / cm 2 The seeds are sown at a density of

[0069] For safety reasons, it is desirable to conduct endotoxin tests, mycoplasma tests, sterility tests, etc. again on the collected stem cell population and its culture supernatant.

[0070] The produced stem cell population may be cryopreserved (e.g., stored in a deep freezer at -152°C) until use, if necessary. Cryopreservation can be performed, for example, by adding an appropriate cryoprotectant to the medium used for the cell culture. Examples of cryoprotectants that can be used include dextran, DMSO, and commercially available cryopreservation solutions.

[0071] 3. Prevention or Treatment of Cerebral Palsy Dental pulp stem cells have the effect of preventing or treating cerebral palsy and promoting neurogenesis in the brain. Therefore, dental pulp stem cells can be used as an active ingredient in a preventive or therapeutic agent for cerebral palsy or a neurogenesis promoter. The dental pulp stem cells used in the present invention can be used, for example, as a medicine. Dental pulp stem cells may be used together with other active ingredients, but preferably, dental pulp stem cells are used as the sole active ingredient.

[0072] Preferably, the dental pulp stem cells used in the present invention are cells that have not been induced to differentiate, and particularly preferably cells that have not been induced to differentiate into neurons. Furthermore, dental pulp stem cells can be genetically modified by various known methods, but the dental pulp stem cells used in the present invention are preferably unmodified. Cerebral palsy is a persistent and variable abnormality in movement, posture, or memory and learning in infants after birth, resulting from a non-progressive lesion involving neuronal cell death in the infant's brain that occurs between conception and the neonatal period (within four weeks of birth). However, this does not include progressive diseases, transient motor disorders, or motor developmental delays that are expected to normalize in the future. Although the symptoms and severity of the disorder vary depending on the location of the brain lesion, they are classified as stiffness of the limbs (spasticity), excessive limb movement (athetosis), and decreased muscle tonus (ataxia). Known complications include mental retardation, epilepsy, joint contractures, and scoliosis.

[0073] Cerebral palsy can be caused by brain abnormalities or intrauterine infections before birth, hypoglycemia, intracranial hemorrhage, premature birth, or neonatal asphyxia (HIE) during the perinatal period, or infections such as encephalitis or meningitis during the neonatal period, but is most often caused by perinatal disorders (perinatal brain damage).

[0074] The type of cerebral palsy is not particularly limited. Cerebral palsy caused by various causes can be prevented or treated. Dental pulp stem cells are preferably effective in preventing and treating cerebral palsy symptoms caused by perinatal brain damage, particularly cerebral palsy caused by hypoxia and / or ischemia. This treatment may be provided, for example, at the time of perinatal neonatal asphyxia and / or a diagnosis of periventricular leukomalacia, or in a more distant stage (after six months of age), with the treatment being particularly preferred for more distant cerebral palsy. The preventive or therapeutic agent of the present invention may be used prophylactically in subjects suspected of having cerebral palsy (e.g., subjects who perinatally experienced neonatal asphyxia and / or a diagnosis of periventricular leukomalacia), or to inhibit or ameliorate the progression of symptoms in subjects with cerebral palsy. The preventive or therapeutic agent of the present invention can preferably be used to improve motor function and / or memory / learning function in subjects with cerebral palsy, and / or to promote neurogenesis in the brain of the subject. More preferably, the preventive or therapeutic agent of the present invention can be used to improve motor function and memory and learning function in a subject with cerebral palsy symptoms, and even more preferably, the preventive or therapeutic agent of the present invention can be used to improve motor function and learning function and promote neurogenesis in the brain in a subject with cerebral palsy symptoms.

[0075] Neurogenesis is the generation of new neurons from neural stem cells, and it is known that neurogenesis occurs not only during mammalian development but also in adults in specific regions such as the dentate gyrus and subventricular zone (Gage, J. of Neuroscience, February 1, 2002, 22(3): 612-613). When brain damage occurs due to perinatal injury or other factors, neural stem cells in the subventricular zone and dentate gyrus proliferate, and some of these cells migrate to the injured area of ​​the brain, forming a compensatory mechanism. However, this proliferation and migration of neural stem cells is known to decrease after a certain period of time (approximately one month on average, based on observations in animal models) after injury (Visco et al., Experimental Neurology 340 (2021) 113643). Therefore, brain damage caused by cerebral palsy cannot be recovered solely through the compensatory mechanisms inherent in the body (Visco et al., Experimental Neurology 340 (2021) 113643, Chen et al., Frontiers in Pediatrics (2022) 10:986452).

[0076] In this application, the phrase "promoting neurogenesis" by the prevention or treatment of the present invention means that the proliferation of neural stem cells and / or the increase in neurons in brain tissue (particularly the subventricular zone and dentate gyrus of the hippocampus as described above) in a subject with cerebral palsy is significantly increased compared to when the preventive or therapeutic agent of the present invention is not administered.

[0077] Neurogenesis in the brain can be detected by combining markers such as bromodeoxyuridine (BrdU), doublecortin (DCX), and Ki-67, and NeuN can detect mature neurons in brain tissue.

[0078] The preventive or therapeutic agent of the present invention may be administered to a subject suspected of developing cerebral palsy even if the subject does not exhibit symptoms of cerebral palsy, to a subject with symptoms of cerebral palsy, or to a subject diagnosed with cerebral palsy. Examples of subjects suspected of developing cerebral palsy include subjects who were hypoxic during the perinatal period, subjects who suffered from ischemia, subjects who suffered from neonatal asphyxia, and subjects diagnosed with periventricular leukomalacia. In the present application, a "subject with symptoms of cerebral palsy" refers to a subject who exhibits one or more symptoms of cerebral palsy, and may or may not have been diagnosed with cerebral palsy.

[0079] The preventive or therapeutic agent of the present invention is not particularly limited as long as it contains the above-mentioned active ingredient, and may further contain other ingredients as necessary. The other ingredients are not particularly limited as long as they are pharmaceutically acceptable. Other ingredients include additives as well as ingredients with pharmacological activity. Examples of other ingredients include carriers or vehicles, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, albumin, culture media, antibiotics, pH adjusters, growth factors, hormones, etc. Examples of carriers or vehicles include physiological saline, Ringer's solution, and appropriate buffer solutions (e.g., phosphate buffer solutions).

[0080] The subject to which the prophylactic or therapeutic agent of the present invention is applied is not particularly limited, and examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer.

[0081] The subject of application is preferably a human. In the case of a human, the agent can be administered from infancy onwards (1 year or older, e.g., 2 years or older, 3 years or older, 4 years or older, 5 years or older, or 6 years or older). The subject of application can also be a subject with symptoms of cerebral palsy, for example, a subject diagnosed with symptoms of cerebral palsy. The preventive or therapeutic agent of the present invention can exert therapeutic effects on cerebral palsy when administered to the above subjects.

[0082] The administration routes of the preventive or therapeutic agent of the present invention include, for example, intravenous injection (including drip infusion), intraarterial injection, intraportal vein injection, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, and nasal administration. Local administration may be used instead of systemic administration. An example of local administration is direct injection into brain tissue. When administering the preventive or therapeutic agent of the present invention as an injection, dental pulp stem cells can be administered by suspending them in cell culture medium, physiological saline, complex electrolyte solution, etc. These suspensions may contain serum, albumin, dextran, dimethyl sulfoxide, cryopreservation solution, etc.

[0083] The content of dental pulp stem cells in the preventive or therapeutic agent of the present invention and the administration schedule may be determined taking into consideration the sex, age, weight, pathological condition, etc. of the subject (patient). The content of dental pulp stem cells per administration is, for example, 1 x 10 5 ~1×10 11 The dosage can be 100 mg / kg body weight. In addition to a single administration, multiple administrations may be administered continuously or periodically. The number of administrations is suitably 2 to 20 times (preferably 2 to 10 times, or 2 to 5 times). The administration interval can be selected from 1 day to 4 weeks, and is preferably 7 to 24 days, or 10 to 18 days. Multiple administrations can more significantly improve motor function and / or memory and learning function and / or significantly promote neurogenesis in the brain in subjects with cerebral palsy symptoms.

[0084] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0085] Test Example 1: Preparation of Human Deciduous Dental Pulp Stem Cells. Deciduous dental pulp stem cells were prepared from human exfoliated deciduous teeth. Specifically, the procedure was as follows: Dental pulp was excised from human deciduous teeth, minced, and then Liberase (Roche) was added at a final concentration of 0.05 mg / ml. The mixture was then treated with stirring at 37°C for 15 minutes. The cell-containing supernatant was then collected, and the remaining pulp tissue was treated with Liberase (0.05 mg / ml) at 37°C for 15 minutes. Cells were collected from the treated pulp tissue through a 70 mm strainer and centrifuged with the previously collected supernatant. The collected cells were seeded into culture vessels using MEMα (Gibco) containing 10% hPL and cultured for at least 9 days until colonies formed, grew, and detached. The medium was changed 2 and 9 days after seeding. Once colonies detached, the cells were collected using TrypLE Select (Gibco) and expanded in the same medium.

[0086] Test Example 2. Evaluation of the therapeutic effect of deciduous tooth dental pulp stem cells on cerebral palsy Test Example 2-1. Preparation of a cerebral palsy model rat An HIE model (Rice-Vannucci model) was prepared. Specifically, 7-day-old male Wistar / ST rats were subjected to left common carotid artery ligation and hypoxia loading (8% O2 for 60 minutes). Simultaneously, a sham-operated group of rats was also prepared.

[0087] Test Example 2-2. Grouping by Behavioral Testing The model rats prepared in Test Example 2-1 were subjected to a horizontal ladder test at 25 to 28 days of age. Specifically, the rats were asked to walk up a horizontal ladder, and the gait of the paralyzed leg was scored from 1 to 4 (0: normal gait; 1: the paralyzed leg slipped on the ladder but did not fall; 2: the paralyzed leg fell off the ladder but the next gait was normal; 3: the paralyzed leg fell off the ladder and the next gait was also abnormal; 4: both legs fell off the ladder). The score per step (score / step) was calculated. Only rats showing significantly abnormal findings compared to the sham group were selected. The selected model rats were divided into two groups (cell administration group and vehicle group) so that the scores in the above test were comparable.

[0088] <Test Example 2-3. Cell Administration> For the cell administration group, 1 × 10 6 In a test using another batch, 1 × 10 cells of deciduous dental pulp stem cells (Test Example 1) were administered via the tail vein (single administration). 6 Cells (Test Example 1) were administered via the tail vein (multiple administrations). On the other hand, the vehicle group received only medium containing no stem cells, just like the cell administration group.

[0089] Test Example 2-4: Evaluation Test The horizontal ladder test, cylinder test, and shuttle avoidance test were performed on the cell administration group, vehicle group, and sham group.

[0090] The method of the Horizontal Ladder test was the same as in Test Example 2-2.

[0091] In the cylinder test, a rat was placed inside a cylinder, and the limb used when the rat stood up and placed its forelimbs against the wall (left (healthy side), right (affected side), or both) was recorded to evaluate the rate of limb use on the affected side. The score was calculated using the formula: (LR) / (L+R+B) (L: left side, R: right side, B: both sides). The more severe the injury, the less the right (affected side) limb was used (the higher the score).

[0092] In the shuttle avoidance test, rats were placed in a cage and warned with a light and sound. If they did not avoid the shuttle, they were given an electrical stimulus. This was repeated to evaluate memory and learning ability. For two consecutive days, 10 x 6 sets (60 trials in total) were conducted per day, and the avoidance rate for each set was evaluated.

[0093] The results of the Horizontal Ladder test are shown in Figures 1 to 3. It was found that the administration of deciduous dental pulp stem cells improved motor function as assessed by the Horizontal Ladder test. Furthermore, the improvement tended to be stronger when administered multiple times.

[0094] The results of the cylinder test are shown in Figure 4. It was found that administration of deciduous dental pulp stem cells improved motor function as assessed by the cylinder test. Furthermore, multiple administrations showed a more significant improvement than single administrations.

[0095] The results of shuttle avoidance are shown in Figure 5. It was found that administration of deciduous dental pulp stem cells improved memory and learning function, as assessed by shuttle avoidance. Furthermore, multiple administrations showed a more significant improvement than single administrations.

[0096] Test Example 3 Measurement of Body Weight and Brain Weight Ratio For each group in Test Example 2, the body weight and the brain weight ratio (cerebrum weight / cerebellum+brainstem weight) at the end of the test (110 days old) were measured.

[0097] The results of body weight measurements are shown in Figure 6, and the results of brain weight ratio measurements are shown in Figure 7. The body weight of the Sham group was significantly higher, but no difference was observed between the Cell and Vehicle groups. The brain weight ratio (cerebrum weight / cerebellum + brainstem weight) was significantly lower in the Vehicle group.

[0098] Experimental Example 4: Mechanism Analysis by Comprehensive Evaluation of Target Proteins. Similar to the multiple administrations in Experimental Example 2, deciduous dental pulp stem cells were administered multiple times. Two weeks after the final cell administration, brains were homogenized, and the extracted proteins were comprehensively analyzed by liquid chromatography-mass spectrometry (LC / MS). Of the total proteins analyzed (n = 1696), proteins with detection limits below the detection limit in five or more of six samples in the vehicle and cell administration groups were excluded (n = 204). Sixty-one proteins showed significant differences between the vehicle and cell administration groups (p-value < 0.05 (Student's t-test) FDR < 0.1 (Storey method)). These proteins are thought to be affected by the administration of deciduous dental pulp stem cells, excluding the effects of HIE. GO analysis using DAVID was performed to extract clusters with an enrichment score > 2, revealing enrichment of proteins related to neurogenesis, cell morphogenesis, axonogenesis, and phosphorylation processes.

[0099] Test Example 5: Evaluation of the in vivo dynamics of deciduous dental pulp stem cells using quantum dots. Quantum dot-labeled deciduous dental pulp stem cells (1 x 10 6 Cells (cells / body) were administered via the tail vein of rats. After intravenous cell injection, evaluation was performed at four time points (A: 1 hour after intravenous injection, B: 1 day, C: 2 days, D: 1 week). At each time point, the target organ (brain) was removed and its fluorescence intensity was evaluated using an in vivo imaging system. Based on the fluorescence intensity, the fluorescence intensity ratio (fluorescence intensity of each individual in the cell group / average fluorescence intensity of the vehicle group at each time point) was calculated. The results are shown in Figure 8.

[0100] Test Example 6. Evaluation of neurogenesis using deciduous tooth dental pulp stem cells Test Example 6-1: Neurogenesis markers As in Test Examples 2-1 to 2-3, deciduous tooth dental pulp stem cells were administered multiple times to HIE model (Rice-Vannucci model) rats. Starting the day after the third cell administration, 50 mg / kg of bromodeoxyuridine (BrdU) was administered intraperitoneally once a day for a total of three days.

[0101] Two weeks after the third cell injection, rats were perfused with paraformaldehyde (PFA), and brain tissue was harvested and embedded in paraffin blocks. These blocks were then thinly sliced ​​to prepare brain tissue sections, which were then subjected to (1) immunohistochemical staining with anti-BrdU antibody and (2) fluorescent double immunostaining with anti-BrdU antibody and anti-doublecortin (DCX) antibody.

[0102] The dentate gyrus and subventricular zone of the injured side of each section were evaluated, and the number of BrdU-positive cells and the proportion of BrdU / DCX double-stained positive cells among the BrdU-positive cells were counted.

[0103] Immunohistological staining using anti-BrdU antibodies revealed BrdU-positive cells in the innermost layer of the dentate gyrus, the subependymal layer of the lateral ventricle, and the striatum of the injured side in the cell-administered group. Furthermore, fluorescent double staining revealed a significant increase in the percentage of BrdU / DCX-positive cells in the dentate gyrus of the injured side in the cell-administered group (Figure 9). Furthermore, the number of BrdU-positive cells significantly increased in the cell-administered group (Figure 10).

[0104] Test Example 6-2: Neural Marker The animals (Vehicle group, Cell group, and Sham group) that underwent behavioral experiments in Test Example 2 were perfused with PFA at 4 months of age, and brain tissue was collected and embedded in paraffin blocks. These blocks were thinly sliced ​​to prepare brain tissue sections, which were then immunohistochemically stained using an anti-NeuN antibody. The number of NeuN-positive cells in the dentate gyrus of the hippocampus on the injured side was counted in these sections using the stereology method.

[0105] As a result, the number of NeuN-positive cells in the dentate gyrus of the hippocampus was significantly higher in the cell administration group than in the vehicle group (FIG. 11).

[0106] The results of this study demonstrated that intravenously administered dental pulp stem cells can migrate into the brain of recipients, and that administration of dental pulp stem cells promotes neurogenesis in the injured brain, particularly in the dentate gyrus of the hippocampus.

[0107] The test results presented in this invention suggest that neurogenesis is involved in the improvement in motor function and memory learning observed when deciduous tooth pulp stem cells are administered to individuals with cerebral palsy.

[0108] Test Example 7. Effect on Perinatal Hypoxic-Ischemic Encephalopathy Test Example 7-1: Preparation of Dental Pulp Stem Cells As in Test Example 1, dental pulp was excised from extracted human deciduous teeth within 48 hours of extraction under a sterile environment, minced, and then treated with Liberase (MNP-S GMP Grade, Roche) at a final concentration of 0.05 mg / ml for 15 minutes at 37°C with stirring. The cell-containing supernatant was collected, and the remaining dental pulp tissue was treated with Liberase at 0.05 mg / ml for 15 minutes at 37°C with stirring. Cells were collected from the treated dental pulp tissue through a 70 mm strainer and centrifuged with the previously collected supernatant. The collected cells were seeded into a culture vessel (CellBind T75 flask) using MEMα (Gibco) containing 10% hPL (AventaCell BioMedical) and cultured for at least 9 days until colonies formed, grew, and detached. The medium was changed 2 and 9 days after seeding. Once colonies were confirmed to have detached, the cells were collected using TrypLE Select (Gibco) and expanded in the same medium.

[0109] Test Example 7-2: Evaluation test in a rat model of cerebral palsy. Seven-day-old Wistar / ST rats were anesthetized, and the left carotid artery was ligated. After 1-2 hours, the rats were placed in a hypoxic (8% O2) environment for 1 hour to create a neonatal rat hypoxic-ischemic encephalopathy model. The next day, 1x10 of either the solvent or the human dental pulp stem cells of the present invention was intravenously administered. 5 The effect of administering 100 mg of acetaminophen to improve motor function in patients with perinatal hypoxic-ischemic encephalopathy was examined. Specifically, a rotarod test was performed 41 days after administration, and the latency to fall from the rod was measured to evaluate motor coordination and endurance of the limbs.

[0110] As a result, compared to the sham group (normal group, Sham), the vehicle-administered group (Vehicle) showed a significant shortening of the latency to fall, indicating a clear impairment of motor function, whereas the cell-administered group (Cell) showed almost no shortening of the latency to fall, indicating a recovery of motor function (Figure 12). Based on these results, it is believed that these human dental pulp stem cells can reverse the symptoms of cerebral palsy in a relatively short period of time when administered via a route and at a time that is clinically feasible for perinatal hypoxic-ischemic encephalopathy.

[0111] Test Example 8. Effect on severe perinatal hypoxic-ischemic encephalopathy. SD rats on the 7th day after birth were anesthetized, and the right common carotid artery was ligated and then cut. The rats were placed in a hypoxic (8% O2) environment for 105 minutes to create a severe hypoxic-ischemic encephalopathy model in neonatal rats. A sham-treated group was also created. At the age of 35 days, the rats were divided into three groups using the rotarod method: a sham-treated group (Sham), a vehicle-administered group (Vehicle), and a cell-administered group (Cell). At the ages of 5, 7, 9, and 11 weeks (four times at 2-week intervals), the human dental pulp stem cells of the present invention (1 x 10 7 The mice were administered a dose of 1000 mg / kg of the drug via the tail vein. At 13 weeks of age, a cylinder test was performed in a blinded manner. After euthanasia at 14 weeks of age, the whole brains of half of the animals in each group (5 in the sham group, 6 in the vehicle-treated group, and 6 in the cell-treated group) were removed and fixed in 4% paraformaldehyde / phosphate buffer. The cerebral tissue specimens were embedded in paraffin and thin-sectioned according to standard methods, and all animals were subjected to HE staining and myelin basic protein (MBP) immunostaining. Because most of the cerebrum on the injured side (right side) was missing in almost all animals in both the vehicle-treated and cell-treated groups, blinded image analysis of the total length of MBP was performed on the uninjured left cerebral tissue.

[0112] The cylinder test results showed that the cell-treated group showed a significantly higher percentage of left forelimb use, which is innervated by the affected side of the brain, compared with the vehicle-treated group, demonstrating the improvement of motor dysfunction with cell administration (Figure 13A). To examine the relationship between cell administration and histological changes in cerebral tissue, MBP immunostaining was performed, and nerve fiber length was calculated from the stained images. A significant increase in total nerve fiber length was observed in the cell-treated group compared with the vehicle-treated group (Figure 13B). Based on these findings, it is possible that under these experimental conditions, these human dental pulp stem cells contribute to the functional recovery of the injured left forelimb by extending (or increasing) nerve fibers in the unaffected cerebrum.

[0113] These results suggest that repeated administration of this drug to patients with severe perinatal hypoxic-ischemic encephalopathy using a clinically feasible route and timing improves motor dysfunction in cerebral palsy, and that nerve elongation on the unaffected side contributes to part of the mechanism of action.

[0114] Test Example 9. Surface Marker Analysis Human dental pulp stem cells were prepared from extracted deciduous teeth of eight donors in the same manner as in Test Example 1. 1x10 5 The cells were incubated with phycoerythrin (PE)-conjugated antibodies against various surface markers (CD117 (#313204), CD31 (#303105), CD34 (#343606), CD45 (#368510), CD90 (#328110), CD105 (#323206), CD73 (#344004), CD325 (#350806), CD49d (#304303), CD51 (#327909), CD29 (#303003), CD44 (#338807), CD166 (#343904), and CD106 (#305805), all from Biolegend). Data were collected by flow cytometry. The analysis was performed by gating so that the positive rate of the isotype control antibody was around 1%, and the positive rates of various surface markers were calculated.

[0115]

[0116] As a result, the positive rates for CD117, CD325, and CD45 were less than 5% for all donor cells, and were judged to be negative. The positive rates for CD73, CD90, CD105, CD49d, and CD51 were more than 96% for all donor cells, and were judged to be positive.

[0117] Test Example 10. Cytokine production After subculture of the human dental pulp stem cells in 10% hPL / MEMα or 10% FBS / MEMα for 7 days, 5.76 x 10 cells were cultured in a 6-well plate. 5 The cells were seeded in the same medium at a concentration of 2.5 ml / well. The following day, the medium was replaced with 2.5 ml / well of hPL-free, FBS-free, phenol red-free MEMα. After 48 hours, the culture supernatant was collected, aliquoted, and stored at -80°C. The frozen samples were thawed, and the concentrations of SCF, ANGPTL4, BIGH3, and IL-6 in the samples were measured using a Multiplex HGF panel (Biolegend, #740180), a Human ANGPTL4 assay kit (IBL, #27749), a Human beta IG-H3 ELISA (abcam, #ab220651), and a Multiplex Neuroinflammatory panel (Biolegend, #740796), respectively. The concentration ratios relative to IL-6 were calculated (Tables 2 and 3). The amount of each factor in the cell culture medium was calculated based on the volume of culture supernatant and the number of cells seeded. 6 The production amount was calculated as the amount produced per cell (Tables 4 and 5).

[0118] As a result, the conditioned medium of dental pulp stem cells subcultured in 10% hPL / MEMα showed a higher concentration ratio of SCF, ANGPTL4, and BIGH3 to IL-6 and a higher concentration of 1x10 compared to the conditioned medium of dental pulp stem cells subcultured in 10% FBS / MEMα. 6 The production amount per cell was high.

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[0123] Test Example 11. Effect on Neurogenesis Test Example 11-1: Co-culture experiment with neural stem cells Neural stem cells (NSCs) (Sigma-Aldrich, SCR022) collected from the hippocampal dentate gyrus of adult rats were primary cultured in a 24-well plate according to the attached protocol (culture medium: DMEM / F-12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12) 100 ml, B-27 Supplement (50X) serum-free 2.0 ml, GlutaMAX Supplement 1.0 ml, basic fibroblast growth factor (0.1 mg / ml) 20 μl) and used in the subsequent experiments.

[0124] Twenty-four hours after primary culture of NSCs, human deciduous tooth-derived dental pulp stem cells (SHED), human bone marrow-derived mesenchymal stem cells (BM-MSC), or human dermal fibroblasts (Fibroblasts) were seeded (2 x 10^4 cells / well) on inserts (Corning Culture Inserts for 24 Wells, 0.4 μm PET, transparent, product number 353095) obtained as in Test Example 1, and co-cultured in serum-free medium (Gibco, MEM α nucleosides no phenol red INV, product number 41061029). NSCs alone were used as a control for co-culture.

[0125] Test Example 11-2: Cell Staining 48 hours after the start of co-culture, the cell supernatant was collected, and the cells on the plate were fixed with PFA. These cells were stained with Hoechst (nuclei), anti-SOX2 antibody (neuronal cell marker), and anti-S-100 antibody (astrocyte marker). As a result of the staining, the cells were SOX2-positive, but very few S-100-positive cells were observed. This confirmed that SHED did not affect the differentiation induction of NSCs during 48 hours of culture.

[0126] Test Example 11-3: Evaluation of cell proliferation rate The cells stained in Test Example 11-2 were counted using the stereology method, and the cell proliferation rate (growth rate) was calculated by normalizing the number of cells with the control. The results showed that SHED showed the highest NSC proliferation effect (Figure 14).

[0127] Test Example 11-4: Evaluation of BCNF concentration in culture supernatant For the co-culture performed in Test Example 11-3, the concentration of brain-derived trophic factor (BDNF) in the culture supernatant collected from the NSC culture side before cell fixation was measured by ELISA. The results showed that the BDNF concentration in the culture supernatant was significantly higher in the co-culture with SHED (Figure 15).

[0128] These results demonstrate that SHED contributes to the proliferation of NSCs in coculture with NSCs, with a greater effect than BM-MSCs and fibroblasts. Furthermore, the culture supernatant of NSCs cocultured with SHED contained high levels of BDNF. BDNF (Brain-Derived Neurotrophic Factor) is a protein produced and secreted by mammalian cells and is widely distributed in the mammalian brain. BDNF is a neurotrophic factor known to promote neuronal development and outgrowth, mediate synaptic function (Kowianski et al., Cell Mol Neurobiol (2018) 38:579-593), and has been reported to have neuroprotective effects (Lau et al., Cell Reports (2015)12:1353-1366; Numakawa et al., Histol Histopathol (2010) 25:237-258). These effects are thought to be useful in the treatment of neurodegenerative diseases as well as various other diseases that require nerve tissue regeneration.

[0129] The results of this study indicated that SHED may contribute to neurogenesis by promoting the proliferation of remaining NSCs in areas of degenerated brain and neural tissue through, for example, the production of BDNF.

[0130] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A preventive or therapeutic agent for cerebral palsy, comprising dental pulp stem cells, administered to a subject exhibiting symptoms of cerebral palsy, wherein the cerebral palsy is in its late-stage stage.

2. The preventive or therapeutic agent according to claim 1, wherein the aforementioned long-term period is from infancy onward.

3. A preventive or therapeutic agent according to claim 1, which is administered multiple times.

4. The preventive or therapeutic agent according to claim 1, wherein the dental pulp stem cells are human deciduous tooth pulp stem cells.

5. The preventive or therapeutic agent according to claim 1, wherein the cerebral palsy is caused by hypoxia and / or ischemia.

6. The preventive or therapeutic agent according to claim 1, wherein the cerebral palsy is caused by perinatal brain injury.

7. A preventive or therapeutic agent according to claim 1, for use in improving motor function and / or memory and learning function in subjects having symptoms of cerebral palsy.

8. The preventive or therapeutic agent according to claim 1, wherein the target is human.

9. A preventive or therapeutic agent according to claim 1, which causes the regeneration of nerve cells in the brain in the subject.

10. The preventive or therapeutic agent according to claim 1, wherein more than 90% of the dental pulp stem cells are characterized by being CD117-negative, CD73-positive, CD90-positive, and CD105-positive, more than 90% are CD325-negative or CD51-positive, and the population consists of stem cells derived from human deciduous tooth pulp.

11. The preventive or therapeutic agent according to claim 10, wherein the stem cell population has at least one of the following characteristics 1) to 3): 1) Produce SCF (stem cell factor) in a weight ratio of more than one-tenth that of IL-6. 2) It produces ANGPTL4 (angiopoietin-like 4) at a weight ratio of more than 5 times that of IL-6. 3) It produces BIGH3 (beta-inducible transforming growth factor) at a weight ratio of more than 450 times that of IL-6.

12. The preventive or therapeutic agent according to claim 10, wherein the stem cell population has at least one of the following characteristics 1) to 3): 1) 1 × 10 6 Each cell produces at least 0.1 ng of SCF (stem cell factor) in 48 hours. 2) 1 × 10 6 Each cell produces at least 500 ng of BIGH3 (beta-inducible transforming growth factor) in 48 hours. 3) 1 × 10 6 Each cell produces at least 5 ng of ANGPTL4 in 48 hours.

13. The preventive or therapeutic agent according to any one of claims 11 to 12, wherein the stem cell population is obtained by culturing cells enzymatically isolated from human dental pulp in a culture medium that does not contain FBS (fetal bovine serum) in the presence of human platelet lysates.