JP2023063011A - Method and kit for activation of mesenchymal cell and/or hair follicle germ - Google Patents

Abstract

To provide a method and a kit for effective activation of mesenchymal cells and/or hair follicle germs.SOLUTION: A method for production of activated mesenchymal cells and/or hair follicle germs includes an activation treatment for bringing mesenchymal cells and/or hair follicle germs comprising mesenchymal cells into contact with an Akt activator, which is a synthetic compound, in a solution comprising the Akt activator.SELECTED DRAWING: None

Description

The present invention relates to methods and kits for activation of mesenchymal cells and/or hair follicle primordia.

Patent Document 1 describes that hair growth was promoted by applying bovine lactoferrin or lactoferricin prepared from bovine lactoferrin to the skin of mice.

Non-Patent Document 1 discloses that by culturing rat dermal papilla cells in a medium containing bovine lactoferrin, the proliferation of the dermal papilla cells is promoted, and phosphorylation of Erk and Akt in the dermal papilla cells is amplified. and that application of bovine lactoferrin to the skin of mice promoted hair growth.

Non-Patent Document 2 describes that SC79 activates Akt in the cytoplasm of HeLa cells, HEK293 cells, HL60 cells, NB4 cells and HsSulton cells, and that Akt phosphorylation is detected by culturing cortical neurons in the presence of SC79. It has been described to amplify oxidation and reduce neuronal cell death due to excitotoxicity.

Patent Document 2 describes an Akt activator containing 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid as an active ingredient. Further, Patent Document 3 describes an Akt activator containing, as an active ingredient, a phospholipid compound containing 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid.

Patent Document 4 discloses (a) a peptide consisting of Leu (leucine) and Ile (isoleucine); (b) a modified form of a peptide consisting of Leu and Ile; and (c) a pharmaceutically acceptable (a) or a salt of (b);

Japanese Patent Application Laid-Open No. 2007-031418 WO2014/129598 WO2014/126191 WO2006/090555

Hsiu-Chin Huang et al., Lactoferrin promotes hair growth in mice and increases dermal papilla cell proliferation through Erk/Akt and Wnt signaling pathways, Archives of Dermatological Research (2019) 311: 411-420 Hakryul Jo et al., Small molecule-induced cytosolic activation of protein kinase Akt rescues ischemia-elicited neuronal death, PNAS (2012) vol.109, No.26, 10581-10586

On the other hand, the inventors of the present invention have studied technical means for activating dermal papilla cells.

The present invention has been made in view of the above problems, and one of its objects is to provide a method and kit for effective activation of mesenchymal cells and/or hair follicle primordia.

One aspect of one embodiment of the present invention for solving the above problems is mesenchymal cells and/or follicle-containing mesenchymal cells in a solution containing an Akt activator, which is a synthetic compound. A method for producing activated mesenchymal cells and/or activated hair follicle primordia, comprising an activation treatment of contacting the substrate with the Akt activator. According to the present invention, an effective method for producing activated dermal papilla cells and/or activated hair follicle primordia is provided.

In the method, the Akt activator may be one or more selected from the group consisting of SC79, DCP-LA, Leu-Ile, and chemical modifications thereof. In this case, the Akt activator may be one or more selected from the group consisting of SC79 and chemical modifications thereof.

In the activation treatment, the mesenchymal cells may be brought into contact with the Akt activator in a solution containing the Akt activator. Moreover, in the activation treatment, the hair follicle primordium may be brought into contact with the Akt activator in a solution containing the Akt activator.

Moreover, the method may include performing the activation treatment to obtain activated mesenchymal cells and/or activated hair follicle primordia for transplantation into a living body. Moreover, in the method, the hair follicle primordium may contain the mesenchymal cells and the epithelial cells. In the above method, the mesenchymal cells may be one or more selected from the group consisting of hair follicle mesenchymal cells and their progenitor cells.

Another aspect of one embodiment of the present invention for solving the above problems is mesenchymal cells and/or hair follicles containing mesenchymal cells in a solution containing an Akt activator that is a synthetic compound. Activation of mesenchymal cells and/or hair follicle primordia, comprising activating said mesenchymal cells and/or said hair follicle primordia by contacting said primordia with said Akt activating agent The method. According to the present invention, an effective method for activating dermal papilla cells and/or hair follicle primordia is provided.

Still another aspect of one embodiment of the present invention for solving the above problems is the use of a synthetic compound Akt activator for mesenchymal cells and/or hair follicle primordia containing mesenchymal cells. It is used for activation. INDUSTRIAL APPLICABILITY According to the present invention, a new use of an Akt activator for activation of mesenchymal cells and/or hair follicle primordia is provided.

Still another aspect of one embodiment of the present invention for solving the above problems is a mesenchymal cell and/or a hair follicle primordium containing a mesenchymal cell, comprising an Akt activator that is a synthetic compound. A kit for activating mesenchymal cells and/or hair follicle primordium, which is used for the activation treatment of hair follicles. According to the present invention, a kit for effectively activating dermal papilla cells and/or hair follicle primordia is provided.

Still another aspect of one embodiment of the present invention for solving the above problems is mesenchymal cells and/or hair containing mesenchymal cells in a solution containing an Akt activator that is a synthetic compound. an activation treatment of contacting the follicle primordium with the Akt activator to obtain activated mesenchymal cells and/or activated hair follicle primordia; and the activated mesenchymal cells and/or and transplanting activated hair follicle primordia into a living body. According to the present invention, an effective hair regeneration method using dermal papilla cells and/or hair follicle primordia is provided.

The present invention provides methods and kits for effective activation of mesenchymal cells and/or hair follicle primordia.

FIG. 2 is an explanatory diagram showing the results of evaluating Versican gene expression levels in dermal papilla cells in Example 1 according to the present embodiment. FIG. 2 is an explanatory diagram showing the results of analyzing the protein phosphorylation level of dermal papilla cells in Example 1 according to the present embodiment. FIG. 2 is an explanatory diagram showing the results of evaluating the expression levels of Versican gene, ALP gene, LEF1 gene, and WNT5A gene in dermal papilla cells in Example 1 according to the present embodiment. FIG. 4 is an explanatory diagram showing a photograph of a site where hair follicle primordia were transplanted in Example 2 according to the present embodiment. FIG. 4 is an explanatory diagram showing the result of counting the number of hairs regenerated at the site where the follicle primordium was transplanted in Example 2 according to the present embodiment. FIG. 10 is an explanatory diagram showing a photograph of a site where the hair follicle primordium was transplanted in Example 3 according to the present embodiment. FIG. 10 is an explanatory diagram showing the result of counting the number of hairs regenerated at the site where the follicle primordium was transplanted in Example 3 according to the present embodiment.

An embodiment of the present invention will be described below. Note that the present invention is not limited to this embodiment.

As one aspect of this embodiment, in a solution containing an Akt activator that is a synthetic compound, mesenchymal cells and/or hair follicle primordia containing mesenchymal cells are treated with the Akt activator. It includes a method for producing activated mesenchymal cells and/or activated hair follicle primordia, comprising an activation treatment of contacting with.

That is, the inventors of the present invention have made intensive studies on technical means for activating dermal papilla cells, and unexpectedly found that dermal papilla cells or dermal follicle-containing dermal papilla cells were produced in vitro. The present inventors have independently found that the dermal papilla cells and hair follicle primordium can be effectively activated by a simple operation of contacting the group with an Akt activator, which is a synthetic compound, and have completed the present invention.

Therefore, as another aspect, the present embodiment provides a solution containing an Akt activator, which is a synthetic compound, in which mesenchymal cells and/or hair follicle primordia containing mesenchymal cells are treated with the Akt It includes a method for activating mesenchymal cells and/or hair follicle primordia, comprising activating said mesenchymal cells and/or said hair follicle primordia by contacting with an activating agent.

In addition, as yet another aspect, this embodiment provides the use of a synthetic compound Akt activator for activation of mesenchymal cells and/or hair follicle primordia containing mesenchymal cells. contain.

In yet another aspect, the present embodiment contains an Akt activator, which is a synthetic compound, and is used for activation treatment of mesenchymal cells and/or hair follicle primordia containing mesenchymal cells. A mesenchymal cell and/or hair follicle primordium activation kit.

In addition, as yet another aspect of the present embodiment, in a solution containing an Akt activator that is a synthetic compound, mesenchymal cells and/or hair follicle primordia containing mesenchymal cells are treated with the Akt Obtaining activated mesenchymal cells and/or activated hair follicle primordia by performing an activation treatment of contacting with an activating agent, and obtaining the activated mesenchymal cells and/or the activated hair follicle primordia into a living body.

An Akt activator is a compound that activates Akt (serine/threonine kinase, also called protein kinase B (PKB)) in cells by causing a conformational change and promoting the phosphorylation of Akt. . Here, in the present invention, an Akt activator that is a synthetic compound is used. That is, an Akt activator, which is a compound (artificial compound) produced by artificial synthesis, is used.

In this regard, the bovine lactoferrin used in the above-mentioned Patent Document 1 and Non-Patent Document 1 is a natural compound derived from animals other than humans, and therefore is used, for example, in the preparation of cells or cell aggregates for transplantation into human patients. When using it, it is difficult to ensure safety. In contrast, Akt activators, which are synthetic compounds, are more likely to be safe when used to prepare cells or cell aggregates for transplantation into human patients.

Akt activators that are synthetic compounds (hereinafter simply referred to as "Akt activators") are not particularly limited as long as the effects of the present invention can be obtained, but examples include SC79, DCP-LA, Leu-Ile and these It is preferably one or more selected from the group consisting of chemical modifications of SC79 and particularly preferably one or more selected from the group consisting of SC79 and its chemical modifications.

SC79 is a synthetic compound identified by CAS number "305834-79-1" (2-amino-6-chloro-α-cyano-3-(ethoxycarbonyl)-4H-1-benzopyran-4-acetic acid ethyl ester: 2-amino-6-chloro-α-cyano-3-(ethoxycarbonyl)-4H-1-benzopyran-4-acetic acid ethyl ester). SC79 is represented by structural formula (I) below. A chemically modified SC79 is a compound obtained by chemically modifying the SC79 without impairing its Akt activation function.

DCP-LA is a synthetic compound identified by CAS number "28399-31-7" (8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid: 8-[2-(2 -pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid). DCP-LA may be a pharmaceutically acceptable salt thereof. A chemically modified form of DCP-LA is a compound obtained by chemically modifying the DCP-LA without impairing its Akt activation function. Specifically, chemical modifications of DCP-LA include, for example, phospholipid modifications of DCP-LA (e.g., 1,2-o-bis-[8-{2-(2-pentyl-cyclopropylmethyl) -cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidylethanolamine, 1,2-o-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn -glycero-3-phosphatidyl-L-serine, 1,2-o-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-3-glycero-3- phosphatidylcholine, 1,2-o-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidyl-D-l-inositol, and 1, 1 selected from the group consisting of 2-o-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidyl-L-l-inositol above). A chemical modification of DCP-LA may be a pharmaceutically acceptable salt thereof.

Leu-Ile is a dipeptide composed of leucine (Leu) and isoleucine (Ile). Chemically modified Leu-Ile is a compound obtained by chemically modifying Leu-Ile without impairing its Akt activation function.

The mesenchymal cells in the present embodiment are not particularly limited as long as the effects of the present invention can be obtained, but are preferably one or more selected from the group consisting of, for example, hair follicle mesenchymal cells and their progenitor cells. . Hair follicle mesenchymal cells are mesenchymal cells that contribute to hair growth (more specifically, for example, mesenchymal cells that contribute to hair growth in cooperation with hair follicle epithelial cells).

Hair follicle mesenchymal cells may be those isolated from living hair follicles, or may be those induced to differentiate from undifferentiated cells in vitro. The undifferentiated cells used for inducing the differentiation of hair follicle mesenchymal cells in vitro are not particularly limited as long as they have the ability to differentiate into the hair follicle mesenchymal cells in vitro. stem cells (e.g., iPS (induced pluripotent stem) cells, ES (embryonic stem) cells, Muse (multilineage-differentiating stress-ending) cells or EG (embryonic germ) cells), and stem cells other than the pluripotent stem cells ( For example, one or more selected from the group consisting of stem cells obtained by reprogramming differentiated cells, and mesenchymal stem cells (e.g., adipose tissue-derived mesenchymal stem cells)) Preferably.

Specifically, the hair follicle mesenchymal cells are preferably one or more selected from the group consisting of dermal papilla cells and dermal sheath cup cells, and are dermal papilla cells. is particularly preferred.

Progenitor cells of hair follicle mesenchymal cells are not particularly limited as long as they are cells that have the ability to differentiate into the hair follicle mesenchymal cells in vitro. , mesenchymal cells derived from the dermal layer of fetal or neonatal skin), and stem cells other than pluripotent stem cells that have the ability to differentiate into the hair follicle mesenchymal cells in vitro (e.g., adipose tissue-derived mesenchymal stem cells). The adipose tissue-derived mesenchymal stem cells are not particularly limited as long as the effects of the present invention can be obtained, but for example, they are isolated from living adipose tissue (subcutaneous adipose tissue and/or other adipose tissue).

In addition, the mesenchymal cells preferably express hair growth-related genes. Genes related to hair growth expressed by mesenchymal cells are not particularly limited as long as they are genes related to hair growth in vivo. It is good also as being 1 or more selected from.

The epithelial cells in this embodiment are not particularly limited as long as the effects of the present invention can be obtained, but are preferably one or more selected from the group consisting of, for example, hair follicle epithelial cells and their progenitor cells. Hair follicle epithelial cells are epithelial cells that contribute to hair growth (more specifically, for example, epithelial cells that contribute to hair growth in cooperation with hair follicle mesenchymal cells).

Hair follicle epithelial cells may be those isolated from hair follicles in vivo, or may be those induced to differentiate from undifferentiated cells in vitro. The undifferentiated cells used for inducing differentiation of hair follicle epithelial cells in vitro are not particularly limited as long as they are cells capable of differentiating into the hair follicle epithelial cells in vitro. For example, pluripotent stem cells (e.g., iPS cells, ES cells, Muse cells, or EG cells), and stem cells other than the pluripotent stem cells (e.g., stem cells obtained by reprogramming differentiated cells). Preferably.

Specifically, the hair follicle epithelial lineage cells are selected from the group consisting of hair follicle epithelial stem cells, hair matrix cells, outer root sheath cells, and inner root sheath cells. is preferably one or more, and particularly preferably one or more selected from the group consisting of hair follicle epithelial stem cells, hair matrix cells, and outer root sheath cells.

The progenitor cells of hair follicle epithelial cells are not particularly limited as long as they are cells that have the ability to differentiate into the hair follicle epithelial cells in vitro. epithelial cells derived from the epidermal layer of neonatal skin), and stem cells other than pluripotent stem cells that have the ability to differentiate into the hair follicle epithelial cells in vitro. is preferred.

The origin of the cells used in this embodiment is not particularly limited as long as the effects of the present invention can be obtained, but the cells are preferably derived from animals having hair follicles. That is, the cells used in this embodiment may be human cells, non-human mammals (e.g., primates (e.g., monkeys), rodents (e.g., mice, rats, hamsters, guinea pigs, or rabbit), carnivorous (eg dog, cat), or ungulate (eg pig, cow, horse, goat or sheep)) cells. However, human cells are used for transplantation into humans.

Cells used for the purpose of transplantation into a living body in the present embodiment are preferably derived from the individual to whom the cells are to be transplanted, but may be derived from an individual other than the individual to which the cells are to be transplanted. . For example, human cells used for transplantation into humans are preferably cells derived from a human patient to whom the human cells are to be transplanted, but cells derived from humans other than the patient (e.g., cells other than the patient) It may be a human cell induced to differentiate in vitro from human-derived pluripotent stem cells (for example, iPS cells, ES cells, Muse cells or EG cells stored in cell banks).

The hair follicle primordium in this embodiment is a cell aggregate containing mesenchymal cells and having hair regeneration ability. The hair regrowth ability of the follicle primordium is the ability to induce hair growth at the site where the follicle primordium is transplanted when the follicle primordium is transplanted into a living body.

A hair follicle primordium containing mesenchymal cells may further contain other cells. That is, the hair follicle primordium may contain, for example, mesenchymal cells and epithelial cells. In addition, the hair follicle primordium may further contain cells other than mesenchymal cells and epithelial cells. Cells other than mesenchymal cells and epithelial cells are not particularly limited as long as the effects of the present invention are obtained, but are, for example, one or more selected from the group consisting of melanocytes, melanocyte precursor cells, and melanocyte stem cells. is preferred.

A hair follicle primordium is formed by in vitro aggregation of cells that should form the follicle primordium. That is, the present method involves aggregating cells containing mesenchymal cells in vitro to obtain hair follicle primordia containing the mesenchymal cells (more specifically, hair containing aggregates of the mesenchymal cells). nucleus) may be included.

In this case, the method comprises aggregating cells containing mesenchymal cells and epithelial cells in vitro to obtain hair follicle primordia containing the mesenchymal cells and epithelial cells (more specifically, It may include forming a hair follicle primordium containing foliar cell aggregates and epithelial cell aggregates.

The method for forming hair follicle primordia by aggregating cells containing mesenchymal cells in vitro is not particularly limited as long as the effects of the present invention can be obtained. to form the hair follicle primordium.

Cell culture for forming a follicle primordium begins by seeding cells that are to constitute the follicle primordium. Cells are seeded by placing the cells in a culture vessel (eg, well for cell culture). The culture vessel is not particularly limited as long as the effects of the present invention can be obtained. It is preferred to form a single hair follicle primordium from cells.

Specifically, the area of the bottom surface of the culture vessel (for example, the bottom surface of one well) may be, for example, 1000 mm ² or less, preferably 500 mm ² or less, and more preferably 100 mm ² or less. , 50 mm ² or less, and particularly preferably 20 mm ² or less.

In addition, the area of the bottom surface of the culture vessel may be, for example, 0.01 mm ² or more, preferably 0.10 mm ² or more, more preferably 0.30 mm ² or more, and 0.50 mm ^{2 or more} . It is more preferably 0.70 mm 2 or more, and particularly preferably 0.70 mm ² or more. The area of the bottom surface of the culture vessel may be specified by arbitrarily combining one of the above lower limits and one of the above upper limits.

When seeding mesenchymal cells and epithelial cells, it is preferable to seed the mesenchymal cells and the epithelial cells at the same time, but one of the mesenchymal cells and the epithelial cells is seeded first. , and then the other may be further seeded.

In seeding cells, it is preferable to seed dispersed cells. That is, when seeding mesenchymal cells and epithelial cells at the same time, a cell suspension in which the mesenchymal cells and epithelial cells are dispersed is placed in a culture vessel. In addition, when one of mesenchymal cells and epithelial cells is first seeded and then the other is further seeded, the cell suspension in which the one of the cells is dispersed is first placed in a culture vessel, and then the A cell suspension in which the other cells are dispersed is additionally put into the culture vessel.

Cells seeded using a cell suspension are dispersed and mixed in a culture medium in a culture vessel. Individual cells dispersed in the culture medium are not substantially bound to other cells, or adhere to other cells, but the culture medium can be fluidized by an operation such as pipetting. It is easily separated from the other cells.

The cell seeding density (the total number of cells seeded per 1 cm ² of the bottom surface of the culture vessel) is not particularly limited as long as the effects of the present invention can be obtained, but for example, it should be 0.1×10 ⁴ cells/cm ² or more. 0.5×10 ⁴ /cm ² or more is preferable, 1.0×10 ⁴ /cm ² or more is more preferable, and 2.5×10 ⁴ /cm ² or more is preferable. more preferably, and particularly preferably 5.0×10 ⁴ /cm ² or more.

In addition, the cell seeding density may be, for example, 1000×10 ⁴ cells/cm ² or less, preferably 700×10 ⁴ cells/cm ² or less, and 500×10 ⁴ cells/cm ² or less. more preferably 400×10 ⁴ particles/cm ² or less, and particularly preferably 300×10 ⁴ particles/cm ² or less. The cell seeding density may be specified by any combination of one of the above lower limits and one of the above upper limits.

When forming a hair follicle primordium containing mainly mesenchymal cells, the ratio of the number of mesenchymal cells to the total number of seeded cells is not particularly limited as long as the effect of the present invention can be obtained. , It may be 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more. .

Similarly, the ratio of the number of mesenchymal cells to the total number of cells contained in the hair follicle primordium mainly composed of mesenchymal cells is not particularly limited as long as the effects of the present invention can be obtained, but for example , It may be 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more. .

In addition, when forming a hair follicle primordium mainly containing mesenchymal cells and epithelial cells, the total ratio of the number of mesenchymal cells and the number of epithelial cells to the total number of seeded cells is Although not particularly limited as long as the effect of the present invention can be obtained, for example, it may be 50% or more, preferably 60% or more, more preferably 70% or more, and 80% or more. is even more preferable, and 90% or more is particularly preferable.

Similarly, the total ratio of the number of mesenchymal cells and the number of epithelial cells to the total number of cells contained in the hair follicle primordium mainly composed of mesenchymal cells and epithelial cells is Although it is not particularly limited as long as the effect of is obtained, for example, it may be 50% or more, preferably 60% or more, more preferably 70% or more, and 80% or more. More preferably, it is particularly preferably 90% or more.

When forming a hair follicle primordium containing mesenchymal cells and epithelial cells, the ratio between the number of seeded mesenchymal cells and the number of seeded epithelial cells (mesenchymal: epithelial seeding ratio) is Although not particularly limited as long as the effects of the present invention can be obtained, for example, it may be in the range of 1:10 to 10:1, preferably in the range of 1:9 to 9:1, It is more preferably within the range of 1:8 to 8:1, even more preferably within the range of 1:7 to 7:1, particularly within the range of 1:6 to 6:1. preferable.

Furthermore, the mesenchymal: epithelial seeding ratio is, for example, preferably in the range of 1:5 to 5:1, more preferably in the range of 1:4 to 4:1, 1:3 to 3 :1 is more preferred, and 1:2 to 2:1 is particularly preferred.

The culture method for forming hair follicle primordia mainly composed of mesenchymal cells is not particularly limited as long as the effects of the present invention can be obtained. By culturing in a state (non-adhesive state), the mesenchymal cells are preferably aggregated to form a floating state (non-adhesive state) hair follicle primordium containing aggregates of the mesenchymal cells. .

The culture method for forming the hair follicle primordium mainly composed of mesenchymal cells and epithelial cells is not particularly limited as long as the effects of the present invention can be obtained. By mixing cells and epithelial cells and co-culturing them in a floating state (non-adhesive state), the mesenchymal cells and the epithelial cells are aggregated to form aggregates of the mesenchymal cells and the epithelium. It is preferable to form the hair follicle primordium in a floating state (non-adhesive state) containing aggregates of lineage cells.

More specifically, in the suspension culture of mesenchymal cells and epithelial cells, as the culture time elapses, the mesenchymal cells aggregate to form mesenchymal cell aggregates, and the epithelium The lineage cells aggregate to form an epithelial cell aggregation portion, and further, a portion of the mesenchymal cell aggregation portion and a portion of the epithelial cell aggregation portion form a bond. As a result, a hair follicle primordium containing mesenchymal cell aggregates and epithelial cell aggregates bound to the mesenchymal cell aggregates is formed.

The suspension culture is performed in a culture medium having fluidity as a whole. For suspension culture, a culture vessel having a non-cell-adhesive bottom surface is preferably used. In this case, the seeded cells are cultured in a non-adherent state on the non-cell-adhesive bottom surface without substantially adhering to the bottom surface. Specifically, for example, cells sedimented on a non-cell-adhesive bottom surface do not adhere to the bottom surface in the culture medium, or are easily removed from the bottom surface by flowing the culture medium by pipetting or the like. It adheres to the bottom surface so weakly that it detaches from the bottom surface. The shape of cells cultured on the non-cell-adhesive bottom surface is maintained approximately spherical.

In addition, the hair follicle primordium is formed on the non-cell-adhesive bottom surface in a state of floating in the culture solution (ie, non-adhesive state) without substantially adhering to the bottom surface. Specifically, for example, the hair follicle primordium formed on the non-adhesive bottom surface does not adhere to the bottom surface in the culture solution, or the culture solution is fluidized by an operation such as pipetting. It adheres to the bottom surface weakly enough to be easily detached from the bottom surface.

As described above, the method according to the present embodiment (hereinafter referred to as the "method") comprises mesenchymal cells and/or follicle-containing mesenchymal cells in a solution containing an Akt activator. An activation treatment is included in which the group is contacted with the Akt activator of interest.

That is, the present method comprises an activation treatment in which mesenchymal cells are contacted with the Akt activator in a solution containing an Akt activator, and a hair follicle primordium in a solution containing the Akt activator. Including one or both of the activating treatments in contact with an activating agent.

A solution containing an Akt activator used for the activation treatment (hereinafter referred to as "treatment solution") is capable of maintaining survival of mesenchymal cells and hair follicle primordia in the solution, and the activation of Akt Although not particularly limited as long as the agent can activate the mesenchymal cells and hair follicle primordia, for example, a physiological saline (e.g., phosphate-buffered saline (PBS)) to which the Akt activator has been added, or A culture medium (a solution having a composition suitable for culturing cells) is preferred.

A method for preparing the treatment solution is not particularly limited as long as the effects of the present invention can be obtained, but the treatment solution is prepared by externally adding an Akt activator to the base solution. Thus, the treatment solution is prepared by adding, for example, a commercially available Akt activator to the base solution.

The basic solution is not particularly limited as long as it can maintain the survival of mesenchymal cells and hair follicle primordia in the solution, but for example, physiological saline (e.g., phosphate-buffered saline (PBS)) or culture solution ( It is preferably a solution having a composition suitable for culturing cells).

In preparing the treatment solution, the kit for activating mesenchymal cells and/or hair follicle primordia according to the present embodiment is preferably used. That is, as described above, this activation kit contains an Akt activator and is used for activation treatment of mesenchymal cells and/or hair follicle primordia containing mesenchymal cells. The Akt activator contained in the activation kit is then used to prepare a treatment solution for activation treatment.

The activation kit may further include components of the base solution (eg, inorganic salts, sugars, amino acids, etc.). In this case, for example, a base solution is prepared using the components of the base solution included in the activation kit, and the Akt activator included in the activation kit is added to the base solution to prepare the treatment solution. do. Note that the treatment solution may be prepared by further adding other components not included in the activation kit.

The form of the Akt activator contained in the activation kit is not particularly limited as long as the effects of the present invention are obtained, and the activation kit may contain a solid composition (e.g., powder) containing the Akt activator. and may include a liquid composition comprising an Akt activator.

When the activation kit contains the components of the basic solution, the form of the components of the basic solution is not particularly limited as long as the effect of the present invention can be obtained. , powder), or a liquid composition comprising the components of the base solution.

In addition, the activation kit may further include an instruction manual describing that the activation kit is used for activation treatment of mesenchymal cells and/or hair follicle primordia.

Conditions for the activation treatment are not particularly limited as long as the effects of the present invention can be obtained. That is, the concentration of the Akt activator in the treatment solution at the start of the activation treatment (at the time of starting contact between the mesenchymal cells and/or hair follicle primordium and the Akt activator in the treatment solution) is Although it is not particularly limited as long as the effects of the present invention can be obtained, it may be, for example, 1.0 μg/mL or more. That is, activation treatment may be initiated in a treatment solution containing Akt activator at a concentration of 1.0 μg/mL or higher.

In addition, the activation treatment is preferably started in a treatment solution containing an Akt activator at a concentration of 1.5 μg/mL or higher, and in a treatment solution containing an Akt activator at a concentration of 2.0 μg/mL or higher. and even more preferably in a treatment solution containing the Akt activator at a concentration of 2.5 μg/mL or greater, and containing the Akt activator at a concentration of 3.0 μg/mL or greater. Starting in the processing solution is particularly preferred.

Alternatively, the activation treatment may be initiated in a treatment solution containing an Akt activator at a concentration of 11.0 μg/mL or less, for example, containing Akt activator at a concentration of 10.0 μg/mL or less Preferably starting in the treatment solution, more preferably starting in a treatment solution containing Akt activator at a concentration of 9.0 μg/mL or less, and Akt activator at a concentration of 8.5 μg/mL or less. Even more preferably, starting in a treatment solution containing Akt activator at a concentration of 8.0 μg/mL or less is particularly preferred. The concentration of the Akt activator in the treatment solution at the start of the activation treatment may be specified by any combination of one of the above lower limits and one of the above upper limits.

In the activation treatment, the mesenchymal cells and/or hair follicle primordia are kept in contact with the Akt activating agent in the treatment solution for a period of time (e.g., the mesenchymal cells and/or hair follicle primordia are continuously The time to hold (activation treatment time) is not particularly limited as long as the effect of the present invention is obtained, but may be, for example, 10 minutes or more, preferably 30 minutes or more, and 40 minutes or more. is more preferable, 50 minutes or more is even more preferable, and 60 minutes or more is particularly preferable.

The activation treatment time may be, for example, 168 hours or less, 144 hours or less, 120 hours or less, 96 hours or less, or 72 hours or less. may be 60 hours or less, may be 48 hours or less, may be 36 hours or less, may be 24 hours or less, may be 18 hours or less, It may be 12 hours or less, 9 hours or less, 6 hours or less, 4 hours or less, 3 hours or less, or 2 hours or less. may The activation processing time may be specified by arbitrarily combining one of the above-described lower limit values and one of the above-described upper limit values.

The temperature at which the activation treatment is performed (activation treatment temperature) is not particularly limited as long as the effects of the present invention can be obtained. It is more preferably 25° C. or higher, and particularly preferably 36° C. or higher. Also, the activation treatment temperature may be, for example, 40° C. or lower, preferably 39° C. or lower, and particularly preferably 38° C. or lower. The activation processing temperature may be specified by arbitrarily combining one of the above lower limits and one of the above upper limits.

In this method, the mesenchymal cells and/or hair follicle primordium activation treatment may be performed only once, or may be performed twice or more. In this method, when the activation treatment is performed intermittently two or more times, the two or more activation treatments may be performed under the same conditions, or all or part of them may be performed under different conditions. good too.

In the mesenchymal cell activation treatment, the mesenchymal cells adhered to the substrate surface (for example, the mesenchymal cells forming a monolayer on the substrate surface) are Akt-activated in the treatment solution. agent.

That is, after performing adhesion culture of mesenchymal cells on the substrate surface in a culture solution that does not contain an Akt activator, the mesenchymal cells adhered to the substrate surface are removed from the substrate surface. By keeping the mesenchymal system in the treatment solution without releasing it (e.g., exchanging the medium without the Akt activator with the treatment solution, or adding the Akt activator to the medium), A cell activation treatment may be performed.

In this case, after the activation treatment, the treatment solution is replaced with a culture medium that does not contain the Akt activator, and the adhesion culture of the mesenchymal cells that have been subjected to the activation treatment is performed without the Akt activator. You may continue in a culture medium. Moreover, after the activation treatment, the mesenchymal cells subjected to the activation treatment may be detached from the surface of the substrate, and the dispersed mesenchymal cells may be recovered.

Alternatively, the mesenchymal cells may be activated by adhering and culturing the mesenchymal cells on the surface of the substrate in a culture solution (ie, treatment solution) containing an Akt activator. In this case, after the activation treatment, the mesenchymal cells subjected to the activation treatment may be detached from the surface of the substrate, and the dispersed mesenchymal cells may be recovered.

The adherent culture of mesenchymal cells may be proliferation culture in which the mesenchymal cells are grown on the substrate surface in order to increase the number of the mesenchymal cells. The substrate surface to which mesenchymal cells adhere in adhesion culture is not particularly limited as long as the effects of the present invention can be obtained. A bottom surface made of plastic or a bottom surface coated with a cell-adhesive component (eg, collagen) is preferably used.

The seeding density of mesenchymal cells (the number of mesenchymal cells seeded per 1 cm ² of the substrate surface) for starting adherent culture is not particularly limited as long as the effects of the present invention can be obtained. The number may be 5×10 2 /cm ² or more, preferably 5×10 ² /cm ² or more, and particularly preferably 5×10 ³ /cm ² or more.

In addition, the seeding density of mesenchymal cells for adhesion culture may be, for example, 1×10 ⁶ cells/cm ² or less, preferably 1×10 ⁵ cells/cm ² or less. 10 ⁴ /cm ² or less is particularly preferable. The seeding density of mesenchymal cells for adherent culture may be specified by arbitrarily combining one of the lower limits described above and one of the upper limits described above.

In the mesenchymal cell activation treatment, the mesenchymal cells suspended in the treatment solution may be brought into contact with an Akt activator. That is, before starting the culture of the mesenchymal cells, the mesenchymal cells to be seeded may be dispersed and held in a treatment solution to activate the mesenchymal cells.

In this case, after the activation treatment, the mesenchymal cells subjected to the activation treatment may be seeded and adherent culture of the mesenchymal cells may be performed, or suspension culture of the mesenchymal cells, Alternatively, the mesenchymal cells and epithelial cells may be co-cultured in a floating culture to form the hair follicle primordium.

In addition, after the mesenchymal cells are adherently cultured, the mesenchymal cells are recovered from the substrate surface, and the mesenchymal cells are dispersed and retained in the treatment solution to obtain the mesenchymal cells. may be activated.

In this case, after the activation treatment, the mesenchymal cells subjected to the activation treatment may be seeded and adherent culture of the mesenchymal cells may be performed, or suspension culture of the mesenchymal cells, Alternatively, the mesenchymal cells and epithelial cells may be co-cultured in a floating culture to form the hair follicle primordium.

In addition, a mesenchymal cell activation treatment may be performed in the suspension culture for forming the hair follicle primordium. That is, first, floating culture of mesenchymal cells or co-culturing of mesenchymal cells and epithelial cells is performed in a culture medium that does not contain an Akt activator, and then hair follicle primordium is formed. Activation treatment of the mesenchymal cells may be performed by retaining the mesenchymal cells, or the mesenchymal cells and the epithelial cells, in a treatment solution prior to treatment.

In addition, in a culture solution (i.e., treatment solution) containing an Akt activator, suspension culture of mesenchymal cells or co-culture of mesenchymal cells and epithelial cells is initiated, and the treatment solution is The activation treatment of the mesenchymal cells may be performed by performing the suspension culture in the medium.

In the suspension culture for forming these hair follicle primordia, after the activation treatment and before the formation of the hair follicle primordium, the treatment solution is replaced with a culture medium containing no Akt activator. A suspension culture of mesenchymal cells that have been subjected to activation treatment, or a suspension culture that is a co-culture of mesenchymal cells and epithelial cells that have been subjected to activation treatment, does not contain the Akt activator. You may continue in a culture medium.

In the hair follicle primordium activation treatment, the hair follicle primordium floating in the treatment solution may be brought into contact with an Akt activator. That is, first, suspension culture of mesenchymal cells or suspension culture of co-culture of mesenchymal cells and epithelial cells is performed in a culture medium that does not contain an Akt activator to form hair follicle primordia, Next, the hair follicle primordia may be activated by holding the hair follicle primordium in a floating state (non-adhesive state) in the treatment solution.

In addition, a suspension culture of mesenchymal cells or a suspension culture of co-culture of mesenchymal cells and epithelial cells is performed in a culture solution (i.e., treatment solution) containing an Akt activator to form a hair follicle primordium. The hair follicle primordium may be activated by forming the .

The activation treatment of the hair follicle primordium containing mesenchymal cells is a mesenchymal cell activation treatment because the mesenchymal cells contained in the hair follicle primordium are brought into contact with the Akt activator. It can also be said. In addition, activation treatment of cell aggregates containing mesenchymal cells other than hair follicle primordium can be performed in the same manner as the above-described activation treatment of hair follicle primordium. In this regard, the activation treatment of contacting cell aggregates containing mesenchymal cells other than hair follicle primordium with an Akt activator in a treatment solution is to convert the mesenchymal cells contained in the cell aggregates into Akt Since it is brought into contact with an activating agent, it can be said that this is an activating treatment for mesenchymal cells.

In this method, a mesenchymal cell and/or hair follicle primordium is activated to activate the mesenchymal cell and/or hair follicle primordium. Therefore, the method includes a method of activating mesenchymal cells or a method of activating hair follicle primordia. The method for activating hair follicle primordium containing mesenchymal cells is also a method for activating mesenchymal cells contained in the hair follicle primordium.

Further, in this method, mesenchymal cells and/or hair follicle primordia are activated to obtain activated mesenchymal cells and/or activated hair follicle primordia. Therefore, the method includes a method for producing activated mesenchymal cells or a method for producing activated hair follicle primordia. The method for producing activated hair follicle primordium containing mesenchymal cells is also the method for producing activated mesenchymal cells contained in the hair follicle primordium.

This method comprises aggregating activated mesenchymal cells obtained by activation treatment of mesenchymal cells, and obtaining hair follicle primordia containing the activated mesenchymal cells (activated hair follicle primordia). may include forming a In addition, the present method comprises aggregating activated mesenchymal cells and epithelial cells obtained by activation treatment of mesenchymal cells, and obtaining hair follicles containing the activated mesenchymal cells and epithelial cells. Forming a primordium (activated follicle primordium) may be included.

According to this method, the mesenchymal cells and/or hair follicle primordium are effectively treated by a simple treatment of contacting the mesenchymal cells and/or hair follicle primordium with an Akt activating agent in vitro. can be activated.

Activated mesenchymal cells obtained by this method including activation treatment have, for example, higher hair regeneration ability than mesenchymal cells obtained by the same method except that the activation treatment is not performed.

That is, for example, in general, dermal papilla cells isolated from a living body (eg, human) are adherently cultured in vitro in order to increase their number, resulting in a marked reduction in their hair regeneration ability. On the other hand, in the present method, by subjecting the mesenchymal cells to an activation treatment, the decline in the hair regrowth ability of the mesenchymal cells can be effectively suppressed and/or the hair regrowth ability, which has once been lowered, can be suppressed. can be effectively restored.

That is, the activated mesenchymal cells obtained in the present method including the activation treatment, for example, compared to the mesenchymal cells obtained by the same method except that the activation treatment is not performed, one or more hair growth The expression level of related genes is increased.

Specifically, for example, the expression level of one or more selected from the group consisting of Versican gene, ALP gene, LEF1 gene and WNT5A gene in activated mesenchymal cells obtained in the present method including activation treatment is It is higher than that of the mesenchymal cells obtained by the same method except that the modification treatment is not applied.

More specifically, the Versican gene expression level of the activated mesenchymal cells obtained by the present method including the activation treatment is the same as that of the mesenchymal cells obtained by the same method except that the activation treatment is not performed. It may be 1.1 times or more, preferably 1.2 times or more, more preferably 1.3 times or more, even more preferably 1.5 times or more, and 1 0.8 times or more is particularly preferred.

In addition, the ALP gene expression level of the activated mesenchymal cells obtained by this method including the activation treatment is 1.0% lower than that of the mesenchymal cells obtained by the same method except that the activation treatment is not performed. It may be 1 time or more, preferably 1.2 times or more, more preferably 1.3 times or more, even more preferably 1.5 times or more, 1.8 times It is particularly preferable that it is above.

In addition, the expression level of the LEF1 gene in the activated mesenchymal cells obtained by this method including the activation treatment is 1.0% lower than that in the mesenchymal cells obtained by the same method except that the activation treatment is not performed. It may be 1 time or more, preferably 1.2 times or more, and particularly preferably 1.3 times or more.

In addition, the WNT5A gene expression level of the activated mesenchymal cells obtained by this method including the activation treatment is 1.0% lower than that of the mesenchymal cells obtained by the same method except that the activation treatment is not performed. It may be 1 time or more, preferably 1.2 times or more, more preferably 1.3 times or more, even more preferably 1.4 times or more, 1.6 times It is particularly preferable that it is above.

Although the mechanism of activation of mesenchymal cells by the Akt activator achieved in the present embodiment has not been fully elucidated, for example, activation of the PI3K-Akt signaling pathway in the mesenchymal cells , is presumed to contribute to the activation of the mesenchymal cells.

The activated hair follicle primordium obtained by this method including activation treatment has a higher hair regrowth ability than the hair follicle primordium obtained by the same method except that the activation treatment is not performed. That is, the activated hair follicle primordium obtained by the present method including the activation treatment is, for example, transplanted into a living body by the same method except that the activation treatment is not performed at the transplantation site of the living body. It has the ability to regenerate a greater number of hairs compared to the resulting follicle primordia.

Specifically, when the activated hair follicle primordium obtained by this method including the activation treatment is transplanted into a living body, the same method is applied to the transplanted site of the living body except that the activation treatment is not performed. 1.1 times or more, preferably 1.2 times or more, more preferably 1.3 times or more, even more preferably 1.4 times or more, particularly preferably 1.5 times that of the hair follicle primordium obtained in It has the ability to regenerate more hairs.

Applications of the activated mesenchymal cells and activated hair follicle primordium obtained by this method are not particularly limited, but as described above, the activated mesenchymal cells and activated hair follicle primordium have high hair regeneration ability. Therefore, it is useful as a transplant to a living body.

Therefore, in the present method, mesenchymal cells and/or hair follicle primordia containing mesenchymal cells are subjected to an activation treatment in which they are brought into contact with an Akt activator in a treatment solution, and then transplanted into a living body. obtaining activated mesenchymal cells and/or activated hair follicle primordia for.

In this case, the present method may be a method for producing mesenchymal cells for transplantation into a living body or a method for producing a hair follicle primordium for transplantation into a living body. In addition, the present method may be a method for activating mesenchymal cells for transplantation in vivo or a method for activating hair follicle primordia for transplantation in vivo.

In addition, the present method includes performing an activation treatment in which mesenchymal cells and/or hair follicle primordia containing mesenchymal cells are brought into contact with an Akt activating agent in a treatment solution to activate mesenchymal cells. It may be a hair regeneration method comprising obtaining cells and/or activated hair follicle primordia, and transplanting the activated mesenchymal cells and/or activated hair follicle primordium into a living body.

A living body into which mesenchymal cells and/or hair follicle primordia are to be transplanted may be a human or a non-human animal, but is preferably a human. Transplantation of mesenchymal cells and/or hair follicle primordia into a living body is preferably transplantation into the skin of the living body, and particularly preferably transplantation into the scalp of the living body.

Transplantation of mesenchymal cells and/or hair follicle primordia into a living body may be for medical or research purposes. Transplantation of mesenchymal cells and/or hair follicle primordia into a living body is preferably for treatment or prevention of a disease associated with hair loss. That is, the transplantation of mesenchymal cells and/or hair follicle primordia into a living body is transplantation into a human patient suffering from a disease accompanied by hair loss or a human patient likely to suffer from a disease accompanied by hair loss. is preferred. Therefore, the hair regeneration method according to the present embodiment is preferably a method for treating or preventing diseases associated with hair loss.

Diseases associated with hair loss are not particularly limited, but for example, androgenetic alopecia (AGA), female androgenetic alopecia (FAGA), postpartum alopecia, diffuse alopecia, seborrhea Composed of alopecia areata, alopecia pityriasis, traction alopecia, metabolic alopecia, alopecia areata compressive, alopecia areata, alopecia areata, alopecia nervosa, trichotillomania, alopecia generalis, and symptomatic alopecia It may be one or more selected from the group.

In addition, the mesenchymal cells and/or hair follicle primordium obtained in the present embodiment can be used, for example, to search for substances that can be used for the treatment or prevention of diseases associated with hair loss, search for substances involved in the disease, search for substances involved in the disease, and It may be used for research on the mechanism of

Next, specific examples according to this embodiment will be described.

[Example 1-1: Activation process]
Commercially available human dermal papilla cells (PromoCell, passage number P4) were mixed with the culture solution at a density of 2.5×10 ⁴ cells/mL to prepare a cell suspension. Dermal papilla cell growth medium (PromoCell) was used as the culture medium. By pouring 2 mL of this cell suspension into each well of a 6-well plate, dermal papilla cells were seeded at a density of 5×10 ⁴ cells (about 5.3×10 ³ cells/cm ² ) per well.

Then, the dermal papilla cells were cultured in an incubator for 24 hours to allow the dermal papilla cells to adhere to the bottom of the well. On the other hand, an SC79 solution was prepared by dissolving commercially available SC79 (SC79, Akt activator, manufactured by Abcam) as an Akt activator in DMSO (dimethylsulfoxide) as a solvent.

Then, SC79 solution in an amount such that the final concentration of SC79 is 0 μM, 10 μM, 20 μM or 30 μM (0 μg/mL, 3.64 μg/mL, 7.28 μg/mL or 10.92 μg/mL respectively) was added to each well. , and the dermal papilla cells were held (cultured) for 1 hour at 37°C in the culture solution after the addition. Only DMSO without SC79 was added to wells with SC79 concentration of 0 μM (0 μg/mL). One hour after the addition of SC79, dermal papilla cells were collected and their gene expression was analyzed.

[Example 1-2: Activation process]
Dermal papilla cells were seeded in each well of a 6-well plate at a density of 5×10 ⁴ cells in the same manner as in Example 1-1 above. Then, the dermal papilla cells were cultured in an incubator for 24 hours to allow the dermal papilla cells to adhere to the bottom of the well. After that, an amount of SC79 solution that makes the final concentration of SC79 0 μM or 10 μM (0 μg / mL or 3.64 μg / mL, respectively) was added to the culture medium of each well, and the hair was heated at 37 ° C. in the culture medium after the addition. Papilla cells were maintained (cultured) for 1 hour, 6 hours, or 24 hours. Only DMSO without SC79 was added to wells with SC79 concentration of 0 μM (0 μg/mL). Then, at 0 hour (immediately after addition), 1 hour, 6 hours, and 24 hours after the addition of SC79, the dermal papilla cells were collected, and their gene expression and protein phosphorylation level were measured. Analyzed.

[Analysis of gene expression]
RNA was extracted from the collected dermal papilla cells, subjected to reverse transcription, and subjected to gene analysis using RT-PCR. The nucleotide sequences of the primers used in RT-PCR were as follows: For Versican, Forward Primer is "5'-GGCACAAATTCCAAGGGCAG-3'", Reverse Primer is "5'-TCATGGCCCACACGATTAACA-3'"; , Forward Primer is “5′-ATTGACCACGGGCACCAT-3′”, Reverse Primer is “5′-CTCCACCGCCTCATGCA-3′”; , Forward Primer is “5′-CTTCCTTGGTGAACGAGTCTG-3′”, Reverse Primer is “5′” -TCTGGATGCTTTCCGTCAT-3 '"; For WNT5A, Forward Primer is '5'-TCCACCTTCCTCTTCACACTGA-3', Reverse Primer is '5'-CGTGGCCAGCATCACATC-3'; And, for GAPDH used as a control, Forward Primer is '5'-TGGAAGGACTCATGACCACAG-3'", Reverse Primer is "5'-GGATGATGTTCTGGAGAGCCC-3'".

[Analysis of phosphorylation level of Akt protein (Western blotting)]
Protein was extracted from the collected dermal papilla cells, and the extracted protein solution was applied to a 10% polyacrylamide gel. Electrophoresis was then performed at 100 V for 15 minutes followed by 150 V for 40 minutes. After electrophoresis, the polyacrylamide gel was layered on the filter and membrane in the order of filter/membrane/polyacrylamide gel/filter, and transferred at 15 V for 75 minutes.

Antibodies were used at the following dilutions: Anti-Phospho-Akt (Ser473) Rabbit mAb (1:2000; Cell Signaling Technology); Anti-Akt (pan) Rabbit mAb (1:1000; Cell Signaling Technology); GAPDH Rabbit mAb (1:1000; Cell Signaling Technology); Anti-rabbit IgG, HRP-linked Antibody (1:2000; Cell Signaling Technology).

[result]
FIG. 1 shows the relationship between the SC79 concentration (μM) in the culture medium and the relative expression level of the Versican gene for the dermal papilla cells obtained in Example 1-1 above (*p<0.05, n=3). In FIG. 1, the gene expression levels at SC79 concentrations of 10 μM, 20 μM and 30 μM are relative values with the expression level at SC79 concentration of 0 μM (when SC79 was not added) being “1”. is shown.

As shown in FIG. 1, when the SC79 concentration in the culture medium was 10 μM and 20 μM, the Versican gene expression level in dermal papilla cells was significantly increased compared to when the SC79 concentration was 0 μM. .

That is, the Versican gene expression level in dermal papilla cells maintained in a culture medium containing SC79 at a concentration of 10 μM was about 1.2 times that of dermal papilla cells maintained in a culture medium containing no SC79. .

In addition, the Versican gene expression level in dermal papilla cells maintained in a culture medium containing SC79 at a concentration of 20 μM was approximately 1.2 times that of dermal papilla cells maintained in a culture medium containing no SC79. .

On the other hand, when the SC79 concentration in the culture was 30 μM, the Versican gene expression level was significantly lower than when the SC79 concentration was 0 μM.

FIG. 2 shows the analysis of the phosphorylation level of Akt protein in dermal papilla cells cultured for 0, 1, 6, and 24 hours in a culture medium containing SC79 at a concentration of 10 μM in Example 1-2 above. The results are shown.

As shown in FIG. 2, the phosphorylation level of Akt protein in dermal papilla cells cultured for 1 hour in a culture medium supplemented with SC79 was significantly increased compared to that in dermal papilla cells immediately after addition of SC79 (0 h). rice field. In addition, the phosphorylation level of Akt protein in these dermal papilla cells decreased as the culture time in the SC79-added culture medium increased from 6 hours to 24 hours. On the other hand, no significant change was observed in the total amount of Akt protein before and after the addition of SC79.

FIG. 3 shows the Versican gene, ALP gene, and LEF1 gene in dermal papilla cells cultured for 0, 1, 6, and 24 hours in culture media with SC79 concentrations of 0 μM and 10 μM in Example 1-2 above. , and the results of evaluating the expression level of the WNT5A gene (*p<0.05, n=3). In FIG. 3, the expression level of each gene is shown as a relative amount, with the gene expression level at 0 hours (immediately after addition) after SC79 was added to the culture solution being set to "1".

As shown in FIG. 3, the expression levels of the Versican gene, ALP gene, LEF1 gene, and WNT5A gene in dermal papilla cells cultured in a culture medium containing SC79 at a concentration of 10 μM were all was significantly increased compared to that of dermal papilla cells cultured in .

That is, the expression level of the Versican gene in dermal papilla cells maintained in a culture medium containing SC79 was approximately 1.3 to 1.9 times that of dermal papilla cells maintained in a culture medium containing no SC79. rice field.

In addition, the expression level of the ALP gene in dermal papilla cells maintained in a culture medium containing SC79 was about 1.3 to 1.9 times that of dermal papilla cells maintained in a culture medium not containing SC79. rice field.

In addition, the expression level of the LEF1 gene in dermal papilla cells maintained in a culture medium containing SC79 was approximately 1.2 to 1.3 times that of dermal papilla cells maintained in a culture medium containing no SC79. rice field.

In addition, the expression level of the WNT5A gene in dermal papilla cells maintained in a culture medium containing SC79 was approximately 1.2 to 1.7 times that of dermal papilla cells maintained in a culture medium containing no SC79. rice field.

[Isolation of epithelial cells]
Back skin tissue was collected from C57BL/6 mouse embryos of 18 days of fetal age, and 48 U/mL dispase treatment was performed at 4° C. for 1 hour under shaking conditions of 20 to 30 rpm, and the epithelial layer and mesenchymal layer of the skin tissue were separated. separated. Thereafter, the epithelial layer was treated with 100 U/mL collagenase for 80 minutes and then treated with trypsin for 10 minutes to isolate epithelial cells.

[Formation/activation treatment of hair follicle primordium]
Commercially available human dermal papilla cells (PromoCell, passage number P4) and the mouse fetal epithelial cells isolated as described above were each mixed in a culture solution at a density of 5×10 ⁴ cells/mL and dispersed. A cell suspension containing dermal papilla cells and epithelial cells was prepared.

In Example 2-1, a basal medium prepared by mixing DMEM(+) and HuMedia-KG2 at a volume ratio of 1:1 was prepared by adding an amount of SC79 solution to a final SC79 concentration of 10 μM. A culture medium was used. In Example 2-2, a culture solution prepared by adding only DMSO to the basal medium without SC79 was used.

By pouring 200 μL of the cell suspension into each well of a 96-well plate, 1×10 ⁴ cells of dermal papilla cells and 1×10 ⁴ cells of epithelial cells (2×10 ⁴ cells in total) were added to each well. sown.

Floating culture, which is a co-culture of dermal papilla cells and epithelial cells, was carried out in an incubator for 3 days. In co-culture, dermal papilla cells and epithelial cells aggregated to form one follicle primordium in each well.

[Transplantation of hair follicle primordium]
30 hair follicle primordia formed by co-culturing for 3 days in Example 2-1 were collected and subcutaneously transplanted to nude mice. Similarly, 30 hair follicle primordia formed by co-culturing for 3 days in Example 2-2 were collected and subcutaneously transplanted to nude mice. Then, on the 21st day after the transplantation of the follicle primordium, a skin piece including the transplantation site was collected from the nude mouse, and the number of hairs regenerated at the transplantation site was counted.

[result]
FIG. 4 shows a photograph of the site of the nude mouse skin where the hair follicle primordium was transplanted. As shown in FIG. 4, the sites transplanted with hair follicle primordia formed in a culture medium containing no SC79 (0 μM) and the hair follicle primordium formed in a culture medium containing SC79 at a concentration of 10 μM. Hair regeneration was confirmed in the skin at any of the transplanted sites.

FIG. 5 shows the results of counting the number of hairs regenerated at the transplanted site of the follicle primordium. As shown in FIG. 5, the number of hairs regenerated at the site where the hair follicle primordium formed in the culture medium containing SC79 at a concentration of 10 μM was transplanted compared with the number of hairs formed in the culture medium without SC79 (0 μM). It was about 1.8 times that at the site where the hair follicle primordium was transplanted.

That is, the hair follicle primordium formed in the culture medium containing SC79 at a concentration of 10 μM has significantly higher hair regeneration ability than the hair follicle primordium formed in the culture medium without SC79 (0 μM). was confirmed to have

[Isolation of epithelial cells]
Epithelial cells were isolated from embryonic mouse skin tissue in the same manner as in Example 2 above.

[Formation of hair follicle primordium]
Commercially available human dermal papilla cells (PromoCell, passage number P4) and the mouse fetal epithelial cells isolated as described above were each mixed in a culture solution at a density of 5×10 ⁴ cells/mL, and a cell suspension was obtained. A turbid solution was prepared. As the culture medium, a medium prepared by mixing DMEM(+) and HuMedia-KG2 at a volume ratio of 1:1 was used.

By pouring 200 μL of the cell suspension into each well of a 96-well plate, 1×10 ⁴ cells of dermal papilla cells and 1×10 ⁴ cells of epithelial cells (2×10 ⁴ cells in total) were added to each well. sown.

Floating culture, which is a co-culture of dermal papilla cells and epithelial cells, was carried out in an incubator for 3 days. In co-culture, dermal papilla cells and epithelial cells aggregated to form one follicle primordium in each well.

[Activation process]
In Example 3-1, 25 hair follicle primordia formed by co-culturing for 3 days were collected, and a treatment solution prepared by adding an amount of SC79 solution to the final concentration of SC79 of 10 μM was added to the culture medium. and kept at 37° C. for 1 hour.

In Example 3-2, 25 hair follicle primordia formed by co-cultivation for 3 days were collected, and an SC79 solution was added to the culture solution to give a final SC79 concentration of 20 μM. and kept at 37° C. for 1 hour.

In Example 3-3, 25 hair follicle primordia formed by co-culturing for 3 days were collected, dispersed in a solution prepared by adding only DMSO without SC79 to the culture medium, and 37 °C for 1 hour.

[Transplantation of hair follicle primordium]
Twenty-five hair follicle primordia treated with SC79 at a concentration of 10 μM in Example 3-1 for 1 hour were collected and subcutaneously transplanted to nude mice. Twenty-five hair follicle primordia treated with SC79 at a concentration of 20 μM in Example 3-2 for 1 hour were collected and subcutaneously transplanted to nude mice. 25 hair follicle primordia that had not been treated with SC79 in Example 3-3 were collected and subcutaneously transplanted to nude mice. On the 29th day after the transplantation of the hair follicle primordium, a skin piece including the transplantation site was collected from the nude mouse, and the number of hairs regenerated at the transplantation site was counted.

[result]
FIG. 6 shows a photograph of the site of the nude mouse skin where the hair follicle primordium was transplanted. As shown in FIG. 6, the site transplanted with the hair follicle primordium treated with SC79 at a concentration of 10 μM for 1 hour, the site transplanted with the hair follicle primordium treated with SC79 at a concentration of 20 μM for 1 hour, and SC79 Hair regeneration was confirmed in the skin at any of the sites where the hair follicle primordium that had not been treated with 2-D was transplanted.

FIG. 7 shows the results of counting the number of hairs regenerated at the implantation sites of nude mice. As shown in FIG. 7, regeneration occurred at the site transplanted with the hair follicle primordium treated with SC79 at a concentration of 10 μM for 1 hour and at the site transplanted with the hair follicle primordium treated with SC79 at a concentration of 20 μM for 1 hour. The number of hairs was about 1.4 times higher than that at the sites implanted with follicle primordia not treated with SC79. That is, it was confirmed that the hair regeneration ability of the follicle primordium was remarkably enhanced by a simple operation of treating the hair follicle primordium with SC79 for 1 hour before transplantation.

Claims (12)

In a solution containing an Akt activator, which is a synthetic compound, mesenchymal cells and/or hair follicle primordia containing mesenchymal cells are contacted with the Akt activator, including an activation treatment,
A method for producing activated mesenchymal cells and/or activated hair follicle primordia. The Akt activator is one or more selected from the group consisting of SC79, DCP-LA, Leu-Ile, and chemical modifications thereof.
The method of claim 1. The Akt activator is one or more selected from the group consisting of SC79 and chemical modifications thereof,
3. The method of claim 2. In the activation treatment, the mesenchymal cells are contacted with the Akt activator in a solution containing the Akt activator;
4. A method according to any one of claims 1-3. In the activation treatment, the hair follicle primordium is brought into contact with the Akt activator in a solution containing the Akt activator;
5. A method according to any one of claims 1-4. performing the activation treatment to obtain activated mesenchymal cells and/or activated hair follicle primordia for transplantation into a living body;
6. A method according to any one of claims 1-5. The hair follicle primordium contains the mesenchymal cells and the epithelial cells,
7. A method according to any one of claims 1-6. The mesenchymal cells are one or more selected from the group consisting of hair follicle mesenchymal cells and their progenitor cells,
8. A method according to any one of claims 1-7. By contacting mesenchymal cells and/or hair follicle primordia containing mesenchymal cells with the Akt activator in a solution containing the Akt activator, which is a synthetic compound, the mesenchymal system A method for activating mesenchymal cells and/or hair follicle primordia, comprising activating cells and/or said hair follicle primordia. Use of an Akt activator that is a synthetic compound for activation of mesenchymal cells and/or hair follicle primordia containing mesenchymal cells. Akt activators, which are synthetic compounds,
Used for activation treatment of mesenchymal cells and/or hair follicle primordia containing mesenchymal cells,
A mesenchymal cell and/or hair follicle primordium activation kit. In a solution containing an Akt activator, which is a synthetic compound, mesenchymal cells and/or hair follicle primordia containing mesenchymal cells are subjected to an activation treatment in which they are brought into contact with the Akt activator, obtaining activated mesenchymal cells and/or activated hair follicle primordia;
transplanting the activated mesenchymal cells and/or the activated hair follicle primordium into a living body;
including,
Hair regrowth method.

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