WO2019221307A1 - Color control method for regenerated hair, regeneration method for hair, and method for producing hair follicle primordia - Google Patents

Abstract

[Problem] To provide: a color control method for regenerated hair which can control the color of hair simply and efficiently; a regeneration method for hair; and a method for producing hair follicle primordia. [Solution] This color control method for regenerated hair comprises: using source cells including epithelial stem cells obtained from adult hair follicle tissues containing a bulge region which is a constant part; mixing and culturing dispersed hair papilla cells and the dispersed source cells to prepare hair follicle primordia; and transplanting the hair follicle primordia into an animal to grow hair from the hair follicle primordia, wherein the weight ratio of a non-constant part which is included in the adult hair follicle tissues is changed to alter the color of hair grown from the hair follicle primordia.

Description

Method for controlling color of regenerated hair, method for regenerating hair and method for producing hair follicle primordium

The present invention relates to a method for controlling the color of regenerated hair, a method for regenerating hair, and a method for producing a hair follicle primordium.

Patent Document 1 discloses that cultured hair papilla cells were obtained from hair follicles of adult mouse hair follicles, bulge region epithelial cells were obtained from bulge regions of the hair follicles, and subbulge region cells from subbulge regions of the hair follicles. And that the base of the hair matrix was obtained from the base of the hair matrix of the hair follicle. Patent Document 1 discloses that bulge region epithelial cells and sub-bulge region cells or hair matrix basal cells are mixed and centrifuged to obtain the bulge region epithelial cells, sub-bulge region cells or hair matrix base. Cell aggregates containing the somatic cells and centrifuging the cultured hair papilla cells to produce cell aggregates of the cultured hair papilla cells, and then in the collagen gel drop, the bulge region epithelium A cell aggregate of the cultured hair papilla cell is adhered to a cell aggregate including the cell and the subbulge region cell or the hair matrix base cell, and further, the cell aggregate of the bulge region epithelial cell It is described that a nylon thread as a guide was inserted into the gel, and then the gel drop was solidified to produce a regenerated hair follicle primordium. Furthermore, Patent Document 1 describes that as a result of transplanting the regenerated hair follicle primordium into the skin of a nude mouse so that the nylon thread guide is exposed on the body surface, regenerated hair of black hair was obtained. ing.
In Patent Document 2, a mixed suspension of epithelial cells and mesenchymal cells collected from a mouse fetus is added to a micro intaglio plate composed of regularly arranged micro concave portions, and mixed culture is performed while supplying oxygen. It is described that a regenerated hair follicle primordium was produced by doing so. Further, Patent Document 2 describes that hair growth was observed as a result of injecting a regenerated hair follicle primordium into the skin of nude mice.

International Publication No. 2012/115079 International Publication No. 2017/073625

However, conventionally, it has been difficult to control the color of hair grown from the regenerated hair follicle primordium using an adult hair follicle as a cell source.

The method for controlling the color of regenerated hair according to an embodiment of the present invention for solving the above-described problem uses a raw material cell containing epithelial stem cells obtained from an adult hair follicle tissue containing a bulge region of a constant part, A hair follicle primordium is produced by mixing and culturing dispersed hair papilla cells and the dispersed raw material cells, and transplanting the hair follicle primordia to an animal to produce the hair follicle primordium. And changing the weight ratio of the non-constant part contained in the adult hair follicle tissue to change the color of the hair growing from the hair follicle primordium. ADVANTAGE OF THE INVENTION According to this invention, the color control method of the reproduction | regeneration hair which can control the color of hair easily and effectively is provided.
In the method, the animal may be a non-human animal. Moreover, in the said method, it is good also as darkening the color of the hair which grows from the said hair follicle primordium by increasing the weight ratio of the said non-constant part contained in the said adult hair follicle structure | tissue. Moreover, in the said method, the said non-constant part is good also as including parts other than a hair nipple and a hair mother.
The method for regenerating hair according to an embodiment of the present invention for solving the above-mentioned problem is achieved by using raw material cells containing epithelial stem cells obtained from an adult hair follicle tissue containing a bulge region of a constant part. The follicular primordial cells and the dispersed raw material cells are mixed and cultured to produce a hair follicle primordial, and the hair follicle primordial is transplanted into an animal to produce hair from the hair follicle primordial By using the adult hair follicle tissue further comprising a non-constant part, so that the color of the hair follicle primordium compared to the case of using the adult hair follicle tissue not containing the non-constant part is used. Grows dark hair. ADVANTAGE OF THE INVENTION According to this invention, the hair regeneration method which can control the color of hair easily and effectively is provided.
In the method, the animal may be a non-human animal. In the method, the first hair follicle primordium produced using the first adult hair follicle tissue containing the non-constant part at a first weight ratio is transplanted into the animal, Produced from a hair follicle primordium and using a second adult hair follicle tissue comprising the non-constant part in a second weight percentage greater than the first weight percentage. Transplanting the second cell aggregate to the animal to produce a second color hair darker than the first color from the second hair follicle primordium. Moreover, in the said method, the said non-constant part is good also as including parts other than a hair nipple and a hair mother.
The method for producing a hair follicle primordium according to an embodiment of the present invention for solving the above-described problem uses a raw material cell containing epithelial stem cells obtained from an adult hair follicle tissue containing a bulge region of a constant part. And producing a hair follicle primordium that grows hair by being transplanted into an animal by mixing and culturing the dispersed hair papilla cells and the dispersed raw material cells, and is non-constant By using the adult hair follicle tissue further comprising a part, the hair follicle primordium that produces darker hair than the case of using the adult hair follicle tissue not containing the non-constant part is produced. ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the hair follicle primordium which can control the color of hair easily and effectively is provided.
The method uses a first hair follicle primordium that produces hair of a first color by being transplanted into the animal by using the adult hair follicle tissue comprising the non-constant part in a first weight ratio. And the first color by being implanted into the animal by using the adult hair follicle tissue comprising the non-constant part in a second weight percentage greater than the first weight percentage. Producing a second hair follicle primordium that produces darker second colored hair. Moreover, in the said method, the said non-constant part is good also as including parts other than a hair nipple and a hair mother.

According to the present invention, there are provided a color control method for regenerated hair, a method for regenerating hair, and a method for producing a hair follicle primordium capable of easily and effectively controlling the color of the hair.

It is explanatory drawing which shows the structure of an adult hair follicle typically. It is explanatory drawing about the structure of the hair follicle extract | collected from the adult mouse | mouth. It is a phase-contrast microscope image of the hair papilla cell which was primarily cultured for 2 days in the presence of 10 ng / mL FGF2 in Example 1 according to this embodiment. It is a phase-contrast microscope image of the dermal papilla cell which was primarily cultured for 2 days in the presence of 100 ng / mL FGF2 in Example 1 according to this embodiment. It is a phase-contrast microscope image of the dermal papilla cell which was primarily cultured for 9 days in the presence of 10 ng / mL FGF2 in Example 1 according to this embodiment. It is a phase-contrast microscope image of the dermal papilla cell which was primary-cultured for 9 days in presence of 100 ng / mL FGF2 in Example 1 which concerns on this embodiment. It is a fluorescence-microscope image of the primary culture cell obtained from the hair papilla of the adult hair follicle structure | tissue which carried out the Versican / DAPI double immunostaining in Example 1 which concerns on this embodiment. It is a fluorescence-microscope image of the cell obtained from the adult hair follicle structure | tissue containing the bulge area | region which carried out CD34 / DAPI double immunostaining in Example 1 which concerns on this embodiment. It is the photograph of the hair grown from the hair follicle primordium formed using the cell obtained from the adult hair follicle structure | tissue which does not contain a non-constant part in Example 2 which concerns on this embodiment, and was transplanted under the mouse | mouth subcutaneously. It is the photograph of the hair grown from the hair follicle primordium formed using the cell obtained from the adult hair follicle structure | tissue which contains a non-constant part in Example 2 which concerns on this embodiment, and was transplanted under the mouse | mouth subcutaneously. It is an example of a phase contrast micrograph of a hair follicle primordium formed in the presence of 10 ng / mL FGF2 using cells obtained from an adult hair follicle tissue containing a non-constant part in Example 3 according to the present embodiment. is there. It is an example of a phase contrast micrograph of a hair follicle primordium formed in the presence of 50 ng / mL FGF2 using cells obtained from an adult hair follicle tissue containing a non-constant part in Example 3 according to the present embodiment. is there. It is an example of a phase contrast micrograph of a hair follicle primordium formed in the presence of 100 ng / mL FGF2 using cells obtained from an adult hair follicle tissue containing a non-constant part in Example 3 according to the present embodiment. is there. Other phase contrast micrograph of hair follicle primordium formed in the presence of 10 ng / mL FGF2 using cells obtained from adult hair follicle tissue containing non-constant part in Example 3 according to this embodiment It is an example. Other phase contrast micrographs of hair follicle primordium formed in the presence of 50 ng / mL FGF2 using cells obtained from adult hair follicle tissue containing non-constant parts in Example 3 according to this embodiment It is an example. Other phase contrast micrographs of hair follicle primordium formed in the presence of 100 ng / mL FGF2 using cells obtained from adult hair follicle tissue containing non-constant parts in Example 3 according to this embodiment It is an example. It is an example of the result of having evaluated the Versican gene expression in the hair follicle primordium formed using the cell obtained from the adult hair follicle structure | tissue which contains a non-constant part in Example 3 which concerns on this embodiment. It is an example of the result of having evaluated the Wng-10b gene expression in the hair follicle primordium formed using the cell obtained from the adult hair follicle structure | tissue which contains a non-constant part in Example 3 which concerns on this embodiment. In the hair follicle primordium formed in the presence of 10 ng / mL FGF2 and transplanted to mice by the patch method using cells obtained from adult hair follicle tissue containing non-constant parts in Example 3 according to this embodiment It is a photograph. In the hair follicle primordium formed in the presence of 100 ng / mL FGF2 using cells obtained from adult hair follicle tissue containing non-constant parts in Example 3 according to the present embodiment and transplanted into mice by the patch method It is a photograph.

Hereinafter, a method according to an embodiment of the present invention (hereinafter referred to as “the present method”) will be described. Note that the present invention is not limited to the present embodiment.
One aspect of this method is to use a raw material cell containing epithelial stem cells obtained from an adult hair follicle tissue containing a bulge region of a homeostatic part, a dispersed hair papilla cell, and the dispersed raw material cell. Including in the adult hair follicle tissue, including producing a hair follicle primordium by mixing and culturing, and transplanting the hair follicle primordia to an animal and growing hair from the follicle primordium A method for controlling the color of regenerated hair, which changes the color of the hair grown from the hair follicle primordium by changing the weight ratio of the non-constant part.
Another aspect of the present method is to use raw material cells containing epithelial stem cells obtained from an adult hair follicle tissue containing a bulge region of a constant part, dispersed hair papilla cells, and the dispersed raw material. Producing a hair follicle primordium by mixing and culturing cells, and transplanting the hair follicle primordia to an animal and growing hair from the hair follicle primordium, Furthermore, by using the adult hair follicle tissue, a hair regeneration method that produces dark hair from the hair follicle primordia as compared to the case of using the adult hair follicle tissue that does not include the non-constant part. Including.
In addition, still another aspect of the present method is the use of raw material cells containing epithelial stem cells obtained from adult hair follicle tissue containing a bulge region of the homeostatic region, and dispersed hair papilla cells and dispersed Using the adult hair follicle tissue further comprising a non-constant part, including producing a hair follicle primordium that grows hair by being transplanted to an animal by mixing and culturing the raw material cells The manufacturing method of the hair follicle primordial which manufactures the said hair follicle primordium which grows a darker hair compared with the case where the adult hair follicle structure | tissue which does not contain the said non-constant part is used is included.
That is, the inventors of the present invention have made extensive studies on technical means for controlling the color of hair grown from a hair follicle primordium reconstructed in vitro using cells obtained from adult hair follicle tissue. As a result, surprisingly, by using an adult hair follicle tissue containing a non-constant part as a cell source, it has been found uniquely that the color of hair growing from the hair follicle primordium can be easily and effectively controlled. It came to be completed.
Here, the structure of the adult hair follicle tissue will be described. FIG. 1A schematically shows the structure of an adult hair follicle. FIG. 1B shows the structure of a hair follicle collected from an adult mouse. As shown in FIGS. 1A and 1B, the adult hair follicle tissue that wraps the hair shaft includes a constant part and a non-constant part.
The constant part is also called a stationary part or an invariant part. The constant part occupies approximately half of the upper side of the adult hair follicle tissue (that is, the tip side of the hair shaft). There is a sebaceous gland in the constant part. The homeostatic part includes a bulge region to which the napped muscles are bonded below the sebaceous glands (that is, opposite to the tip of the hair shaft). As shown in FIG. 1B, in the mouse, there is a ring wurst (Ringwurst) at a position corresponding to the napped muscle. The bulge region contains epithelial stem cells.
For this reason, the raw material cell obtained from the adult hair follicle tissue containing the bulge region of the constant part contains the epithelial stem cell obtained from the bulge region. Epithelial stem cells are identified as cells expressing CD34 obtained from, for example, adult hair follicle tissue (particularly the bulge region). The constant part further includes a sub-bulge region below the bulge region. The sub-bulge area constitutes the lowermost part of the constant part.
The non-constant part is also called an unsteady part or a variable part. The non-constant part occupies the lower half of the adult hair follicle tissue. That is, the non-constant part is, for example, a part of the adult hair follicle tissue that is below the sub-bulge region of the constant part. The non-constant part includes a hair bulb part. The hair bulb portion includes a hair papilla and a hair mother. The dermal papilla contains dermal papilla cells. The hair matrix includes hair matrix cells and pigment cells. The non-constant part includes a part other than the hair bulb part (that is, a part between the constant part and the hair bulb part). In addition, it is preferable to use the adult hair follicle tissue in the growth phase of the hair cycle as the cell source of the source cells.
One of the characteristics of this method is that an adult hair follicle tissue containing a non-constant part is used as a cell source of source cells containing epithelial stem cells. The non-constant part contained in the adult hair follicle tissue is not particularly limited as long as it is a part or all of the non-constant part of the adult hair follicle tissue. It is good also as including a part other than a hair ball part, and it is good also as including parts other than a hair nipple and a hair mother.
The weight ratio of the portion other than the hair papilla, the portion other than the hair matrix, the portion other than the hair papilla and the hair matrix, or the portion other than the hair bulb relative to the non-constant portion contained in the adult hair follicle tissue is, for example, 50% by weight. The above may be sufficient, 70 weight% or more may be sufficient, and 90 weight% or more may be sufficient.
The non-constant part contained in the adult hair follicle tissue may not include the hair papilla, may not include the hair matrix, may not include the hair papilla and the hair matrix, and includes the hair bulb part. It's okay to not.
The non-constant part contained in the adult hair follicle tissue may include a part or all of a part between the sub-bulge region of the constant part and the hair bulb part of the non-constant part.
The weight ratio of the part between the subbulge region of the permanent part and the hair bulb part of the non-constant part with respect to the non-constant part contained in the adult hair follicle tissue may be, for example, 50% by weight or more, 70 It may be 90% by weight or more.
As described above, an adult hair follicle tissue containing a non-constant part includes a bulge region of the constant part in order to be used as a cell source of source cells including epithelial stem cells. The adult hair follicle tissue as the cell source of the source cells may further include a subbulge region.
The adult hair follicle tissue used as the cell source of the source cells is a hair follicle tissue collected from an adult animal. The adult animal is not particularly limited as long as it is an adult having a hair follicle as a skin appendage, and may be a human or a non-human animal (an animal other than a human). The non-human animal is not particularly limited, but is preferably a non-human vertebrate (a non-human vertebrate). The non-human vertebrate is not particularly limited, but is preferably a non-human mammal. Non-human mammals are not particularly limited, for example, primates (eg, monkeys), rodents (eg, mice, rats, hamsters, guinea pigs, rabbits) carnivores (eg, dogs, cats), or ungulates (Eg, pig, cow, horse, goat, sheep).
The raw material cells used in this method are cells that are mixed and cultured with hair papilla cells in the production of the hair follicle primordium described below. A raw material cell is obtained by performing an enzyme treatment to the adult hair follicle tissue containing a bulge region and a non-constant part.
The enzyme used for the enzyme treatment is an enzyme effective for releasing the raw material cells from the adult hair follicle tissue (for example, decomposing a matrix around the raw material cells in the adult hair follicle tissue and / or the raw material cells). For example, it may be one or more selected from the group consisting of collagenase, dispase, and trypsin.
The source cells are separately prepared independently from the hair papilla cells. That is, an adult hair follicle tissue used as a cell source of raw material cells is prepared separately from a cell source of hair papilla cells (for example, a hair papilla separated from a living hair follicle tissue). Separately from the preparation of dermal papilla cells, raw material cells are prepared from an adult hair follicle tissue containing a bulge region and a non-constant part.
The source cells include cells obtained from non-constant parts of the adult hair follicle tissue in addition to epithelial stem cells obtained from the bulge region of the living hair follicle tissue. That is, the raw material cells may contain cells obtained from non-constant parts other than the hair papilla, may contain cells obtained from non-constant parts other than the hair matrix, and other than the hair papilla and hair matrix. It is good also as including the cell obtained from non-constant part of this, and good also as including the cell obtained from non-constant part other than a hair bulb part.
In addition, the raw material cells may not include cells obtained from the hair papilla separated from the non-constant part, or may not include cells obtained from the hair matrix separated from the non-constant part, The cells obtained from the hair papilla and hair matrix separated from the non-constant part may not be included, and the cells obtained from the hair bulb part separated from the non-constant part may not be included.
The source cells may not contain cells obtained from the hair papilla, may not contain cells obtained from the hair matrix, and do not contain cells obtained from the hair papilla and hair matrix. It is good also as not including the cell obtained from the hair bulb part.
The hair papilla cell is not particularly limited as long as it can form a part of the hair follicle primordium. For example, it may be a cell derived from the hair papilla of an adult hair follicle tissue, and skin tissue (fetus, juvenile body) Cells derived from any adult skin tissue), cells cultured in advance, or stem cells (eg, induced pluripotent (iPS) stem cells) in vitro. , Embryonic stem (ES) cells, or embryonic germ (EG) cells). Papilla cells are identified as, for example, cells that express Versican.
In this method, a hair follicle primordium is produced by mixing and culturing dispersed hair papilla cells and dispersed raw material cells. That is, mixed culturing of the hair papilla cells and the raw material cells is performed by seeding a mixed cell suspension containing the dispersed hair papilla cells and the dispersed raw material cells in a culture vessel. The mixed cell suspension is prepared by mixing the dermal papilla cell suspension containing dispersed dermal papilla cells and the raw material cell suspension containing dispersed raw material cells.
Note that the dispersed cells are cells that are not bound to other cells or are only bound to several cells, and are dispersed and suspended in the solution. For example, when the cell suspension containing dispersed cells is centrifuged to separate the cells and the solvent contained in the cell suspension, the cell precipitate formed after the centrifugation is The cells that are aggregates and constitute the precipitate are not dispersed cells.
The dermal papilla cell suspension is prepared by dispersing the dermal papilla cells in a culture medium suitable for culturing the dermal papilla cells. A culture solution suitable for culturing hair papilla cells is not particularly limited, but for example, a culture solution containing FGF2 (bFGF) is preferably used.
The concentration of FGF2 contained in the culture medium used for culturing hair papilla is not particularly limited, but may be, for example, 10 ng / mL or more, preferably 30 ng / mL or more, and preferably 50 ng / mL. More preferably, it is more preferably 75 ng / mL or more, and particularly preferably 100 ng / mL or more.
The raw material cell suspension is prepared by dispersing the raw material cells in a culture solution suitable for culturing the raw material cells. The culture solution suitable for culturing the source cells is preferably a culture solution suitable for culturing epithelial stem cells. A culture solution suitable for culturing epithelial stem cells is not particularly limited, and a known culture solution used for culturing epithelial stem cells may be used.
The number ratio between the dermal papilla cells and the raw material cells contained in the mixed cell suspension is not particularly limited as long as the hair follicle primordium is finally produced. The ratio of the number of source cells may be, for example, 0.10 or more and 10.00 or less, preferably 0.20 or more and 5.00 or less, and 0.50 or more and 2.00 or less. More preferably, it is 0.75 or more and 1.50 or less.
The culture medium for mixing and culturing the dispersed dermal papilla cells and the dispersed raw material cells is not particularly limited. For example, it is preferable to use a culture medium containing FGF2. The concentration of FGF2 contained in the culture medium used for the mixed culture is not particularly limited, but may be, for example, 10 ng / mL or more, preferably 30 ng / mL or more, preferably 50 ng / mL or more. More preferably, it is more preferably 75 ng / mL or more, and particularly preferably 100 ng / mL or more.
Cell density (total number of dermal papilla cells and raw material cells contained per unit volume of the mixed cell suspension) in the mixed culture of dermal papilla cells and raw material cells is within the range in which the hair follicle primordium is formed If there is no particular limitation, for example, 5.0 × 10 ⁵ cells / mL or more, 1.0 × 10 ⁸ cells / mL or less, 2.5 × 10 ⁶ cells / mL or more, 5.0 × 10 ⁷ cells / mL or less, preferably 2.5 × 10 ⁶ cells / mL or more, 2.5 × 10 ⁷ The cell / mL or less is particularly preferable.
The culture vessel used for the mixed culture of the hair papilla cells and the raw material cells is not particularly limited. For example, a cell non-adhesive culture vessel is preferably used. A non-cell-adhesive culture vessel is a culture vessel having a non-cell-adhesive surface. That is, for example, when the culture vessel is a recess (bottomed hole) formed on the surface of the substrate, the recess has a cell non-adhesive bottom surface and further has a cell non-adhesive side surface. Is preferred.
A cell non-adhesive surface is a surface to which cultured cells do not adhere and extend. That is, cells cultured in a culture vessel having a non-cell-adhesive surface may adhere weakly to the surface but do not spread on the surface. For this reason, the cells adhered to the non-cell-adhesive surface are detached from the surface and floated, for example, by flowing the culture solution without performing enzyme treatment or chelation treatment.
The culture vessel used for the mixed culture of the hair papilla cells and the raw material cells is preferably, for example, a recess formed on the surface of the base material having a relatively large oxygen permeability. That is, as a culture container, for example, the oxygen permeability is 100 cm. ³ / (M ² It is preferably a recess formed in a base material of 24 h · atm) or more, and the oxygen permeability is 500 cm. ³ / (M ² More preferably, it is a recess formed in a base material of 24 h · atm) or more, and the oxygen permeability is 1000 cm. ³ / (M ² It is particularly preferable that the recess is formed on a base material of 24 h · atm) or more.
For example, a silicon-based material (for example, polydimethylsiloxane (PDMS)) is preferably used as a material constituting the base material having high oxygen permeability.
When a recess formed on the surface of the substrate is used as a culture container used for mixed culture of follicular papilla cells and raw material cells, the volume of the recess and the shape and area of the bottom surface of the recess are not particularly limited. . The volume of the recess may be, for example, 1 nL or more and 1 mL or less, preferably 10 nL or more and 100 μL or less, and particularly preferably 100 nL or more and 10 μL or less. The shape of the bottom surface of the recess may be, for example, a circle, an ellipse, or a polygon. The area of the bottom surface of the recess is, for example, 1 μm ² 100 mm ² It may be less than 10 μm ² 10 mm ² Or less, preferably 100 μm ² 1mm or more ² It is particularly preferred that
The culture vessel used for the mixed culture of follicular dermal papilla cells and raw material cells includes a plurality of recesses regularly formed on the surface of the substrate (for example, a plurality of linearly or meshedly arranged at regular intervals). It is good also as using a recessed part.
The mixed culture time of the hair papilla cells and the raw material cells is not particularly limited as long as the follicle primordium can be formed. For example, it may be 12 hours or more, 10 days or less, or 1 day or more, It is preferable that it is 7 days or less. The culture temperature is not particularly limited as long as it is within the range in which the hair follicle primordium can be formed. For example, the culture temperature may be 25 ° C. or more and 40 ° C. or less, and preferably 35 ° C. or more and 39 ° C. or less. .
A hair follicle that is a composite cell aggregate comprising the aggregated portion of the hair papilla cell and the aggregated portion of the raw material cell by culturing the mixed hair papilla cell and the raw material cell in the mixed cell suspension A primordial is formed.
That is, the hair follicle primordium includes a hair papilla cell aggregation part formed by binding and aggregation of hair follicle cells to each other, and a raw material cell aggregation part formed by binding and aggregation of raw material cells to each other. . The source cell aggregated part may include an epithelial stem cell aggregated part formed by combining and aggregating epithelial stem cells.
In the hair follicle primordium, a part of the hair papilla cell aggregation part and a part of the raw material cell aggregation part are bonded. That is, a part of hair papilla cells contained in the hair papilla cell aggregation part and a part of raw material cells contained in the raw material cell aggregation part are bonded. In this case, a part of the hair papilla cells contained in the hair papilla cell aggregation part and a part of the epithelial stem cells contained in the raw material cell aggregation part may be combined.
The formation of the hair follicle primordium in this method is carried out after the dermal papilla cells and the raw material cells are dispersed and mixed, and then the dermal papilla cells bind to each other and spontaneously aggregate, Achieved by combining source cells (for example, epithelial stem cells) and spontaneously agglutinating, and combining some of the dermal papilla cells and some of the source cells (for example, some epithelial stem cells). Is done.
The shape of the hair follicle primordium formed in this method is not particularly limited, and may be, for example, spherical or oblong. The volume of the hair follicle primordium formed in the present method is not particularly limited. For example, 1 × 10 ^-5 mm ³ 10 mm ³ 1 × 10 ^-4 mm ³ 1mm or more ³ Preferably, it is 1 × 10 ^-3 mm ³ 0.1 mm ³ It is particularly preferred that
The density of cells contained in the hair follicle primordium (the total number of cells contained per unit volume of the hair follicle primordium) is not particularly limited. ⁶ cells / cm ³ 7.0 × 10 ¹⁰ cells / cm ³ It may be the following, 5.0 × 10 ⁶ cells / cm ³ 6.5 × 10 ¹⁰ cells / cm ³ Or less, preferably 1.0 × 10 ⁷ cells / cm ³ Or more, 5.0 × 10 ¹⁰ cells / cm ³ It is particularly preferred that
In this method, the hair follicle primordium which grows hair by being transplanted to an animal is produced by mixing and cultivating dispersed hair papilla cells and dispersed raw material cells. That is, the hair follicle primordium formed by the mixed culture in this method can be transplanted to an animal as described later, and hair is grown from the hair follicle primordium after being transplanted to the animal.
In the present method, when the produced hair follicle primordium is transplanted into an animal to produce hair from the hair follicle primordium, the animal is not particularly limited and may be a human or a non-human animal. Good. The non-human animal is not particularly limited, but is preferably a non-human mammal. Non-human mammals are not particularly limited, for example, primates (eg, monkeys), rodents (eg, mice, rats, hamsters, guinea pigs, rabbits), meat (eg, dogs, cats), or It may be a hoof (eg, pig, cow, horse, goat, sheep).
The transplantation of the hair follicle primordium to the animal is preferably a transplantation to the skin of the animal. The skin transplantation may be, for example, a subcutaneous transplantation or an intradermal transplantation.
The production of the hair follicle primordium and its implantation into animals in this method may be for medical use or research use. That is, in this method, for example, for the treatment or prevention of diseases associated with hair loss, a hair follicle primordium is produced for the purpose of transplanting to a human patient suffering from or likely to suffer from the disease, or The hair follicle primordium may be transplanted into the human patient.
Diseases associated with hair loss are not particularly limited. For example, androgenetic alopecia (AGA), female androgenetic alopecia (FAGA), postpartum alopecia, diffuse alopecia, seborrhea Alopecia, alopecia areata, traction alopecia, metabolic alopecia, pressure alopecia, alopecia areata, alopecia areata, alopecia, systemic alopecia, and symptomatic alopecia It may be one or more selected from the group.
In this method, for example, a hair follicle primordium is produced in order to search for a substance that can be used for treatment or prevention of a disease associated with hair loss and / or to search for a substance involved in the mechanism of the disease. Alternatively, the hair follicle primordium may be transplanted into a non-human animal.
And in the color control method of regenerated hair according to the present embodiment, the weight ratio of the non-constant part contained in the adult hair follicle tissue (that is, the weight relative to the weight of the adult hair follicle tissue used as the cell source of the source cells) By changing the ratio of the weight of the non-constant part contained in the adult hair follicle tissue, the color of the hair growing from the hair follicle primordium is changed.
Specifically, for example, by increasing the weight ratio of the non-constant part contained in the adult hair follicle tissue, the color of hair growing from the transplanted hair follicle primordium is increased. Here, the darkness of the hair color can be evaluated visually, but for example, it may be quantitatively evaluated as the amount of the melanin pigment contained per unit weight of the hair. The melanin pigment contained in the hair is quantified by, for example, incubating the hair to be measured in a sodium hydroxide solution at 60 ° C. for 30 minutes to extract melanin, and then measuring the absorbance at 405 nm using an absorptiometer. By doing so, the extracted melanin can be quantified.
Further, in the hair regeneration method according to the present embodiment, by using an adult hair follicle tissue containing a non-constant part, an adult hair follicle tissue not containing the non-constant part is obtained from the transplanted hair follicle primordia. Produces darker hair than when used.
That is, for example, a non-constant part including a part other than the hair papilla, a non-constant part including a part other than the hair matrix, a non-constant part including a part other than the hair papilla and the hair matrix, or a non-constant part including a part other than the hair bulb part It is obtained from an adult hair follicle tissue that does not contain the non-constant part by transplanting the first hair follicle primordium produced using the raw material cells obtained from the adult hair follicle tissue containing the constant part to an animal. Compared with the case where the second hair follicle primordium produced using the raw material cells is transplanted into the animal, dark hair is grown from the transplanted first hair follicle primordium.
In addition, the first hair follicle primordium produced using the first adult hair follicle tissue containing the non-constant part at a first weight ratio is transplanted into an animal, A second cell aggregate produced using a second adult hair follicle tissue that grows hair of the first color and includes the non-constant part at a second weight percentage greater than the first weight percentage. May be transplanted into an animal to grow second colored hair darker than the first color from the second hair follicle primordium.
In this case, by adopting a plurality of weight ratios as the weight ratio of the non-constant part contained in the living hair follicle tissue used as the cell source of the source cells, the color concentration is increased from the plurality of transplanted hair follicle primaries. A plurality of different hairs can be grown.
Further, in the method for producing a hair follicle primordium according to the present embodiment, by using an adult hair follicle tissue containing a non-constant part, compared to a case where an adult hair follicle tissue not containing the non-constant part is used. Produces a hair follicle primordium that produces dark hair.
That is, for example, a non-constant part including a part other than the hair papilla, a non-constant part including a part other than the hair matrix, a non-constant part including a part other than the hair papilla and the hair matrix, or a non-constant part including a part other than the hair bulb part By using a raw material cell obtained from an adult hair follicle tissue containing a constant part, it is transplanted to an animal as compared with a case where a raw material cell obtained from an adult hair follicle tissue not containing the constant part is used. To produce a hair follicle primordium that produces dark hair.
In addition, by using a first adult hair follicle tissue containing a non-constant part in a first weight ratio, a first hair follicle primordium that produces hair of the first color by being transplanted to an animal is produced. And using the second adult hair follicle tissue containing the non-constant part at a second weight ratio that is greater than the first weight ratio, so that the non-constant part can be transplanted into the animal by the first color. A second follicle primordium that produces dark second colored hair is produced.
In this case, by adopting a plurality of weight ratios as the weight ratio of the non-constant part contained in the living hair follicle tissue used as the cell source of the raw material cells, hair having different color densities can be produced when transplanted. Multiple hair follicle primordia can be produced.
The weight ratio of the non-constant part contained in the living hair follicle tissue is not particularly limited as long as it is in the range of 0% or more and 100% or less. That is, when the first weight ratio is 0% (that is, when an adult hair follicle tissue not including a non-constant part is used), the second weight ratio may be, for example, 5% or more. It may be 10% or more, may be 20% or more, and may be 30% or more. Usually, when the non-constant part is not removed from the living hair follicle tissue (that is, when the living hair follicle tissue includes both the constant part and the non-constant part), the weight of the non-constant part relative to the weight of the adult hair follicle tissue The ratio is about 40%.
In this method, when the weight ratio of the non-constant part contained in the living hair follicle tissue is 0%, that is, when an adult hair follicle tissue that does not contain a non-constant part is used as the cell source of the source cells, Thus, by mixing and cultivating the dispersed hair papilla cells and the dispersed raw material cells, a hair follicle primordium that produces white hair when transplanted to an animal can be produced. it can.
That is, according to this method, by using raw material cells obtained from an adult hair follicle tissue containing a sub-bulge region in addition to a bulge region, a hair follicle primordium in which hairs that are substantially uncolored grow is produced. The
Therefore, for example, when adopting the conditions under which white hair grows and the conditions under which colored hair grows, the present method can be used as a research tool for obtaining knowledge relating to hair color control. it can. The method can also be used to artificially produce white hair.
Further, when the first weight ratio is greater than 0%, the ratio of the second weight ratio to the first weight ratio is not particularly limited as long as it is greater than 1, but is, for example, 1.5 or more. It is good also as being 2.0 or more, it is good also as being 5.0 or more, and good also as being 10.0 or more.
Next, specific examples according to the present embodiment will be described.

[Collecting dermal papilla cells]
Papilla cells were collected from the dermal papilla of adult hair follicle tissue. That is, first, pupae were collected from 7-week-old adult C57BL / 6 mice, and adipose tissue around the pupae was removed. Next, the hair papilla was cut out from the eyelash follicle. The hair papilla was immersed in an enzyme solution containing 100 U / mL collagenase and 4.8 U / mL dispase and subjected to enzyme treatment in a CO ₂ incubator at 37 ° C. for 40 minutes. Cells isolated from the hair papilla by enzyme treatment were seeded in a culture dish and cultured in a medium containing 10 ng / mL or 100 ng / mL FGF2. The cultured cells were collected using a trypsin-EDTA solution and used for experiments.
[Collection of source cells]
Raw material cells containing epithelial stem cells were collected from an adult hair follicle tissue containing a constant part including a bulge region and a non-constant part from which the hair papilla and hair matrix were removed. That is, first, pupae were collected from 7-week-old adult C57BL / 6 mice, and adipose tissue around the pupae was removed. Next, the hair papilla and hair matrix were removed from the eyelash follicle. The hair follicle tissue from which the hair papilla and hair matrix have been removed is immersed in an enzyme solution containing 100 U / mL collagenase and 4.8 U / mL dispase, and the enzyme treatment is performed at 37 ° C. for 10 minutes in a CO ₂ incubator. Went. Thereafter, the hair follicle tissue was transferred to a DMEM / HEPES medium prepared by mixing HEPES in a DMEM medium, and the collagen sheath was removed from the hair follicle tissue. Furthermore, it was immersed in a trypsin solution in a hair follicle tissue, and an enzyme treatment was performed at 37 ° C. for 60 minutes in a CO ₂ incubator. Thereafter, the cells isolated from the hair follicle tissue by the enzyme treatment were dispersed by pipetting, and further subjected to a unification treatment with a 70 μm cell strainer, and the obtained cells were used for experiments.
[Microscopic observation of cultured dermal papilla cells]
2A and 2B show phase contrast micrographs of hair papilla cells cultured for 2 days in a medium containing 10 ng / mL and 100 ng / mL FGF2, respectively. 2C and 2D show phase contrast micrographs of hair papilla cells cultured for 9 days in a medium containing 10 ng / mL and 100 ng / mL FGF2, respectively. In any of FIGS. 2A to 2D, it was observed that the cells proliferated so as to spread concentrically from the dermal papilla.
[Identification of dermal papilla cells and epithelial stem cells]
FIG. 3A shows a result obtained by applying immunohistochemical staining of Versican, a marker protein of hair papilla cells, to cells collected from the hair papilla of adult hair follicle tissue and cultured as described above, and observed with a fluorescence microscope. Indicates. As shown in FIG. 3A, the presence of cells expressing Versican was confirmed by fluorescent double staining of Versican / DAPI (the arrows in the figure indicate one of the cells expressing Versican). That is, it was confirmed that dermal papilla cells were obtained from the dermal papilla of the adult hair follicle tissue.
FIG. 3B shows that cells collected from the adult hair follicle tissue from which the hair papilla and hair matrix were removed as described above were subjected to immunohistochemical staining of CD34, which is a marker protein of hair follicle epithelial stem cells. The result observed with the microscope is shown. As shown in FIG. 3B, the presence of cells expressing CD34 was confirmed by fluorescent double staining of CD34 / DAPI (the arrow in the figure indicates one of the cells expressing CD34). That is, it was confirmed that epithelial stem cells were contained in the raw material cells obtained from the adult hair follicle tissue including the constitutive part including the bulge region and the non-constant part from which the hair papilla and hair matrix were removed. .

[Production of multiwell culture vessel]
A multi-well culture container for co-culturing hair papilla cells and raw material cells was prepared in the same manner as in Patent Document 2 described above. That is, first, CAD software (V CarvePro 6.5) was used to design a multiwell pattern to be produced by a computer. Next, a concave mold having the pattern was manufactured by cutting the olefin resin substrate according to the designed pattern using a cutting machine. An epoxy resin (Crystal lysine, manufactured by Nissin Resin Co., Ltd.) was poured into this mold, cured for 1 day, and then released to form a convex mold having the above designed pattern. The formed convex mold is fixed to the bottom surface of a 24-well plate, and polydimethylsiloxane (PDMS) is poured and solidified, and then released to form a multiwell (each well formed in a regular pattern on the PDMS substrate). Was prepared in a multiwell culture container (hereinafter referred to as “PDMS spheroid chip”) having a diameter of 1 mm and a depth of 1 mm.
In this PDMS spheroid chip, since a well is formed on a PDMS substrate having excellent oxygen permeability, an appropriate amount of oxygen is contained in cells and cell aggregates cultured in the well throughout the culture period. Was supplied.
[Collecting dermal papilla cells]
As in Example 1 above, dermal papilla cells were collected from the dermal papilla of adult hair follicle tissue and cultured.
[Collection of source cells]
Similar to Example 1 above, from an adult hair follicle tissue containing a constitutive part containing a bulge region and a non-constant part from which the hair papilla and hair matrix were removed, a source cell containing epithelial stem cells (in this example, “ "Source cells (Full)") were collected.
In addition, from an adult hair follicle tissue from which a non-constant part has been removed (that is, an adult hair follicle tissue including a bulge region and a sub-bulge region but not including a non-constant part), a source cell containing epithelial stem cells (in this example, “source material” Cells (1/2) ") were collected. That is, first, pupae were collected from 7-week-old adult C57BL / 6 mice, and adipose tissue around the pupae was removed. Subsequently, the non-constant part including a hair papilla and a hair mother was removed from the eyelash follicle by cutting a part below the constant part of the eyelash follicle (that is, a part below the subbulge region).
Next, the hair follicle tissue from which the non-constant parts have been removed is immersed in an enzyme solution containing 100 U / mL collagenase and 4.8 U / mL dispase, and the enzyme treatment is performed at 37 ° C. for 10 minutes in a CO ₂ incubator. Went. Thereafter, the hair follicle tissue was transferred to a DMEM / HEPES medium prepared by mixing HEPES in a DMEM medium, and the collagen sheath was removed from the hair follicle tissue. Furthermore, it was immersed in a trypsin solution in a hair follicle tissue, and an enzyme treatment was performed at 37 ° C. for 60 minutes in a CO ₂ incubator. Thereafter, the cells isolated from the hair follicle tissue by enzyme treatment are dispersed by pipetting, and further subjected to a singularization treatment by a 70 μm cell strainer, and the obtained cells are used as “source cells (1/2)” for the experiment. used.
[Manufacture of hair follicle primordium]
As a medium used for culturing hair follicle primordia, a mesenchymal cell culture medium (DMEM + 10% FBS + 1% P / S (Penicillin-Streptomycin)) and HuMediaKG2 medium are mixed at a volume ratio of 1: 1, and FGF2 is mixed with 10 ng / mL. A mixed medium was prepared by addition.
The dermal papilla cells obtained as described above were dispersed in a mixed medium to prepare a dermal papilla cell suspension. Moreover, the raw material cells obtained as described above were dispersed in a mixed medium to prepare a raw material cell suspension. Then, the dermal papilla cell suspension and the raw material cell suspension were mixed to prepare a mixed cell suspension containing the dispersed dermal papilla cells and the raw material cells.
In each well of the multi-well culture container prepared as described above, the cell density of the hair papilla cells and hair follicle epithelial stem cells is 1 × 10 ⁴ cells / well (total number of cells 2 × 10 ³ cells / well). As described above, the mixed cell suspension was seeded and cultured in a CO ₂ incubator at 37 ° C. for 3 days.
When the inside of the well was observed with a phase-contrast microscope on the third day of culture, it was confirmed that the cells seeded in the well aggregated to form a spherical cell aggregate (so-called spheroid). . In a preliminary study, this cell aggregate is mainly composed of a hair papilla cell aggregate part containing hair papilla cells, and a epithelial cell aggregate part mainly binding to the hair papilla cell aggregate part and mainly containing epithelial stem cells. It was confirmed to contain.
As described above, such cell aggregates can be formed by mixing dispersed hair papilla cells and dispersed raw material cells and culturing them in a non-adhesive culture vessel. This is presumed to be achieved by spontaneously gathering cells together to form each cell aggregate.
[Transplantation of hair follicle primordia]
The hair follicle primordium formed by culturing for 3 days was collected, and the hair follicle primordium was transplanted by direct injection into a 5-week-old SCID-nu mouse subcutaneously using a syringe.
[Observation of regenerated hair]
Three weeks after transplantation, the hair follicle primordium implanted subcutaneously in the mouse was observed with a digital microscope.
FIG. 4A and FIG. 4B show the results of observing the hair follicle primordium produced using the raw material cells (1/2) and the hair follicle primordium produced using the raw material cells (Full), respectively. Show. In each figure, the arrow points to the hair grown from the transplanted hair follicle primordium.
As shown in FIG. 4A, white hair grows from the hair follicle primordium produced using the raw material cells (1/2) obtained from the adult hair follicle tissue not containing the non-constant part. Was confirmed. On the other hand, as shown in FIG. 4B, from a hair follicle primordium produced using raw material cells (Full) obtained from an adult hair follicle tissue containing a non-constant part from which the hair papilla and hair matrix are removed, It was confirmed that colored dark hair grew.
That is, by using a raw material cell (Full) obtained from an adult hair follicle tissue containing non-constant parts other than the hair papilla and hair matrix, a raw material cell obtained from an adult hair follicle tissue containing no non-constant part ( It was confirmed that a hair follicle primordium having darker hair than that in the case of using 1/2) can be produced.
Thus, the hair follicle formed by using the raw material cells for the content of non-constant parts (especially non-constant parts other than the hair papilla and hair matrix) in the adult hair follicle tissue that is the cell source of the raw material cells. Although the mechanism for changing the color of hair growing from the primordia is not clear, for example, depending on the content of the non-constant part, the content of cells that control hair color such as pigment cells contained in the hair follicle primordium and It is presumed that the distribution is changed.

[Collecting dermal papilla cells]
As in Example 1 above, dermal papilla cells were collected from the dermal papilla of adult hair follicle tissue and cultured.
[Collection of source cells]
In the same manner as in Example 1 above, raw material cells containing epithelial stem cells were collected from an adult hair follicle tissue containing a constant part including the bulge region and a non-constant part from which the hair papilla and hair matrix were removed.
[Manufacture of hair follicle primordium]
As described above, except that FGF2 containing mixed medium in which 10 ng / mL, 50 ng / mL, or 100 ng / mL of FGF2 was added to the mixed medium used in Example 2 was used as the medium used for culturing hair follicle primordium. In the same manner as in Example 2, a hair follicle primordium was produced using a multiwell culture vessel.
[Microscopic observation of hair follicle primordium]
FIG. 5A, FIG. 5B, and FIG. 5C show the results of observation of the hair follicle primordium formed by culturing in a mixed medium containing 10 ng / mL, 50 ng / mL, and 100 ng / mL of FGF2 with a phase contrast microscope, respectively. Show. The scale bar in FIGS. 5A to 5C indicates 500 μm.
FIG. 5D, FIG. 5E, and FIG. 5F show the results of observation of the hair follicle primordium formed by culturing in a mixed medium containing 10 ng / mL, 50 ng / mL, and 100 ng / mL FGF2 with a phase contrast microscope, respectively. Show. The scale bar in FIGS. 5D to 5F indicates 1 mm.
As shown in FIGS. 5A to 5F, the form of the hair follicle primordium formed by the culture for 3 days did not differ depending on the FGF2 content of the mixed medium.
[Analysis of gene expression related to hair growth]
RT-PCR analysis was performed on the expression of hair growth marker genes (Versican and Wnt-10b) in the hair follicle primordium formed by 3 days of culture. That is, RNA was first extracted from the hair follicle primordium. Specifically, the hair follicle primordium was collected in a 15 mL tube, and when the hair follicle primordium was precipitated, the supernatant was removed so that the volume of the solution was 1 mL. Next, 1 mL of the solution containing the hair follicle primordium was transferred to a 1.5 mL microtube and centrifuged at 4 ° C. and 5000 rpm for 3 minutes.
After discarding the supernatant of the solution after centrifugation, 350 μL of Buffer RLT was added and well pipetted. Further, the solution after pipetting was collected on a QIA Shredder spin column and centrifuged at 4 ° C. and 10,000 rpm for 2 minutes. The top of the QIAShredder spin column was discarded and 350 μL of 70% ethanol was added to the solution in the collection tube. The solution was transferred to an RNeasy spin column and centrifuged at 10000 rpm at 4 ° C. for 15 seconds.
The filtrate in the collection tube was discarded, 700 μL of Buffer RW1 was added, and the mixture was centrifuged at 4 ° C. and 10,000 rpm for 15 seconds. The filtrate in the collection tube was discarded, 500 μL of Buffer RPE was added, and the mixture was centrifuged at 4 ° C. and 10,000 rpm for 15 seconds. The filtrate in the collection tube was discarded, 500 μL of Buffer RPE was added, and the mixture was centrifuged at 4 ° C. and 10,000 rpm for 2 minutes. The column after centrifugation was transferred to a new 2 mL collection tube and centrifuged at 4 ° C. and 10,000 rpm for 1 minute. This was done to remove any remaining Buffer RPE.
The column after centrifugation was transferred to a 1 mL microtube, 30 μL of RNase free water was added, and the mixture was centrifuged at 10000 rpm at 4 ° C. for 1 minute. 30 μL of RNase free water was again added to the 1 mL microtube on which the column after centrifugation was installed, and centrifuged at 10000 rpm at 4 ° C. for 1 minute to obtain an RNA solution.
The RNA concentration was then measured with a spectrophotometer. That is, the spectrophotometer (Nano Vue) was turned on and the dilution rate was set to 60.0. Here, the dilution rate is the final volume in RNA extraction. After the measurement plate was wiped with 70% ethanol, 2 μL of RNase free water was applied to the center of the measurement plate, and the [OA / 100% T] button was pressed. A baseline was obtained by this operation.
2 μL of RNA lysate obtained from the hair follicle primordium as described above was applied to the center of the measurement plate, and the measurement button was pressed. A260 / A280 represents the purity of the sample and is preferably close to 2.0.
Thereafter, RT-PCR was performed. That is, the RNA lysate whose RNA concentration was measured as described above was diluted so that the RNA concentration became 150 μg / mL. The diluted RNA lysate was incubated at 65 ° C. for 5 minutes and cooled on ice.
Thereafter, 2 μL of RNA lysate, 12 μL of Nuclear free water, 4 μL of 5 × RT Buffer, 1 μL of Primer mix, and 1 μL of Enzyme mix were placed in a microtube and covered with a transparent film.
The microtube was set on a thermal cycler and confirmed to be tightly closed. Thereafter, reverse transcription was performed at 37 ° C. for 15 minutes and at 98 ° C. for 5 minutes to obtain cDNA, which is a reverse transcription product of RNA derived from hair follicle primordia.
Thereafter, 1 μL of cDNA (reverse transcript of RNA derived from hair follicle primordium), 10 μL of SYBR Green master mix, 0.4 μL of Forward Primer, 0.4 μL of Reverse Primer, 0.4 μL of Dye, , 7.8 μL of Nuclear free water was placed in a microtube and covered with a transparent film.
In addition, the base sequences of the primers used in the PCR are Versican, Forward Primer is “5′-GACGACTGTTCTTGGTGGG-3 ′”, Reverse Primer is “5′-ATATCCAAACAAGCCTG-3 ′”, and Wnt-10b is Forward Prime “ 5'-CCAAGAGCCGGGCCCGAGTGA-3 '", Reverse Primer is"5'-AAGGGCGGAGGGCCGAGACCG-3'", Forward Primer is"5'-AGAACATCATCCCTCATCSECC-3CT" -3 ′ ”.
The microtube was set on a thermal cycler and confirmed to be tightly closed. Thereafter, PCR was performed with a protocol of 95 ° C. for 4 minutes, (95 ° C. for 5 seconds, 60 ° C. for 60 seconds) × 45 cycles, 72 ° C. for 10 minutes. As a control, the expression level of GAPDH was measured, and the relative expression levels of Versican and Wnt-10b with respect to the expression level of GAPDH were calculated.
6A and 6B show the relative gene expression levels of Versican and Wnt-10b in the hair follicle primordia, respectively. 6A and 6B confirmed that the gene expression levels of Versican and Wnt-10b in the hair follicle primordia increased depending on the FGF2 concentration of the medium.
[Transplantation of hair follicle primordia]
The hair follicle primordium formed by the culture for 3 days was collected and transplanted to the skin of a 5-week-old SCID-nu mouse by the patch method. That is, a plurality of hair follicle primordia were injected in the vicinity of the dermis or fascia of mouse skin.
[Observation of regenerated hair]
Six weeks after the transplantation, the hair follicle primordium transplanted into the mice was observed with a digital microscope. FIG. 7A and FIG. 7B show the results of observing hair follicle primordia formed in a medium containing 10 ng / mL and 100 ng / mL FGF2, respectively. In each figure, the arrow points to the transplanted hair follicle primordium.
From FIG. 7A and FIG. 7B, the hair follicle primordium formed in the medium containing 100 ng / mL FGF is more preferable than the hair follicle primordium formed in the medium containing 10 ng / mL FGF. It was confirmed to show high hair regeneration ability.
This application claims the priority on the basis of Japanese application Japanese Patent Application No. 2018-094498 for which it applied on May 16, 2018, and takes in those the indications of all here.

Claims (11)

1. Using raw material cells containing epithelial stem cells obtained from adult hair follicle tissue containing the bulge region of the homeostasis,
   A hair follicle primordium is produced by mixing and culturing the dispersed hair papilla cells and the dispersed raw material cells, and transplanting the hair follicle primordia to an animal to produce the hair follicle primordium. Grow hair from
   Including
   A method for controlling the color of regenerated hair, wherein the color of hair grown from the hair follicle primordium is changed by changing the weight ratio of the non-constant part contained in the adult hair follicle tissue.
2. The method for controlling the color of regenerated hair according to claim 1, wherein the animal is a non-human animal.
3. The method for controlling the color of regenerated hair according to claim 1 or 2, wherein the color of the hair grown from the hair follicle primordium is increased by increasing the weight ratio of the non-constant part contained in the adult hair follicle tissue.
4. The non-constant part includes parts other than the hair papilla and the hair mother,
   The method for controlling the color of regenerated hair according to claim 1, comprising:
5. Using raw material cells containing epithelial stem cells obtained from adult hair follicle tissue containing the bulge region of the homeostasis,
   A hair follicle primordium is produced by mixing and culturing the dispersed hair papilla cells and the dispersed raw material cells, and transplanting the hair follicle primordia to an animal to produce the hair follicle primordium. Grow hair from
   Including
   By using the adult hair follicle tissue further comprising a non-constant part, hair having a darker color than the case of using the adult hair follicle tissue not containing the non-constant part from the hair follicle primordium is obtained. How to play.
6. The hair regeneration method according to claim 5, wherein the animal is a non-human animal.
7. Transplanting the first hair follicle primordium produced using the first adult hair follicle tissue containing the non-constant part in a first weight ratio into the animal, Growing a first color hair, and a second cell aggregation produced using a second adult hair follicle tissue comprising the non-constant part in a second weight percentage greater than the first weight percentage. Transplanting a lump into the animal to grow a second colored hair darker than the first color from the second follicle primordium,
   The method for regenerating hair according to claim 5 or 6, comprising:
8. The non-constant part includes parts other than the hair papilla and the hair mother,
   The method for regenerating hair according to any one of claims 5 to 7, comprising:
9. Use of raw material cells including epithelial stem cells obtained from adult hair follicle tissue containing a bulge region in the homeostatic region, and mixing and culturing the dispersed hair papilla cells and the dispersed raw material cells To produce a hair follicle primordium that grows hair by being transplanted into an animal,
   Including
   By using the adult hair follicle tissue further comprising a non-constant part, the hair follicle primordium that produces darker hair than when using the adult hair follicle tissue not containing the non-constant part is produced. Manufacturing method of hair follicle primordium.
10. By using the adult hair follicle tissue containing the non-constant part in a first weight ratio, a first hair follicle primordium that produces hair of the first color by being transplanted to the animal is produced. And a second darker than the first color by being transplanted into the animal by using the adult hair follicle tissue comprising the non-constant part in a second weight percentage greater than the first weight percentage. Producing a second hair follicle primordium that produces
    The manufacturing method of the hair follicle primordium of Claim 9 containing this.
11. The non-constant part includes parts other than the hair papilla and the hair mother,
    The manufacturing method of the hair follicle primordium of Claim 9 or 10 containing this.

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