U.S. patent application number 12/782284 was filed with the patent office on 2010-09-09 for preparation method of a hair dermal papilla cell preparation, composition and method for regenerating hair follicles, and animal having regenerated hair follicles.
This patent application is currently assigned to SHISEIDO COMPANY, LTD.. Invention is credited to Takayuki Arai, Ritsuko Ehama, Ritsuro Ideta, Jiro Kishimoto, Tsutomu Soma, Kiichiro Yano.
Application Number | 20100227397 12/782284 |
Document ID | / |
Family ID | 34426691 |
Filed Date | 2010-09-09 |
United States Patent
Application |
20100227397 |
Kind Code |
A1 |
Kishimoto; Jiro ; et
al. |
September 9, 2010 |
Preparation method of a hair dermal papilla cell preparation,
composition and method for regenerating hair follicles, and animal
having regenerated hair follicles
Abstract
The present invention provides a method for preparing a hair
dermal papilla cell preparation comprising preparing a cell
suspension by removing epidermal tissue from skin tissue and
subjecting the resulting dermal tissue fraction to collagenase
treatment, and cyropreserving the cell suspension to kill the
follicular epidermal cells. The present invention also provides a
composition for regenerating hair follicles comprising hair dermal
papilla cell and epidermal cells, wherein the ratio of the number
of hair dermal papilla cell to the number of epidermal cells is
from 1:10 to 10:1.
Inventors: |
Kishimoto; Jiro; (Kanagawa,
JP) ; Ehama; Ritsuko; (Kanagawa, JP) ; Ideta;
Ritsuro; (Kanagawa, JP) ; Arai; Takayuki;
(Kanagawa, JP) ; Yano; Kiichiro; (Kanagawa,
JP) ; Soma; Tsutomu; (Kanagawa, JP) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
SHISEIDO COMPANY, LTD.
Tokyo
JP
|
Family ID: |
34426691 |
Appl. No.: |
12/782284 |
Filed: |
May 18, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10574697 |
Apr 5, 2006 |
7718426 |
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PCT/JP2004/014779 |
Sep 30, 2004 |
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12782284 |
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Current U.S.
Class: |
435/353 ;
435/354; 435/366; 435/374 |
Current CPC
Class: |
C12N 2502/094 20130101;
C12N 5/0698 20130101; C12N 5/0627 20130101; C12N 2502/1323
20130101; A61P 17/14 20180101; A61K 35/36 20130101; A01K 67/0271
20130101; A01K 2267/03 20130101 |
Class at
Publication: |
435/353 ;
435/374; 435/354; 435/366 |
International
Class: |
C12N 5/071 20100101
C12N005/071 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 6, 2003 |
JP |
2003-346937 |
Oct 6, 2003 |
JP |
2003-346939 |
Feb 24, 2004 |
JP |
2004-048322 |
Claims
1. A method for preparing a hair dermal papilla cell preparation
comprising: providing a cell suspension by removing epidermal
tissue from skin tissue and subjecting the resulting dermal tissue
fraction to collagenase treatment, and cryopreserving the cell
suspension to kill the follicular epidermal cells.
2. A method according to claim 1, wherein the cryopreservation is
carried out after adjusting the cell density of the cell suspension
to 1.times.10.sup.5 to 1.times.10.sup.8/ml.
3. A method according to claim 1, wherein the cryopreservation is
carried out at a temperature of -80.degree. C. or lower.
4. A method according to claim 1, wherein the cryopreservation is
carried out in liquid nitrogen.
5. A method according to claim 1, wherein the cryopreservation is
carried out for a period of 1 week or more.
6. A method according to claim 1, wherein the skin tissue is from a
mouse.
7. A method according to claim 1, wherein the skin tissue is from a
rat.
8. A method according to claim 1, wherein the skin tissue is from a
human.
9-36. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention provides a method for preparing a hair
dermal papilla cell preparation containing active hair dermal
papilla cell and in which epidermal cells contained therein are
deactivated, a composition containing hair dermal papilla cell and
epidermal cells for regenerating hair follicles, a method for
regenerating hair follicles using the same, and an animal or
three-dimensional skin equivalent having hair follicles regenerated
by such a method.
BACKGROUND ART
[0002] Hair follicles are exceptional organs that repeatedly
self-regenerate in a mature body throughout nearly the entire
lifetime. Elucidation of the mechanism of this self-regeneration is
expected to lead to clinical applications for which there are
considerable needs, including hair loss therapy by tissue and cell
transplant, and the construction of natural and functionally
superior hair sheets containing hair follicles and sebaceous
glands. Accompanying the growing interest in stem cell research in
recent years, research on follicular epithelial stem cells
(epidermal cells) has progressed rapidly, and the properties of
hair dermal papilla cell serving as mesenchymal cells specific to
hair follicles have gradually been determined. Hair dermal papilla
cell fulfill the role of a so-called control tower, sending out
activating signals to follicular epithelial stein cells for
self-regenerating hair follicles, and have been determined to be
indispensable along with follicular epithelial stem cells in
evaluation of follicle reconstitution (Kishimoto et al., Proc.
Natl. Acad. Sci. USA (1999), Vol. 96, pp. 7336-7341).
[0003] Hair follicle reconstitution experiments in animal models
have been conducted using various methods for the purpose of hair
follicle regeneration. Weinberg et al., J. Invest. Dermatol.
(1993), Vol. 100, pp. 229-236 describes a hair follicle
reconstitution method using a cell transplant method. The
transplant system of Weinberg at al. has a complex constitution
consisting of hair dermal papilla cell, newborn animal epidermal
cells (including follicular epithelial stem cells) as well as the
addition of mouse 3T3 cells. According to the method of Weinberg et
al., hair follicles are regenerated without adding newborn animal
epidermal cells containing follicular epithelial stem cells to the
transplant system. However, this is thought to be phenomenon that
occurred due to the difficulty in completely removing
undifferentiated epidermal cells (follicular epithelial stem cells)
and primordial hair follicles from the dermal cell fraction.
Subsequently, Kishimoto et al. (op. cit.) succeeded for the first
time in isolating and purifying hair dermal papilla cell, and as a
result of conducting a hair follicle reconstitution experiment
according to a cell transplant method in an animal model using the
isolated and purified hair dermal papilla cell, hair follicles were
reorganized and hair growth was observed when a cell fraction
containing a combination of hair dermal papilla cell and epidermal
cells was transplanted. However, it was found that hair growth is
not observed when a cell fraction that does contain either hair
dermal papilla cell or epidermal cells is transplanted.
[0004] The hair dermal papilla cell purification method according
to Kishimoto et al. utilizes the fact that hair dermal papilla cell
have the property of specifically expressing versicans (chondroitin
sulfate proteoglycans). However, it carries out isolation and
concentration by using the expression of versicans by a transgenic
mouse model produced using DNA in which a reporter gene is linked
to a versican gene as an indicator. Thus, this method requires the
production of a transgenic mouse and purification of hair dermal
papilla cell by a cell sorter. A large amount of hair dermal
papilla cell are required to actually regenerate hair follicles and
cause hair growth in hair follicle reconstitution methods in
particular (for example, 5 million cells per transplant).
[0005] Consequently, this method requires the production of a large
number of transgenic mice and the long-term use of a high-speed
cell sorter, thereby resulting economic problems as well as
problems in terms of work time and labor. Although the isolation of
hair dermal papilla cell is also described in, for example, Prouty
et al., American J. Pathol. (1996) Vol. 148, No. 6, pp. 1871-1885,
this method has a complex procedure due to repeated fractionation
by centrifugal separation, has a low level of purity and generates
a low yield.
[0006] Thus, in previous methods for purifying hair dermal papilla
cell, it was difficult to acquire an amount of isolated and
purified active hair dermal papilla cell sufficient for transplant,
for example, and the role of active hair dermal papilla cell in the
regeneration of hair follicles was unable to be completely
elucidated. In particular, it has been virtually impossible with
purification methods of the prior art to determine the suitable
ratio of hair dermal papilla cell and epidermal cells in follicle
reconstitution systems for regenerating hair follicles.
DISCLOSURE OF THE INVENTION
[0007] An object of the present invention is to provide a method
for easily preparing a hair dermal papilla cell preparation
containing only hair dermal papilla cell as the active cellular
component without using a transgenic mouse.
[0008] The inventors of the present invention surprisingly found
that, when a cell suspension of a dermal tissue fraction collected
from skin tissue was cryopreserved, contaminated follicular
epidermal cells in the suspension specifically died, while the
majority of the hair dermal papilla cell continued to be active. As
a result, it was possible to obtain a cell preparation that
contains only hair dermal papilla cell as the active cellular
component. In addition, since a cell preparation obtained in this
manner contains only hair dermal papilla cell as the active
cellular component, it can be used to determine the ratio of hair
dermal papilla cell to epidermal cells effective for regenerating
hair follicles. As a result, the inventors of the present invention
were able to determine the ratio of hair dermal papilla cell to
epidermal cells in a hair follicle reconstitution system considered
to be optimum for regenerating hair follicles.
[0009] Thus, in a first aspect thereof, the present invention
provides a method for preparing a hair dermal papilla cell
preparation comprising: providing a cell suspension by removing
epidermal tissue from skin tissue and subjecting the resulting
dermal tissue fraction to collagenase treatment, and cryopreserving
the cell suspension to kill the follicular epidermal cells.
[0010] Cryopreservation is preferably carried out after adjusting
the cell density of the cell suspension to 1.times.10.sup.5 to
1.times.10.sup.8/ml. More preferably, cryopreservation is carried
out at a temperature of -80.degree. C. or lower in, for example,
liquid nitrogen, preferably over a period of 1 week or more.
[0011] In a preferable embodiment, the skin tissue is from a mouse,
rat or human.
[0012] In a different aspect, the present invention provides a
composition for regenerating hair follicles comprising: hair dermal
papilla cell and epidermal cells; wherein, the ratio of the number
of hair dermal papilla cell to the number of epidermal cells is
from 1:10 to 10:1.
[0013] More preferably, the present invention provides a
composition for regenerating hair follicles comprising a hair
dermal papilla cell preparation and epidermal cells; wherein, said
preparation is prepared by providing a cell suspension by removing
epidermal tissue from skin tissue and subjecting the resulting
dermal tissue fraction to collagenase treatment, and then
cryopreserving the cell suspension to kill the follicular epidermal
cells, in which the ratio of the number of hair dermal papilla cell
to the number of epidermal cells is from 1:10 to 10:1.
[0014] Preferably, the ratio of the number of the hair dermal
papilla cell to the number of the epidermal cells is from 1:3 to
10:1, more preferably from 1:1 to 10:1, even more preferably from
1:1 to 3:1, and most preferably 1:1.
[0015] Moreover, the present invention provides a method for
regenerating hair follicles in an animal or three-dimensional skin
equivalent using the aforementioned composition, and an animal or
three-dimensional skin equivalent in which hair follicles have been
regenerated in this manner.
[0016] According to the present invention, a method is provided for
easily preparing a hair dermal papilla cell preparation that
contains only hair dermal papilla cell as the active cellular
component without using a transgenic mouse. This hair dermal
papilla cell preparation can be used in transplant surgery for
regenerating hair follicles and in research and development on
follicle reconstitution. Since the hair dermal papilla cell
preparation is free of contamination of active epithelial stem
cells, it is particularly advantageous in situations requiring the
ratio of the number of active hair dermal papilla cell and the
number of active epithelial stem cells used for follicle
regeneration to be precisely adjusted as well as requiring a large
number of cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows the results of FACS analysis of a frozen/thawed
dermal cell fraction based on LacZ and 7-AAD.
[0018] FIG. 2 shows the results of FACS analysis of a frozen/thawed
dermal cell fraction based on CD-49 and 7-AAD.
[0019] FIG. 3 shows the results of follicle regeneration by a mixed
transplant of frozen/thawed hair dermal papilla cell and epidermal
cells.
[0020] FIG. 4 shows the results of follicle reconstitution in the
case of using hair dermal papilla cell and epidermal cells derived
from different species (mouse-rat system).
[0021] FIG. 5 shows the results of Hoechst nuclear staining of
follicle reconstitution in the case of using hair dermal papilla
cell and epidermal cells derived from different species (rat-human
system).
[0022] FIG. 6 shows the formation of primordial hair follicles in
the case of having transplanted a follicle regeneration system of
the present invention into a three-dimensional skin equivalent.
BEST MODE FOR CARRYING OUT THE INVENTION
[0023] The present invention provides a method for preparing a hair
dermal papilla cell preparation.
[0024] In addition, the present invention provides a composition
containing hair dermal papilla cell and epidermal cells for
regenerating hair follicles, a method for regenerating hair
follicles using the same, and an animal or three-dimensional skin
equivalent having hair follicles regenerated in such a manner.
[0025] "Hair dermal papilla cell" refer to cells located at the
bottom of hair follicles in the form of mesenchymal cells that
fulfill the role of a so-called control tower by sending out
activation signals to follicular epithelial stem cells for
self-regeneration of hair follicles. A hair dermal papilla cell
preparation containing only active hair dermal papilla cell can be
prepared according to, for example, the method of Kishimoto et al.
(op. cit.) using transgenic mice. With respect to yield, however,
it is preferable to, for example, remove epidermal tissue from the
skin tissue to prepare a cell suspension by subjecting the
resulting dermal tissue fraction to collagenase treatment, followed
by cryopreserving the cell suspension to kill the follicular
epidermal cells.
[0026] The method as claimed in the present invention using the
aforementioned cryopreservation can be specifically carried out,
for example, in the manner described below.
1. Mammalian epidermis is prepared. 2. This skin is allowed to
stand undisturbed for a suitable amount of time, such as overnight,
in a protease solution such as a trypsin solution as necessary,
followed by removing the epidermal portion with a tweezers and so
forth, and treating the remain dermis with collagen to prepare a
cell suspension. 3. The suspension is filtered through a cell
strainer as necessary to remove the sediment by allowing to stand
undisturbed. 4. The number of cells is counted and then
re-suspended in a cryopreserving liquid at a suitable cell density
of preferably about 1.times.10.sup.5 to 1.times.10.sup.8/ml, the
suspension is then divided into smaller aliquots as necessary, and
then cryopreserved in accordance with ordinary cell storage
methods. 5. The frozen suspension is then thawed after a suitable
storage period and used.
[0027] Although there are no particular limitations on the
cryopreserving method, the suspension is stored in an
ultra-low-temperature freezer or in liquid nitrogen at -20.degree.
C. or lower, preferably -50.degree. C. or lower, and more
preferably -80.degree. C. or lower. Although there are also no
particular limitations on the cryopreservation period, it is, for
example, 1 day or more, preferably 3 days or more, and more
preferably 1 week or more so as to destroy the epidermal cells.
Furthermore, hair dermal papilla cell have been confirmed to
continue to survive even after being stored for 4 months in liquid
nitrogen. Conventional storage liquids used for the storage of
cells, such as the Cell Banker 2 Cell Cryopreservation Liquid
(Catalog No. BLC-2, Nippon Zenyaku Kogyo), can be used as the
preservating liquid.
[0028] Cells can be counted by a method known among persons with
ordinary skill in the art. For example, cells can be counted by
placing a cell suspension diluted with an equal volume of 0.4%
Trypan glue stain (No. 15250-061, Invitrogen) on a hemocytometer
(Eosinophil Counter, SLGC) and calculating the number of cells
according to the method described in the instruction manual
provided with the hemocytometer.
[0029] The mammalian skin serving as the source of hair dermal
papilla cell used in the present invention may be from any mammal
without limitation, examples of which include humans, chimpanzees
and other primates, domestic animals such as dogs, cats, rabbits,
horses, sheep, goats, cows and pigs, and laboratory test animals
such as rats, mice and guinea pigs, and preferably nude mice, SCID
mice and nude rats. In addition, there are no limitations on the
animal strain.
[0030] The "epidermal cells" in the composition for regenerating
hair follicles as claimed in the present invention are cells that
compose the majority of the epidermis or epithelium of the skin,
and arise from a single layer of basal cells in contact with the
dermis. For example, in mouse, although epidermal cells originating
in a newborn (or fetus) can be used preferably for the epidermal
cells, they may also be cells originating in the epidermis of
mature skin such as the epidermis of dormant hair, cells
originating in the epidermis of growing hair, or a culture of cells
in the keratinocyte state. Such cells can be prepared from the skin
of a desired donor animal according to methods known among persons
with ordinary skill in the art.
[0031] In a preferable aspect thereof, the epidermal cells can be
prepared in the following manner.
1. Mammalian skin tissue is prepared. 2. This epidermis is treated
with trypsin as necessary by allowing to stand overnight at
4.degree. C. in 0.25% trypsin/PBS. 3. After separating only the
epidermal portion with a tweezers and so forth and cutting into
sections, the sections are suspended for about 1 hour at 4.degree.
C. in a suitable culture medium (such as keratinocyte culture
liquid). 4. This suspension is passed through a cell strainer
having a suitable pore size following by applying to a centrifuge
to recover the epidermal cells. 5. This cell preparation is then
suspended to a desired cell density in KGM or SFM medium and kept
on ice until just prior to use.
[0032] Similar to the aforementioned hair dermal papilla cell, the
epidermal cells of the present invention may originate in the skin
of all mammals, examples of which include humans, chimpanzees and
other primates, domestic animals such as dogs, cats, rabbits,
horses, sheep, goats, cows and pigs, and laboratory test animals
such as rats, mice and guinea pigs, and preferably nude mice, SCID
mice and nude rats. In addition, the site of the epidermis may be a
pilose site such as the scalp or a glabrous site such as the
foreskin.
[0033] When the inventors of the present invention mixed a hair
dermal papilla cell preparation containing only hair dermal papilla
cell as the active cells and in which epidermal cells were killed,
which was obtained according to the aforementioned
cryopreservation, with an epithelial cell preparation containing
only active epidermal cells from which dermal cells had been
removed, at various cell ratios, and the mixtures were transplanted
into recipient animals to examine regeneration of hair follicles,
there was found to be a constant relationship between the ratio of
the number of hair dermal papilla cell to the number of epidermal
cells and the regeneration of hair follicles. Namely, in the case
of desiring regeneration of more hair follicles, it was found that
the ratio of the number of active hair dermal papilla cell to the
number of active epidermal cells should be 1:3 to 10:1, preferably
1:1 to 10:1, more preferably 1:1 to 3:1, and most preferably 1:1.
In other words, regeneration of hair follicles can be adjusted by
suitably adjusting the ratio of the number of active hair dermal
papilla cell to the number of active epidermal cells and
transplanting into a recipient animal.
[0034] Hair dermal papilla cell and epidermal cells may be combined
from the same species or different species. For example, in the
case of a hair dermal papilla cell preparation originating in mice,
the epidermal cells may originate in mice (homologous) or they may
originate in another species such as rats or humans (heterologous).
Thus, a composition for regenerating hair follicles of the present
invention may be a combination in which the hair dermal papilla
cell and epidermal cells both originate in mice, a combination in
which they both originate in rats, or a combination in which they
both originate in humans (examples of homologous), or a combination
in which the hair dermal papilla cell originate in mice while the
epidermal cells originate in rice, a combination in which the hair
dermal papilla cell originate in rats while the epidermal cells
originate in mice, a combination in which the hair dermal papilla
cell originate in mice while the epidermal cells originate in
humans, a combination in which the hair dermal papilla cell
originate in rats while the epidermal cells originate in humans, a
combination in which the hair dermal papilla cell originate in
humans while the epidermal cells originate in mice, or a
combination in which the hair dermal papilla cell originate in
humans while the epidermal cells originate in rats (examples of
heterologous).
[0035] The method for implanting a composition for regenerating
hair follicles as claimed in the present invention can be carried
out using a known transplant method, an example of which is
described in Weinberg et al., J. Invest. Dermatol. Vol. 100 (1993),
pp. 229-236. In the case of transplanting into nude mice, for
example, the prepared cells are mixed from immediately before to 1
hour before transplant, the culture liquid is removed by
centrifugation (9000.times.g, 10 min.) to form a cell mass of about
50 to 100 .mu.l followed by promptly injecting into a silicon
dome-shaped chamber embedded in the skin on the backs of the nude
mice. The chamber is carefully removed 1 week later, and the
presence of hair formation at the transplant site can be observed
macroscopically starting in the second week. Transplantation for
the purpose of hair growth can be carried out in a similar manner
in animals, including humans, and an appropriate method is suitably
selected by the physician or veterinarian.
[0036] Transplantation is preferably carried out by, for example,
transplanting to a circle area having a diameter of about 1 cm in a
transplanted amount in which the number of hair dermal papilla cell
is 1.times.10.sup.6 to 1.times.10.sup.8/cm.sup.2, preferably
1.0.times.10.sup.7 to 1.5.times.10.sup.7/cm.sup.2, and more
preferably 1.27.times.10.sup.7/cm.sup.2.
[0037] In the case of transplanting the aforementioned composition
into a recipient animal, the transplant may be a homologous
transplant, namely an autotransplant, an isotransplant or
allotransplant, or a heterologous transplant. In the case of a
homologous transplant, the hair dermal papilla cell preparation and
the epidermal cells are both of the same species as the recipient.
In a heterologous transplant, either the hair dermal papilla cell
preparation or the epidermal cells are of a different species from
the recipient and the other is of the same species as the
recipient, or both are of a different species from the recipient.
Any animal may be used for the recipient, examples of which include
humans, chimpanzees and other primates, domestic animals such as
dogs, cats, rabbits, horses, sheep, goats, cows and pigs, and
laboratory test animals such as rats, mice and guinea pigs, and
preferably nude mice, SCID mice and nude rats.
[0038] In addition, a chimeric animal having regenerated hair
follicles can be provided by transplanting the aforementioned
composition as claimed in the present invention into a suitable
recipient animal. This animal can serve as a valuable animal model
for researching and elucidating the mechanism of hair follicle
regeneration or for screening for pharmaceuticals and herbal
medicines effective for hair follicle regeneration, hair growth or
hair removal. The recipient animal is preferably an
immunosuppressed animal irrespective of the source of each cell
contained in the system transplanted into the animal. In addition,
any animal species used as a laboratory test animals may be used,
provided that it coincides with the object of the present
invention, examples of which include mice and rats. Among these
animals, examples of animals having a suppressed immune system in
the case of mice include those having a trait such as a missing
thymus gland in the manner of nude mice. Furthermore, in
consideration of the object of the present invention, particularly
preferable examples of recipient animals include commercially
available nude mice (e.g., Balb-c nu/nu strain), SCID mice (e.g.,
Balb/c-SCID strain) and nude rats (e.g., F344/N Jcl-rnu).
[0039] Moreover, a three-dimensional skin equivalent having
regenerated hair follicles can be provided by incorporating the
composition as claimed in the present invention in a
three-dimensional skin equivalent. A three-dimensional skin
equivalent can be produced, for example, in the manner described
below according to methods known among persons with ordinary skill
in the art (Exp. Cell Res., Amano S. et al., (2001), Vol. 271, pp.
249-262). The three-dimensional skin equivalent contains
1.times.10.sup.6 to 1.times.10.sup.8 hair dermal papilla
cell/cm.sup.2, preferably 1.0.times.10.sup.7 to
1.5.times.10.sup.7/cm.sup.2, and more preferably about
1.27.times.10.sup.7/cm.sup.2.
[0040] Production Method of Three-Dimensional Skin Equivalent
[0041] Human fibroblasts are dispersed in a suitable amount of a
mixture of 0.1% collagen solution, DMEM and 10% FBS, transferred to
a Petri dish, and immediately allowed to stand undisturbed in a
CO.sub.2 incubator at 37.degree. C. After gelling, the gel is
scraped from the sides and bottom of the Petri dish so as to
suspend in the Petri dish. It was cultured with shaking, and a
dermis equivalent was obtained when the gel contracts to
approximately one fifths of the size. The dermis equivalent is
placed on a stainless steel grid, a glass ring is placed thereon,
and 0.4 ml of cultured human epidermal cells (1.0.times.10.sup.6
cells/ml) dispersed in KGM (epidermal cell culture medium) are
injected into the glass ring and cultured. At this time, a dermal
cell fraction is simultaneously mixed and injected. Newborn mouse
epidermal cells can also be used instead of human cultured
epidermal cells. Medium consisting of DMEM, KGB and 5% FBS
Ca.sup.2+ is added to the Petri dish so that the upper portion of
the dermis equivalent is exposed to air followed by culturing. The
dermis equivalent is observed after about one week and assessed for
the presence of primordial follicle formation and
reproducibility.
[0042] Similar to the aforementioned chimeric animal having
regenerated hair follicles, this three-dimensional skin equivalent
having reorganized hair follicles can be used for research and
elucidation of the mechanism of hair follicle regeneration as well
as screening for pharmaceuticals and herbal medicines effective for
hair growth and hair removal.
[0043] The following provides a more detailed explanation of the
present invention through examples thereof.
Example 1
[0044] In order to confirm that epidermal cells die as a result of
cryopreservation of a dermal cell fraction, and that a cell
preparation containing only hair dermal papilla cell as the active
cells is obtained as a result thereof, a cell fraction obtained
from skin tissue collected from transgenic mice, in which an
expression vector linked to a structural gene of LacZ marker
protein was inserted downstream from a versican promoter (to be
referred to as Versican-LacZ TG mice), was placed in
cryopreservation followed by analysis of the thawed cell
preparation by flow cytometry.
(1) Preparation of Cryopreserved Hair Dermal Papilla Cell
Preparation from Dermal Cells of Versican-LacZ TG Mice (1-1)
Individuals positive for LacZ were selected from the newborns (used
within 4 days after birth) of the Versican-LacZ TG mice.
Versican-LacZ TG mice can be produced according to, for example,
the method described in Kishimoto et al. (op. cit.). (1-2) After
washing each individual with ethanol and phosphate-buffered
physiological saline (abbreviated as "PBS"), the skin of the back
was removed and allowed to stand overnight at 4.degree. C. in a
mixture of 0.25% trypsin and PBS. (1-3) On the following day, the
epidermis and dermis were separated with a tweezers, and the dermis
portion was treated for about 1 hour at 37.degree. C. with a
mixture of 0.35% collagenase and DMEM (Dulbecco's Modified Eagle's
Medium). (1-4) After carefully suspending the cells obtained in
(1-3), the suspension was passed through a cell strainer followed
by collecting the cells with a centrifuge (900.times.g, 10
minutes). (1-5) The number of cells was counted, the cells were
re-suspended in cell cryopreservating liquid (Cell Banker 2
(BLC-2), Nippon Zenyaku Kogyo), transferred to a cryopreservating
tube and then stored in liquid nitrogen in accordance with
conventional cell storage methods. (1-6) The frozen cells were
thawed after about one week and then used in the flow cytometry
analysis described below.
(2) FluoroReporter LacZ Flow Cytometry Analysis Materials:
[0045] FluoroReporter LacZ Flow Cytometry Kit (Molecular Probe,
Catalog No. F-1930 (50 reactions)/F-1931 (250 reactions))
[0046] Reagent Preparation:
[0047] Reaction liquid: The FDG reagent contained in the kit
(Component A) was diluted 1:10 with MiliQ water. 50 .mu.l were used
per sample.
[0048] Quencher: The PI reagent contained in the kit (Component D)
was diluted 1:100 with the buffer provided with the kit. 0.9 ml
were used per sample. The quencher was chilled at 4.degree. C. on
ice until the time of use. The staining media consisted of CD49f
monoclonal antibody (Serotec), which is a specific antibody that
specifically stains epidermal cells, and 7-ADD (Beckman-Coulter,
PN-IM 3422), which specifically stains dead cells.
(2-1) The suspension of dermal cells from the Versican-LacZ TG mice
was adjusted to 1.times.10.sup.7 cells followed by the addition of
750 .mu.l of staining media and transferred to a 1.5 ml Eppendorf
tube. (2-2) The cell suspension was centrifuged for 5 minutes for
3000 revolutions and the supernatant was discarded. The cell pellet
was re-suspended in 100 .mu.L of staining media. This suspension
was pre-incubated for 10 minutes in a constant temperature bath at
37.degree. C. (2-3) 50 .mu.L of reaction liquid that had been
pre-incubated for 10 minutes in a constant temperature bath at
37.degree. C. were then added and the reaction was carried out at
37.degree. C. for exactly 1 minute. (2-4) 0.9 ml of quencher were
added after which the suspension was stored in ice. (2-5) 10
minutes later, 40 .mu.L of 50 mM PETG (Component B) were added to
completely inhibit the reaction. (2-6) The fluorescence intensity
of the cell suspension was promptly measured with the FACS. The
operating procedure of the flow cytometer (FACS) was in accordance
with the manufacturer's instruction manual. The XL-MCL manufactured
by Beckman-Coulter, for example, can be used for the FACS measuring
apparatus. The distribution of fluorescence intensity of the cells
was measured using a detection setting suitable for the fluorescein
used in this kit.
(3) Analysis Results
[0049] FACS analysis based on LacZ and 7-AAD indicated that the
majority of the LacZ.sup.+ cells among the cryopreserved cells were
viable cells even after cryopreservation (FIG. 1). Thus, hair
dermal papilla cell are clearly not killed by cryopreservation. In
addition, FACS analysis based on CD-49 and 7-AAD indicated that the
majority of the CD-49.sup.+ cells (epidermal cells) were present in
the 7-AAD.sup.+ fraction (dead cell fraction) after thawing (FIG.
2). Thus, the viable cells in the thawed cell preparation were
LacZ.sup.+, CD-49.sup.- and 7-AAD.sup.-, namely hair dermal papilla
cell (LacZ.sup.+) and non-epidermal cells (CD-49.sup.-), and were
viable cells (7-AAD.sup.-). In summary of these results, it was
clear that the majority of the viable cells were not epidermal
cells, but rather hair dermal papilla cell. Accordingly, it was
also clearly demonstrated that epidermal cells can be specifically
killed by freezing/thawing, and that a hair dermal papilla cell
preparation can be prepared that contains only hair dermal papilla
cell as the active cells.
Example 2
[0050] Hair follicles were attempted to be regenerated by
transplanting a mixture of frozen/thawed hair dermal papilla cell
and epidermal cells.
I. Cell Preparation
[0051] (1) Mouse Epidermal cells (1-1) Each individual selected
from newborn ICR strain mice was washed with ethanol and
phosphate-buffered physiological saline (abbreviated as "PBS") on
the day before surgery followed by removal of skin from the back
and treating the skin with trypsin by allowing to stand undisturbed
overnight at 4.degree. C. in a mixture of 0.25% trypsin and PBS.
(1-2) After peeling off only the epidermis portion with a tweezers
and cutting into sections, the epidermis was suspended for about 1
hour at 4.degree. C. in keratinocyte culture liquid (referred to as
"KGM"). (1-3) The suspension of (1-2) was passed through a cell
strainer and centrifuged (900.times.g, 10 minutes) to recover the
epidermal cells. (1-4) An amount of epidermal cells equivalent to
two newborns were used for surgery for each recipient animal (cell
count of about 1.times.10.sup.7 cells). An equivalent amount of
cells were suspended in KGF or SFM medium and allowed to stand
undisturbed on ice until the time of use. This was designated as
the "mouse epithelial cell preparation".
(2) Preparation of "Fresh" Mouse Dermal Cell Preparation
(Comparative Example)
[0052] (2-1) Skin from the newborns of ICR strain mice was treated
with trypsin in the same manner as the aforementioned (1-1) and
(1-2) above on the day before surgery. (2-2) After peeling off the
epidermis portion with tweezers and cutting the remaining dermis
into sections, the dermis was suspended and treated for about 1
hour at 37.degree. C. in a suitable culture liquid of DMEM and 10%
PBS containing 0.35% collagenase. (2-3) The suspension of (2-2) was
passed through a cell strainer and centrifuged to recover the
dermal cells. (2-4) About 1.times.10.sup.7 dermal cells in terms of
the cell count were used for surgery for each recipient animal. An
equivalent amount of cells were suspended in a mixture of DMEM and
10% FBS and so forth, and the suspension was allowed to stand
undisturbed on ice until the time of use. This suspension was
designated as "fresh mouse dermal cell preparation".
(3) Preparation of "Cryopreserved" Mouse Dermal Cell Preparation
Containing Dermal Cell Fraction
[0053] (3-1) Skin from the backs of newborn ICR strain mice was
removed to collect the epidermis. (3-2) After allowing to stand
undisturbed overnight in trypsin solution, the epidermis was
removed with a tweezers on the following day and the remaining
dermis was treated with collagenase to prepare a cell suspension.
(3-3) The sediment was removed by filtering the suspension with a
cell strainer and allowing to stand. (3-4) After counting the
number of cells, the cells were re-suspended in cryopreserving
liquid to a cell density of 1.times.10.sup.5 to
1.times.10.sup.8/ml, the suspension was transferred to freezing
tubes and then stored in liquid nitrogen according to conventional
cell storage methods. (3-5) The suspension was thawed about one
week later and 1.times.10.sup.7 cells/transplant were used in the
transplant experiment. This was designated as the "cryopreserved"
mouse dermal cell preparation.
II. Hair Follicle Reconstitution Method (Method for Transplanting
to Animals)
[0054] The aforementioned mouse epithelial cell preparation of
(1-4) was mixed with either the "fresh" mouse dermal cell
preparation of (2-4) or the "cryopreserved" mouse dermal cell
preparation of (3-5). These mixtures were used as cell suspensions
in the reorganized hair follicle production procedure described
below.
<Reorganized Hair Follicle Production Procedure>
[0055] Materials:
[0056] Recipient animals (Balb-c nu/nu strain nude mice, age 5
weeks or older),
[0057] silicon dome-shaped cap having a diameter of about 1
centimeter (to be referred to as "bulb"),
[0058] anesthetic,
[0059] surgical scissors, tweezers, suture,
[0060] micropipetter, and
[0061] cell suspensions (suspended in about 150 .mu.l of a mixture
of DMEM culture liquid and 10% FBS).
[0062] <Procedure>
(i) The nude mice were anesthetized. (ii) Skin was cut away from
the back over an area of just under 1 centimeter in diameter. (iii)
The bulb was inserted into the wound opening and fastened with the
suture. (iv) A cell suspension was injected into the bulb using the
pipetter. (v) The animals were housed for about 1 week in this
state followed by removal of the bulb. (vi) After having removed
the bulb, the growth of reorganized hair follicles was observed at
the scar where the scab had come off about 1 to 6 weeks later
(normally after 2 weeks).
[0063] The results of the hair follicle reconstitution experiment
are shown in Table 1 and FIG. 3. In contrast to regeneration of
hair follicles being observed even in the case of transplanting
only the "fresh" mouse dermal cell preparation, regeneration of
hair follicles was not observed in the case of transplanting only
the "cryopreserved" mouse dermal cell preparation. These results
agree with the results obtained by Kishimoto et al. (op. cit.)
using hair dermal papilla cell purified with a cell sorter from a
dermal cell fraction originating in transgenic mice, and verified
that only hair dermal papilla cell are contained as active cells in
the "cryopreserved" mouse dermal cell preparation".
TABLE-US-00001 TABLE 1 Dermal cell Epithelial cell Hair follicle
(No. of hairs/no. fraction fraction regeneration of transplants)
Fresh (not frozen) Fresh ++ (4/4) Fresh (not frozen) -- ++ (4/4)
Frozen/thawed Fresh ++ (10/10) Frozen/thawed -- -- (0/10) -- Fresh
-- (0/10)
Example 3
Effect of Cell Ratio of Papilla Cell Fraction and Epithelial Cell
Fraction on Hair Follicle Reconstitution Efficiency
[0064] A "cryopreserved" mouse dermal cell preparation and
epidermal cells originating in the skin of newborn rats prepared
using the same treatment as the mouse epidermal cells described in
Example 2 were respectively adjusted to cell counts of
0.1.times.10.sup.6, 3.3.times.10.sup.6 and 1.times.10.sup.7 cells
and then mixed. The mixtures were transplanted into skin on the
back of nude mice according to the reorganized hair follicle
production procedure previously described to investigate the
presence of hair follicle reconstitution. Those results are shown
in Table 2 below.
TABLE-US-00002 TABLE 2 Effects of Cell Ratio of Dermal Cell
Fraction and Epithelial Cell Fraction on Hair Follicle
Reconstitution Efficiency Epithelial cell Hair follicle Dermal cell
fraction fraction regeneration 0 1.0 .times. 10.sup.7 - 1.0 .times.
10.sup.6 1.0 .times. 10.sup.7 .+-. 3.3 .times. 10.sup.6 1.0 .times.
10.sup.7 + 1.0 .times. 10.sup.7 1.0 .times. 10.sup.7 +++ 1.0
.times. 10.sup.7 3.3 .times. 10.sup.6 +++ 1.0 .times. 10.sup.7 1.0
.times. 10.sup.6 ++ 1.0 .times. 10.sup.7 0 -
[0065] According to the results of Table 2, hair follicle
regeneration was observed when the mixing ratio of hair dermal
papilla cell and epidermal cells was within the range of 1:10 to
10:1, and when cell mixtures having a ratio of 1:1 to 3:1, and
particularly about 1:1, were transplanted, hair follicle
regeneration was determined to occur prominently.
Example 4
Hair Follicle Reconstitution when Using Epidermal Cells Originating
in Mature Mouse Skin
[0066] Preparation of epidermal cells originating in mature mouse
skin (mice age 10 weeks or older) was carried out in compliance
with the preparation of newborn mouse epidermal cells described in
Example 2. Two types of epidermal cells were used for the epidermal
cells originating in mature mouse skin. These consisted of
epidermal cells prepared from epidermis of dormant hair of mice age
10 weeks or older (mixture containing dormant hair epidermal
cells), and epidermal cells prepared from the epidermis of mice in
which hair growth was promoted (mixture containing active
growth-induced hair epidermal cells). The epidermal cells
originating in mature mouse skin were each mixed with a
"cryopreserved" mouse dermal cell preparation so that the ratio of
the number of cells was 1:1, and the mixtures were transplanted to
the backs of mice according to the previously described reorganized
hair follicle production procedure. Those results are shown in
Table 3 below.
TABLE-US-00003 TABLE 3 (No. of primordial Dermal cell Epithelial
cell Hair follicle follicles formed/no. fraction fraction
reconstitution of transplants) Mouse Mouse (newborn + (2/2)
epidermal cells) Mouse Mouse (mature ++ (3/3) mice: dormant) Mouse
Mouse (mature ++ (3/3) mice: active) +++: High-density hair growth
observed ++: Hair growth observed +: Follicle formation observed in
transplanted tissue
[0067] The presence or absence of the formation of primordial hair
follicles was evaluated by preparing thin sections from tissue at
the transplant site, staining the sections using a typical stain
such as hematoxylin-eosin stain, and then observing the morphology
and evaluating based on indentation of the epidermal layer and
aggregation of dermal granulocytes directly beneath the indented
portions of the epidermis.
[0068] As is clear from these results, hair growth was observed in
both the case of transplanting the mixture containing dormant hair
epidermal cells and the mixture containing active growth-induced
epidermal cells. Thus, the present invention was determined to be
effective for hair follicle regeneration not only in the case of
the epidermal cells originating in newborn epidermis, but also in
the cases of originating in mature epidermis such as the epidermis
of dormant hair or actively growing hair.
Example 5
Hair Follicle Reconstitution when Using Different Species of Hair
Dermal Papilla Cell and Epidermal Cells
[0069] A "cryopreserved" mouse dermal cell preparation and
epidermal cells originating in the skin of newborn rats mixed at
about 1.times.10.sup.7 cells each, and the reconstitution of hair
follicles was investigated by transplanting into the skin on the
backs of nude mice according to the previously described
reorganized hair follicle production procedure. Those results are
shown below in Table 4 and in FIG. 4.
TABLE-US-00004 TABLE 4 (No. of primordial Dermal cell Epithelial
cell Hair follicle follicles formed/no. fraction fraction
reconstitution of transplants) Mouse Mouse (newborn) +++ (12/12)
Rat Rat (newborn) +++ (3/3) Mouse Rat (newborn) +++ (4/4) +++:
High-density hair growth observed ++: Hair growth observed +:
Follicle formation observed in transplanted tissue
[0070] As is clear from these results, the present invention was
determined to be effective for regenerating hair follicles even if
the mixture of hair dermal papilla cell and epidermal cells
consists of cells from different species, and the effects are the
same as when using cells from the same species.
Example 6
Hair Follicle Reconstitution when Using Epidermal Cells Originating
in Human Newborn Foreskin
[0071] Epidermal cells were prepared from human newborn foreskin
obtained by circumcision and so forth and mixed with a
cryopreserved mouse hair dermal papilla cell preparation followed
by investigating for the presence of hair follicle reconstitution
by transplanting the mixture into skin on the backs of nude mice
according to the previously described reorganized hair follicle
production method.
[0072] Foreskin tissue can be stored for approximately 1 to 3 weeks
by placing in a refrigerator in a conventional fibroblast culture
medium (such as DMEM) and keratinocyte culture medium (such as
keratinocyte SMF medium, Invitrogen).
[0073] Cell cultures derived from foreskin were prepared in the
manner described below.
[0074] The foreskin tissue in the aforementioned media (which can
be used regardless of race) was allowed to react for 30 minutes in
a Petri dish filled with a solution to which an antibiotic such as
streptomycin or penicillin had been added to PBS (for tissue
culturing, calcium and magnesium-free). The tissue was further
allowed to react for 10 minutes after transferring to a Petri dish
filled with fresh PBS and antibiotic. After removing excess fat
tissue, the tissue was cut into skin sections of about 1 cm.sup.2
and allowed to react for about 18 hours at 4.degree. C. while
suspended in Dispase solution (Godo Seishu Co., concentration: 1000
to 5000 U). After reacting, the epithelium portion was grabbed and
peeled off with a tweezers after having washed again with PBS. The
removed epithelium sheet was suspended in 5 ml of 0.5 mN EDTA
solution containing 0.05% trypsin and allowed to react for 15
minutes at 37.degree. C.
[0075] After stopping the reaction by adding trypsin inhibitor, the
suspension was centrifuged for 10 minutes and 900 revolutions
followed by discarding the supernatant. The pellet was re-suspended
in 10 ml of keratinocyte SFM medium (Invitrogen) and the number of
cells was counted. About 1.times.10.sup.6 to 1.times.10.sup.7 cells
were used for a single transplant experiment. In the case of
preparing subcultured epidermal cells for transplant, about
1.times.10.sup.6 epidermal cells were seeded in a 100 mm Petri dish
or 75 cm.sup.2 flask coated with type I or type IV collagen
solution, and cultured according to conventional methods in a
CO.sub.2 incubator using keratinocyte SFM medium. Once the cells
had reached confluence, the cells were separated with trypsin,
collected and again prepared to a cell density of 1.times.10.sup.6
followed by subculture until the required number of cells or
passage was reached.
[0076] The results of the above transplants are shown in Table 5
below.
TABLE-US-00005 TABLE 5 (No. of primordial Dermal cell Epithelial
cell Hair follicle follicles formed/No. fraction fraction
reconstitution of transplants) Mouse Human (newborn + (3/3)
foreskin) Mouse Human (newborn + (2/2) foreskin cultured cells) +:
Hair follicle formation observed in transplant tissue
[0077] It was determined from these results that the present
invention is effective for regeneration of hair follicles even if
epidermal cells of human origin are combined with mouse hair dermal
papilla cell. In addition, it is interesting to note that foreskin
cells resulted in regeneration of hair follicles when combined with
hair dermal papilla cell even though they are epidermal cells
originating at skin location that does not have hair follicles or
hair roots. Thus, it was clearly demonstrated that epidermal cells
capable of being combined with hair dermal papilla cell in hair
follicle regeneration systems are not limited to those from pilose
sites, but also from glabrous skin sites as well.
Example 7
Hair Follicle Reconstitution when Using Epidermal Cells Originating
in Human Adult Foreskin
[0078] Epidermal cells were prepared from adult foreskin tissue
(age 20) obtained by phimosiectomy and so forth and mixed with a
cryopreserved mouse hair dermal papilla cell preparation followed
by investigating for the presence of hair follicle reconstitution
by transplanting the mixture into skin on the backs of nude mice
according to the previously described reorganized hair follicle
production method.
[0079] Adult foreskin tissue can be stored for about 1 week by
placing in cryopreservation in a conventional fibroblast culture
medium (such as DMEM) and keratinocyte culture medium (such as
keratinocyte SMF medium, Invitrogen).
[0080] Preparation of cultured cells originating in adult foreskin
tissue was carried out in compliance with the preparation method
for newborn foreskin cultured cells of Example 6.
[0081] The results of the above transplants are shown in Table 6
below.
TABLE-US-00006 TABLE 6 (No. of primordial Dermal cell Epithelial
cell Hair follicle follicles formed/No. fraction fraction
reconstitution of transplants) Mouse Human (adult + (1/1) foreskin)
Mouse Human (adult + (4/6) foreskin cultured cells) +: Hair
follicle formation observed in transplant tissue
[0082] It was determined from these results that the present
invention is effective for regeneration of hair follicles even if
epidermal cells of adult human origin are combined with mouse hair
dermal papilla cell. This clearly demonstrated that epidermal cells
capable of being combined with hair dermal papilla cell in hair
follicle regeneration systems are not limited to those of the
course of development but also of mature tissue.
Example 8
Effects of Passage of Cultured Cells from Human Foreskin on Hair
Follicle Reconstitution
[0083] The results of assessing the efficiency of reconstitution
are shown in Table 7 below in the case of combining epidermal cells
from different races of human newborn or adult foreskin with varied
numbers of passage in an experiment on hair follicle
reconstitution.
TABLE-US-00007 TABLE 7 (No. of Hair dermal Hair primordial papilla
Epidermal No. follicle follicles/no. of cell cells Race of
subcultures reorganization transplants) Mouse Human Negro 0 + (3/3)
(newborn foreskin) Mouse Human Negro 1 + (2/2) (newborn foreskin
cultured cells) Mouse Human Negro 2 + (3/3) (newborn foreskin
cultured cells) Mouse Human Caucasian 1 + (1/1) (newborn foreskin
cultured cells) Mouse Human Caucasian 2 + (1/4) (newborn foreskin
cultured cells) Mouse Human Caucasian 3 - (0/2) (newborn foreskin
cultured cells) Mouse Human Caucasian 5 - (0/2) (newborn foreskin
cultured cells) Mouse Human Japanese 0 + (1/1) (adult foreskin
cultured cells) Mouse Human Japanese 1 + (2/2) (adult foreskin
cultured cells) Mouse Human Japanese 2 + (2/4) (adult foreskin
cultured cells) Mouse Human Japanese 3 + (0/1) (adult foreskin
cultured cells) *Hair follicle formation observed in transplant
tissue -: Hair follicle formation not observed in transplant
tissue
[0084] It was clearly demonstrated from these results that hair
follicles are reorganized to a greater extent the fewer the number
of subcultures of the epidermal cells regardless of race.
Example 9
Study of Reorganized Hair Follicles
[0085] A method that uses, for example, species-specific antibodies
or a species-specific gene sequence (such as human Alu sequence)
can be used to confirm whether or not reorganized hair follicles
are composed of combinations hair dermal papilla cell and epidermal
cells. Most conveniently, this can be distinguished easily by
histological observations using an heterologous type composition
for regeneration of hair follicles as claimed in the present
invention (e.g., using mouse hair dermal papilla cell and rat or
human epidermal cells or the opposite) and staining with Hoechst
#33258 reagent (Molecular Probe) used for nucleus staining (Miller
G. J. and Ferrara J. A., Stain Technol. 63 (1): 15-21, 1988), by
which a plurality of dots (points) being clearly observed in the
nucleus in the case of murine while not being observed in humans
and rats.
[0086] <Hoechst #33258 Nuclear Staining>
[0087] A paraffin section obtained by thinly slicing tissue from
the transplant site was placed on a slide glass (preferably 4 to 6
.mu.m thick), subjected to a conventional paraffin removal
treatment (consisting of washing twice with xylene, washing with
99.9% ethanol, washing with 80% ethanol, washing with 70% ethanol
and finally washing with water) and transferred to PBS solution
(the section can be allowed to remain in this state for a short
period of time).
[0088] 4 mg of Hoechst #33258 (Molecular Probe) were dissolved in 1
mL of PBS solution (and blocked from light with aluminum foil). 10
.mu.l of the resulting Hoechst #33258 solution were diluted with 10
ml of PBS (1000-fold dilution, final concentration: 4 .mu.g/mL),
and several drops were added to the tissue section on the
horizontally positioned slide glass so as to completely cover the
section. Subsequently, the solution was allowed to react for 15
minutes at room temperature while blocking from light, and after
washing with water for 5 minutes, the slide glass was sealed with
GVA Mounting Solution (glycerol-based sealant, Zymed Lab, available
from Funakoshi Yakuhin) and a cover glass. The slide glass was
observed with a fluorescent microscope allowing observation of
excitation in the UV band.
[0089] According to this method, the nuclei of mouse cells appear
bright and a plurality of dots are observed. Since dots are not
visible in the other rat and human cells, the source species of the
tissue can be distinguished.
[0090] FIG. 5 shows the results of a fluorescent micrograph in the
case of having transplanted a hair follicle reconstitution system
in which the hair dermal papilla cell originate in mice while the
epidermal cells originate in humans (human newborn foreskin) in
comparison with the image of tissue following conventional tissue
staining, namely hematoxylin-eosin (HE) staining (Complete
Reference to Staining Methods, Ishiyaku Publishing Co., Ltd., p.
2-7, 1988). It can be clearly seen from this figure that the hair
follicles are composed both of hair dermal papilla cell (portions
having large numbers of bright dots originating in mice) and
epidermal cells (portions not having dots originating in
humans).
Example 10
Primordial Hair Follicle Regeneration in a Three-Dimensional Skin
Equivalent
[0091] Human fibroblasts were dispersed in a suitable amount of a
mixture of 0.1% collagen solution, DMEM and 10% FBS, transferred to
a Petri dish and immediately allowed to stand undisturbed in a
CO.sub.2 incubator at 37.degree. C. After gelling, the gel was
scraped from the sides and bottom of the Petri dish so as to float
in the Petri dish. It was cultured with shaking, a dermis
equivalent was obtained when the gel contracts to approximately one
fifth of the size. The dermis equivalent was placed on a stainless
steel grid, a glass ring was placed thereon, and 0.4 ml of cultured
human epidermal cells (1.0.times.10.sup.6 cells/ml) dispersed in
KGM (epidermal cell culture medium) were injected into the glass
ring and cultured. At this time, a cryopreserved mouse dermal cell
preparation was simultaneously mixed and injected. Medium
consisting of DMEM, KGB and 5% FBS Ca.sup.2+ was added to the Petri
dish so that the upper portion of the dermis equivalent was exposed
to air followed by culturing. The dermis equivalent was observed
after about one week and assessed for the presence of primordial
follicle formation and reproducibility by hematoxylin-eosin
staining and the aforementioned Hoechst staining. Those results are
shown in FIG. 6.
[0092] As is clear from the results of FIG. 6, primordial hair
follicle formation was observed even when a hair follicle
regeneration system as claimed in the present invention is
transplanted into a three-dimensional skin equivalent.
INDUSTRIAL APPLICABILITY
[0093] A composition for regenerating hair follicles as claimed in
the present invention can be used for transplanting hair follicles
and research and development on hair follicle reconstitution.
* * * * *