Assignee: 1. SEOUL NATIONAL UNIVERSITY INDUSTRY FOUNDATION, Korea
2. RNL BIO CO., LTD, Korea
3. Kang, Kyung Sun, Korea
4. Jeong Chan RA, Korea
Publication date: 01/10/2008
Abstract:
The present invention relates to a method for isolating hair follicle stem cells and a composition for inducing hair growth. More specifically, relates to a method for isolating hair follicle stem cells showing a positive immunological response to CD34, by chemically degrading hair follicle-containing scalp tissue and then culturing the degraded tissue in a serum-containing medium and a serum-free medium, as well as a composition for inducing hair growth, which contains, as an active ingredient, CD34-positive hair follicle stem cells isolated by the method. The hair follicle-derived stem cells, which are obtained according to the disclosed method, are classified as autologous adult stem cells, have self-renewal capability, the ability to differentiate into adult hair follicle cells and the ability to induce hair growth, and can be used as a novel cell therapeutic agent against hair loss. In addition, the present invention relates to a method for culturing hair follicle cells, which has high yield compared to that of the prior art, as well as a method for identifying hair follicle stem cells.
First Claim:
A method for isolating hair follicle stem cells, the comprising the steps of: (a) cutting hair folliclecontaining scalp tissue into fine pieces and chemically degrading the cut tissue; (b) collecting the chemically degraded tissue and culturing the collected tissue in a medium containing 130 vol% of serum, in an incubator; (c) replacing the medium with a serum free medium, when the tissue adheres to the incubator and then reculturing the tissue; and (d) collecting the cultured and proliferated scalp cells from the recultured tissue, and isolating hair follicle stem cells showing a positive immunological response to CD34, from the collected cells
Background:
The present invention relates to a method for isolating hair follicle stem cells and a composition for inducing hair growth, and more particularly to a method of isolating hair follicle stem cells showing a positive immunological response to CD34, by chemically degrading hair follicle-containing scalp tissue and then culturing the degraded tissue in each of serum-containing medium and serum- free medium, as well as a composition for the induction of hair growth, which contains, CD34-positive hair follicle stem cells isolated by the method, as an active ingredient.
Recently, with an increasing interest in beauty, an interest in the treatment of alopecia has also increased. Alopecia refers to hair loss in areas of skin that normally have hair. Alopecia can be divided into scarring alopecia where the skin scars, and non-scarring alopecia where only hair falls out. In scarring alopecia, hair follicles are permanently destroyed and hair never regrows. Hair is made in hair follicles, and each follicle undergoes repeated cycles of active growth and rest and has about 10-20 hair follicle growth cycles in a person's lifetime (Cotsarelis, G. et ah, Cell, 61(7): 1329, 1990). Generally, 85-95% of hairs are in the anagen stage, and the number of anagen-phase follicles decreases as a person grows old. Thus, 10-15% of hairs are in the catagen phase or telogen phase, and an average of about 50-60 hairs normally fall out daily. A hair loss of more than 100 hairs a day can lead to alopecia.
Various methods for the treatment of hair loss have been suggested, and among them, a surgical method, which is currently most frequently used, is autologous hair transplantation, in which follicles are removed from a nonbalding region of the scalp and transplanted to the balding region. However, this method has a problem in that the transplanted hair often looks floppy.
With respect to drug therapy, only two drugs, Propecia for oral administration and Minoxidil for skin application, have been approved for use by the US FDA. Propecia shows therapeutic effects during the administration period, but if the administration thereof is stopped, hair loss develops again like before the drug is administered (Bouhanna, P., Dermatol. Surg., 29(11): 1130, 2003; Thiboutot, D., Arch. Dermatol, 135(11): 1417, 1999). Meanwhile, Minoxidil is a lair loss treatment drug which can be used for males and females (Bouhanna, P., Dermatol. Surg., 29(l l): 1130, 2003; Messenger, A.G. & Rundegren, J., Br. J. Dermatol, 150(2): 186, 2004). However, it has been reported that such drugs have a lot of side effects, including sexual dysfunction (Messenger, A.G. & Rundegren, J., Br. J. Dermatol, 150(2): 186, 2004).
Meanwhile, gene therapeutic methods have recently received attention. Since genes, which is involved in diseases causing general hair loss, were found and reported (Ahmad, W. et al, Science, 279(5351):720, 1998), therapeutic methods of delivering target DNA codes directly to hair follicles using such gene structures or inhibiting the expression of the genes have been actively developed. However, the efficiency, therapeutic cost, safety, effects on future generations, etc. of such therapeutic methods are not yet sufficiently studied, and thus, even if genes, which are involved in hair loss, are found, a considerable period of time will be required to achieve the practical application of therapeutic methods which employ the genes.
Also, US Patent 6,399,057 discloses a method for the regeneration of hair, comprising removing hair in the anagen phase from donor regions by plucking so that a bulb is still attached to the hair removed, culturing the hair follicle cells of the removed hair, and then implanting the cultured cells into pores of receptor regions. However, this method is considered to be insignificant.
Meanwhile, adult stem cells have received a great deal of attention as cell therapeutic agents against many diseases. As used herein, the term "adult stem cells" refers to cells from all adult organs, which exhibit self-renewal, self- maintenance and multipotency. Up to date, the characteristics of adult stem cells and operation thereof have been applied in a wide range of clinical areas. For example, the identification of corneal epithelial stem cells inside the eyeball led to the development of new techniques for corneal transplantation (Cotsarelis, G. et al, Cell, 57:201, 1989; Tsai, RJ. et al, N. Engl. J. Med., 343:86, 2000) and resulted in autologous stem cell transplantation and gene therapy due to the characteristics of hematipoietic stem cells (Bernstein, LD. et al., Blood Cells, 20: 15, 1994). Also, the concept of epidermal stem cells has been studied 30 years ago (Potten, C.S., Cell Tiss. Kinet., 1:11, 1974). More recent in vitro studies based on cell culture studies (Barrandon, Y. & Green, H., Proc. Natl. Acad. Sci. USA, 84:2303, 1987; Rochat, A. et al, Cell, 76:1063, 1994) and in vivo studies in mice have suggested a somewhat more complex organization and distribution of stem cells in skin, with stem-like cells implicated at specific locations in the interfollicular epidermis, in the upper regions of the outer root sheath of the hair follicle (the so-called bulge region), and in the germinal matrix of growing hair follicles.
The interrelationship between these three separate skin stem cell compartments remains obscure, although it can be hypothesized that the bulge region stem cells represent the most potent reserve population of ultimate stem cells. Also, it has been consistently reported that follicle stem cells in the bulge region express
CD34 cell surface protein in rats (Trempus, CS. et ah, J. Invest. Dermatol., 120(4): 501, 2003). However, it has been reported that the cells of the bulge region in human do not express CD34. Also, whether such CD34-negative cells cause hair growth in hair follicles is not yet found. Furthermore, a culture method for such follicle stem cells was not clearly established, and a marker for the stem cells was unclear. Moreover, although it is known that follicle stem cells are present in some hair follicles, a large amount of stem cells are required for the practical treatment of human baldness, but technology of proliferating isolated stem cells as much as they can be clinically applied is still unsatisfactory. In addition, a marker protein for the stem cells is not yet clearly identified, and thus a method for treating hair loss using the stem cells is still unsatisfactory.
Accordingly, the present inventors have made many efforts to develop a method for the culture of hair follicle cells and a method for the identification of hair follicle stem cells and to use stem cells for the treatment of alopecia, atrichia and the like in beauty and medical fields. As a result, the present inventors have isolated hair follicle stem cells by culturing hair follicle-containing scalp tissue and found that it is a more efficient method to obtain a high yield of hair follicle stem cells, compared to the prior art, and that a composition containing the isolated hair follicle stem cells is effective in the induction of hair growth, that is, the treatment of alopecia, thereby completing the present invention.
Summary:
It is an object of the present invention to provide a method for isolating hair follicle stem cells by culturing hair follicle-containing scalp tissue.
Another object of the present invention is to provide a composition for the induction of hair growth, which contains the hair follicle stem cells obtained using said method, as an active ingredient.
To achieve the above objects, the present invention provides a method for isolating hair follicle stem cells, the method comprising the steps of: (a) cutting hair follicle-containing scalp tissue into fine pieces and chemically degrading the cut tissue; (b) collecting the chemically degraded tissue and culturing the collected tissue in a medium containing 1-30 vol% of serum, in an incubator; (c) replacing the medium with a serum-free medium, when the tissue adheres to the incubator, and then re-culturing the tissue; and (d) collecting the cultured and proliferated scalp cells from the re-cultured tissue, and isolating hair follicle stem cells showing a positive immunological response to CD34, from the collected cells.
The present invention also provides a composition for the induction of hair growth, which contains said isolated CD34-positive hair follicle stem cells as an active ingredient. |
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