KR101749218B1 - Human adipocyte conditioned media extract derived from adipose stem cells having hair growth-promoting effects and uses thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for hair loss prevention or wool induction, which comprises culturing human adipose-derived stem cells in a serum-free medium and then containing the conditioned medium obtained by filtration as an active ingredient, A method of inducing wool by applying or intradermally injecting the composition, a hair loss therapeutic agent containing the above-mentioned pharmaceutical composition as an active ingredient, and an anti-hair remedy.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a human adipocyte stem cell having a hair follicle effect and a hair growth promoting effect and uses thereof,

The present invention relates to a culture solution of human adipose-derived stem cells having a wool effect and, more particularly, to a method for culturing human adipose-derived stem cells in a serum-free medium, A method of inducing wool by topically applying or intradermally injecting the pharmaceutical composition to a hair loss site or a hairless area, a hair loss treatment agent containing the pharmaceutical composition as an active ingredient, and an anti-hair treatment agent will be.

Due to recent interest in beauty, public interest in the treatment of alopecia is also growing. Alopecia (alopecia) refers to symptoms of abnormal hair loss, usually a lot of hair loss symptoms refers to. Hair has a cycle of development, growth, degeneration and dormancy, and it is known that the hair in the dormant period normally suffers from about 50 to 100 times a day.

There are many causes of alopecia, which include maternal aftereffects, stress, hyperthyroidism or hypothyroidism, excessive diet, iron deficiency, sequelae of chemotherapy, male hormonal hair loss androgenetic alopecia) and skin diseases such as scalp ringworm are known.

Currently, the most commonly used method of treating hair loss is oral administration of propecia and topical application of minoxidil. In addition, surgical methods such as hair transplantation using their own hair Transplantation is widely known. Propecia is a synthetic anti-androgen that inhibits the enzyme 5-alpha reductase, which converts testosterone to dihydrotestosterone (DHT). The drug has been originally used for the treatment of benign prostatic hyperplasia and prostate cancer and has been known to be effective against male hormonal hair loss afterwards. In 1997, the drug was approved by the US Food and Drug Administration . However, continued use of Propecia may have adverse effects such as erectile dysfunction and sexual dysfunction in men, and may result in fetal male reproductive anomalies when absorbed into the skin of pregnant women and women of childbearing age. Minoxidil is used for the treatment of vasodilators and alopecia, with side effects such as scalp itching. Treatment with the drug regimen is effective at the time of administration, but it turns out that hair loss resumes once treatment is stopped. Although hair transplantation is also semi-permanent, it has a disadvantage that it is relatively expensive.

Korean Patent Publication No. 2008-0097593 discloses a cell therapy agent containing human adipose tissue-derived adult stem cells and hair follicle cells. In Korean Patent No. 10-0771171, a composition for treating baldness comprising follicular stem cells as an active ingredient .

The present inventors have found that stem cells derived from human adipose are cultured in a serum-free medium, and then conditioned medium obtained by filtration is composed of a non-oxidative keratin-forming cell, which is a constituent cell of epidermis and dermis, Human papillary papilloma cells, thereby completing the present invention.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing hair loss or inducing wool, which comprises culturing human adipose-derived stem cells in a serum-free medium and then filtering the hair-derived stem cells as an active ingredient.

In addition, the present invention provides a method of inducing wool characterized in that the pharmaceutical composition is locally applied or injected intradermally at a hair loss site or a hairless area.

The present invention also provides a therapeutic agent for depilation comprising the above pharmaceutical composition as an active ingredient.

The present invention also provides an anti-aspiring agent comprising the pharmaceutical composition as an active ingredient.

The conditional medium of human adipose-derived stem cells of the present invention is expected to be effectively used for the treatment and prevention of alopecia due to the regeneration of skin tissue and the activity of inducing wool.

FIG. 1 shows the results of measurement of cell proliferation by MTT assay after treatment of human dermal papilla cells with human adipose-derived stem cell-conditioned medium and adipocyte-conditioned medium. Separated adipose tissue-derived stem cells (ADSCs) were maintained in α-MEM for 9, 12 and 15 days. Differentiation into adipocytes from human adipose-derived stem cells was induced by adipocyte differentiation medium (insulin, dexamethasone, isobutyl-methyl-xanthine and indomethacin) for 9, 12 and 15 days . Human adipose-derived stem cells-conditioned medium and differentiated adipocyte-conditioned medium were collected after 48 hour incubation in serum-free alpha-MEM, respectively. Human dermal papilla cells were cultured in human adipose-derived stem cell-conditioned medium and adipocyte-conditioned medium, and cell proliferation was measured by MTT assay at 24 and 48 hours. The proliferation of human dermal papilla cells was significantly increased in the group treated with human adipose - derived stem cell - conditioned media for 48 hours, while the treatment of adipocyte - conditioned media inhibited proliferation. Student's T test was used for statistical analysis and all results were expressed as an average over at least three independent experiments. Different sources of human adipose derived stem cells were also used for each independent experiment. *: Significance test for negative control at p < 0.05, d: duration of treatment in each medium.
Figure 2 shows the expression changes of signal transduction proteins in human dermal papilla cells after human adipose-derived stem cell-conditioned medium treatment. After human adipose-derived stem cell-conditioned medium treatment, the expression of phosphorylated Akt was increased slightly and the expression of phosphorylated Erk was clearly increased.
FIG. 3 shows the results of analysis of cell cycle and cell cycle-related proteins of human dermal papilla cells after treatment with human adipose-derived stem cell-conditioned medium. Human adipose-derived stem cell-conditioned media increased the proliferation of human dermal papilla cells through regulation of the cell cycle. Treatment of human adipose-derived stem cell-conditioned medium for 48 hours caused an increase in S phase and a decrease in G1 arrest (upper) compared to the control group. After 24 h of human adipose-derived stem cell-conditioned medium, the expression of Cyclin D and CDK2 was clearly up-regulated (bottom). CNT: control, ADSC-CM: human adipose-derived stem cell-conditioned medium treated with human dermal papilla cells.
FIG. 4 shows the results of measuring the proliferation of the immortalized keratin-forming cells by MTT assay after treating the human-derived stem cell-conditioned medium with the non-keratinized cells. Immortalized keratin-forming cells are starved for 24 hours and then cultured in human adipose-derived stem cell-conditioned medium for 48 hours. Cell proliferation was then measured by MTT assay. Treatment of human adipose-derived stem cell-conditioned media significantly increased the proliferation of non-keratinized cells at concentrations of 10-30%. All results were expressed as averages over at least 3 independent experiments. Different sources of human adipose derived stem cells were also used for each independent experiment. Student's T test was used for statistical analysis. *: Significance test for negative control at p &lt; 0.05.
FIG. 5 is a graph showing the relationship between ex (n = 5, average age = 34.8 years) collected at the occiput of a male induced adipocyte stem cell-conditioned media for hair growth in vivo growing anagen hair organs. 12.5% human adipose-derived stem cell-conditioned medium was added to Williams E medium. As a negative control, 1/2 volume of? -MEM was mixed in Williams E medium and 1 mM of minoxidil as a positive control was added to Williams E medium. Significant hair shaft elongation was observed in the group treated with 12.5% human adipose-derived stem cell-conditioned medium. The results are expressed as mean ± standard error (cumulative growth) of more than 15 hair follicles per growth condition for 6 days. Student's T test was used for statistical analysis. Five human adipose-derived stem cell-conditioned media were applied to five male scalp samples, respectively. *: Significance test for negative control at p &lt; 0.05.
Figure 6 shows hair growth stimulation by human adipose-derived stem cells in C3H / HeN nude mice. The induction of hair growth was measured using a 7 week old C3H / HeN nude mouse and the hair follicles were synchronized to the resting period. Human adipose-derived stem cells (5 x 10 ⁵ cells) and control group PBS (phosphate buffered saline) were injected intradermally into the dorsal skin of mice at intervals of 3 days for 9 days. Hair growth was measured for 12 weeks after the first injection. As a result, hair growth was observed in the group treated with human adipose tissue stem cells compared to the control group treated with PBS. In the topical application group, a 0.2 mm meso roller (Moohan, Korea) was used 10 times before treatment to increase the uptake of the skin. The hair growth stimulating effect was measured by darkening of the skin. After 12 weeks, hair growth was increased in the topically applied group of human adipose derived stem cell-conditioned medium compared to the control group.
Figure 7 shows H & E staining results for dorsal skin of C3H / HeN nude mice treated with control and human adipose-derived stem cell-conditioned medium. Histological evaluation was performed 2 and 4 weeks after the first topical application of human adipose-derived stem cell-conditioned medium. As a result, we observed an increase in the number of hair follicles after 4 weeks of human fat-derived stem cell-conditioned medium treatment.

In order to accomplish the object of the present invention, the present invention relates to a pharmaceutical composition for preventing hair loss or for inducing wool, which comprises culturing human adipose-derived stem cells in a serum-free medium and then filtering the conditioned medium obtained as an active ingredient .

In the pharmaceutical composition of the present invention, the conditioned medium is preferably cultured human serum-derived stem cells in serum-free modified Eagle medium (α-MEM) for 40 to 60 hours, centrifuged and then filtered with 0.2 to 0.25 μm filter Filtration. More preferably, the human adipose-derived stem cells are cultured in serum-free modified Eagle medium for 48 hours, centrifuged, and then filtered through a 0.22-μm filter.

In the pharmaceutical composition of the present invention, the conditioned medium can promote scalp and hair growth by increasing the proliferation of human dermal papilla cells and human keratinocytes.

The stem cells are undifferentiated cells capable of differentiating into various types of tissue cells, and they are separated from embryonic stem cells and adult tissues isolated from inner cell mass of blastocyst. (Adult stem cells) can be roughly classified into. Adult stem cells refer to undifferentiated cells having multipotency derived from mammals including humans, preferably human adult tissues. For example, adult stem cells include various cells such as bone marrow, blood, brain, skin, Can be derived from adult cells.

The human adipose-derived stem cells are a kind of adult stem cells isolated from human adipose tissue. The acquisition of adipose tissue can be obtained incidentally during the liposuction process, which is usually performed, and thus it is possible to easily obtain and cultivate a sufficient amount of stem cells, and also has superior safety compared to bone marrow harvesting.

The human adipose-derived stem cells can be cultured by a conventional method using a medium for stem cell culture, for example, a serum medium. Preferably, human adipose-derived stem cells are cultured in α-modified Eagle medium containing 10% fetal bovine serum and finally cultured in serum-free α-modified Eagle medium to increase protein content in the obtained culture medium . The serum-free medium step minimizes differentiation by exposing the stem cells isolated from adipose tissue to a specific extreme environment, and enables the recovery of the proteins secreted by the stem cells from the culture medium as much as possible.

The pharmaceutical compositions comprising the conditioned media of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the preparation of pharmaceutical compositions.

The pharmaceutical dosage forms of the conditioned media of the present invention may also be used in the form of their pharmaceutically acceptable salts and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.

The pharmaceutical composition comprising the conditioned medium according to the present invention may be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions Can be used. Examples of carriers, excipients and diluents that can be contained in the composition containing the conditioned medium include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Various compounds or mixtures including silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferable dosage of the condition medium of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the condition medium of the present invention is preferably administered at 0.0001 to 100 mg / kg per day, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

Conditioned media of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, muscular, subcutaneous, topical application or intradermal injection.

In addition, the present invention provides a method of inducing wool characterized in that the pharmaceutical composition is locally applied or injected intradermally at a hair loss site or a hairless area. The dosage form may be, but is not limited to, topical application or intradermal injection.

In addition, the present invention provides a depilatory remedy comprising the above-mentioned pharmaceutical composition as an active ingredient, and an anti-hair remedy.

The hair loss therapeutic agent and the treatment for alopecia of the present invention can be applied not only to the scalp but also to any part of the body where hair growth is necessary for the treatment of female haploid alopecia that may occur after male hormone alopecia and postmenopausal or ovariectomy surgery have.

The pharmaceutically acceptable carrier to be included in the depilatory remedy and the asymmetry treatment agent of the present invention is one which is commonly used in the present invention and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, Gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, But is not limited thereto.

The depilatory remedy and the asymmetry remedy of the present invention can be formulated into a unit dose form and / or an asymmetrical form by using a pharmaceutically acceptable carrier and / or an excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. Or may be prepared by entrapping in a multi-dose container, and may additionally include a dispersant or stabilizer.

The hair loss remedy and the asymmetry remedy of the present invention may be used as a single therapy but may be used together with other conventional hair growth induction drug therapy or surgery therapy and the maximized effect can be exhibited when such concurrent therapy is performed.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1: human adipose-derived stem cells ( ADSCs ) And conditioned medium ( Conditioned  media collection

Human adipose derived stem cells (ADSCs) were cultured as previously described (Lee et al., 2004, Cell Physiol Biochem 14, 311-324). Human adipose derived stem cells were collected from subcutaneous adipose tissue obtained by liposuction from five women of average age 43.8 years. Human fat-derived stem cells were treated with 0.075% collagenase type I collagenase type I (Sigma-Aldrich, St. Louis, Mo., USA) for 45 minutes at 37 ° C, mesh filter, and then the filtrate was mixed with α-modified Eagle medium (α-MEM; Invitrogen, Carlsbad, CA, USA) and centrifuged. The obtained pellet portion, that is, a human adipose-derived stem cell fraction, was centrifuged at 1200 xg for 5 minutes, and then an α-lactamase containing 10% fetal bovine serum (FBS) The fractions were resuspended in MEM and incubated at 37 ° C in 5% CO ₂ . Adipogenic differentiation from human adipose-derived stem cells into adipocyte differentiation leads to insulin, dexamethasone, isobutyl-methyl-xanthine, and indomethacin. RTI ID = 0.0 &gt; 9-15 &lt; / RTI &gt;

In order to harvest the condition medium (conditioned media, CM) from the cultured cells, human adipose derived stem cells ⁽⁵ × 10 5 cells) and mature fat cells (adipocytes) were cultured in serum-free (serum-free) α-MEM . Human adipose-derived stem cell-conditioned medium (ADSC-CM) and adipocyte-CM medium were harvested 48 hours after culture. The collected conditioned mediums were centrifuged and resuspended in 0.22 μm syringe filter. Lt; / RTI &gt;

Example  2: human Dermal papilla cells ( human dermal papilla cells ) And Immortalization  Proliferation of keratinocytes proliferation ) And cell cycle analysis

Since the periodic change of hair involves rapid remodeling of epidermal and dermal components, the proliferation of cultured human dermal papilla cells (hDPCs) and immortalized keratinocyte (HaCaT cells) ) Were investigated. Human dermal papilla cells and non-keratinized cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen-Gibco-BRL) containing 10% fetal bovine serum. The cell proliferation was assessed by MTT assay using 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide after treating human adipose stem cell-conditioned medium Lt; / RTI &gt; Different sources of human adipose derived stem cells were also used for each independent experiment. Proliferation of human dermal papilla cells was increased up to 130% in the groups treated with human fat-derived stem cell-conditioned medium at 25, 50 and 75%, while addition of adipocyte-conditioned medium inhibited proliferation 1).

To clarify the underlying mechanism, signaling proteins associated with cell proliferation were analyzed by Western blot. (Cell Signaling Tech., Inc., MA), Akt (Cell Signaling Tech., Inc., MA) and phosphorylated Erk (pErk; Thr202 / Tyr204, Cell Signaling Tech. MA) was used. In the Western blot results, the levels of phosphorylated Akt expression were increased 24 and 48 hours after the human adipose-derived stem cell-conditioned medium compared to total Akt. Phosphorylated Erk expression was also significantly increased following treatment with human adipose-derived stem cell-conditioned media, while the expression of total Erk was unchanged (Figure 2).

Next, cell cycle analysis of human dermal papilla cells treated with human adipose-derived stem cell-conditioned medium was performed using a flow cytometer (FACScan flow cytometer, Beckton-Dickinson, Calif.). To induce G1 arrest, human dermal papilla cells (3.2 x 10 ⁵ cells / dish) were starved and the medium was then grown in serum-free medium or 50% human adipose-derived stem cell-conditioned And then cultured for 48 hours. In the FACS cell cycle analysis, treatment of human adipose-derived stem cell-conditioned medium increased the growth of the human dermal papilla cells (G1) while increasing the DNA synthesizer (S) and mitotic factor (G2-M) 3, top). Cyclin D1 (Serotec, UK) and CDK2 (Santa Cruz biotechnology), important molecules involved in the cell cycle, did not change the expression of p21, p27 and CDK4 in subsequent western blots Was clearly up-regulated 24 hours after stem cell-conditioned medium treatment (Fig. 3, bottom). These results indicate that human adipose-derived stem cell-conditioned medium increases the proliferation of human dermal papilla cells through regulation of the cell cycle. In addition, human adipose-derived stem cell-conditioned medium appears to promote survival and proliferation of dermal papillary cells by activating the Erk and Akt signaling pathways. The size of the dermal papilla is closely related to the hair growth cycle and the number of dermal papillary cells is reported to increase in the anagen phase (Elliott et al., 1999, J Invest Dermatol 113, 873-877). In addition, the human adipose-derived stem cell-conditioned medium significantly promotes the proliferation of the immortalized keratin-forming cells at a concentration of 10-30% (FIG. 4).

Example  3: Moraine kidney ( hair shaft elongation ) Human adipose-derived stem cells-conditioned medium of  Effect analysis

The effect of human adipose-derived stem cell-conditioned medium on hair shaft elongation was investigated as the human adipose-derived stem cell-conditioned medium exhibits a marked mitogenic effect. A total of 370 anagen hair follicles collected from 5 men (mean age 34.8 years old, 70 to 80 hair follicles per person) received 10 ng / mL hydrocortisone, 10 g / mL insulin, L- glutamine (Gibco BRL, Gaithersburg, Md., USA) containing 5% CO ₂ , 2 mM and penicillin 100 U / Lt; / RTI &gt; Human adipogenic stem cell-conditioned medium was added to Williams E medium at the level of 12.5%, 25%, 37.5% or 50%, and cultured for 6 days. As a result, the hair follicle length was significantly increased by 40% in the 12.5% treated group compared with the control group treated with minoxidil 1 mM (Fig. 5).

Example  4: In vivo  Analysis of the wool effect of human adipose-derived stem cells-conditioned media

Seven week old male C3H / HeN nude mice (n = 48, Orient Bio, Korea) shaved the dorsal skin and synchronized the hair follicle with the telogen stage. Subsequently, intradermal injection of human adipose-derived stem cells (5 x 10 ⁵ cells / 50 mL PBS) or PBS (phosphate buffered saline) into the dorsal skin was performed at intervals of 3 days for 9 days. At the same time, 1 mL of human fat-derived stem cell-conditioned medium or control medium was topically applied to the back of another C3H mouse. As a result, the hair follicle of the C3H / HeN nude mouse injected intracutaneously with human adipose-derived stem cells was faster in the transition from dormant to growth phase than the control (Fig. 6, top) (Fig. 6, bottom). It was also found that when the dorsal skin of mice treated with human adipose-derived stem cell-conditioned medium was histologically examined, the number of hair follicles was increased (Fig. 7). These results indicate that locally injected human adipose-derived stem cells and human adipose-derived stem cell-conditioned medium can promote hair growth in vivo. It is thought that factors secreted from human adipose derived stem cells have somewhat therapeutic potential for hair growth.

The results of the present invention show that human adipose-derived stem cells promote hair growth by increasing proliferation of human dermal papilla cells, and also enable epidermal cells to regulate the cell cycle and activate the anagen phase of the hair cycle . Therefore, rational manipulation of human adipose-derived stem cells is considered to be a good means for promoting hair growth.

Claims (6)

Human adipose-derived stem cells were cultured in a serum-free medium, and then 12.5% by weight of a conditioned medium obtained by filtration was contained as an active ingredient,
The conditional medium was obtained by culturing human adipose-derived stem cells in serum-free modified medium (α-MEM) for 40 to 60 hours, followed by centrifugation followed by filtration through a 0.2 to 0.25 μm filter. &Lt; / RTI &gt; delete 2. The method according to claim 1, wherein the conditioned medium promotes scalp and hair growth by increasing the proliferation of human dermal papilla cells and human keratinocytes. &Lt; / RTI &gt; Characterized in that the pharmaceutical composition of claim 1 is topically applied or intradermal injected to the hair loss site or hairless area of an animal other than a human. A hair rest treatment agent comprising the pharmaceutical composition of claim 1 as an active ingredient. A therapeutic agent for asymmetry comprising the pharmaceutical composition of claim 1 as an active ingredient.

Images