Hair regeneration

Abstract

To provide compositions for regenerating hair follicles, and to provide uses thereof.SOLUTION: Provided is a composition for use in a method for regenerating hair follicles, the composition comprising an expanded population of androgen-unsuppressed cells produced by culturing in vitro androgen-unsuppressed cells obtained from hair follicle tissue under conditions suitable for proliferation, and the use comprising a step of transplanting the composition proximal to a miniaturized hair follicle and thereby regenerating the miniaturized hair follicle.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to methods and formulations for hair restoration. [Background technology]

[0002] Treatment of hair loss represents a large unmet public health need. Hair loss can have a serious impact on self-confidence and, consequently, on an individual's quality of life. It has also become clear in recent years that male pattern baldness is highly associated with an increased risk of squamous cell carcinoma and basal cell carcinoma, especially in the scalp region (Li et al., 2016, Int J Cancer 139(12):2671-2678). Both men and women suffer from hair loss, with approximately 40% of men having significant hair loss by age 35 and approximately 80% of women experiencing significant hair loss by age 60. Androgenetic alopecia (AGA) is the most common form of hair loss in both men and women, also known as male pattern and female pattern baldness, respectively.

[0003] Hair grows from the follicular tissue, a multi-layered, angular invagination of the superficial epithelium. The follicular unit is composed of a cell population of mesenchymal and epithelial-derived cells that are closely bound together. Germline epithelial cells in the hair bulb proliferate and differentiate to give rise to the mature hair shaft. The mesenchymal component consists of fibroblast-like cells that form a morphological unit known as the dermal papilla (DP), located in the substructure of the follicular unit, and the dermal sheath (DS), which resides around the outer border of the epithelial follicular component. The DP is essential for hair follicle growth and circulation. Biochemical signaling by DP cells controls the cellular mechanics of the epidermal component and the physical size of the entire hair follicle.

[0004] Hair has other tissues such as nerves, sebaceous glands, blood supply, and the attached erect pili muscle that can change the angle of the follicle in the skin. Where this muscle attaches to the follicle itself, there is a bulge region that contains a reservoir of multipotent epithelial stem cells called bulge cells.

[0005] Most of the human body surface, except the soles of the feet and palms of the hands, and the lips, contains hair follicles; an adult may have roughly 5 million hair follicles, of which about 100,000 are on the scalp. Hair follicle density varies by region, with about 100,000 per cm on the thighs. 2 Approximately 50 per cm of scalp 2 The number of hair follicles varies from approximately 600 per follicle. The shaft diameter varies as does the density of the hair follicles, and the total length is highly variable, ranging from approximately 10 to 30 μm in diameter for the fine vellus hairs covering the forehead, close to the "terminal hairs" that cover the scalp and are approximately 200 μm in diameter.

[0006] Hair follicle growth begins when epidermal basal cells form the visible hair placode and dermal fibroblasts begin to congregate and differentiate beneath the placode to form the bulbous DP. Epidermal cells form a multi-layered and elongated column called the hair plug, which thickens at the bottom to form the hair bulb and surround the elongated DP, and the growing hair follicle extends into the dermis. Differentiation of epithelial cells in the hair plug within the defined layer continues, generating the hair shaft. Finally, this hair shaft protrudes from the skin surface and the hair follicle reaches its full length. Hair follicle morphogenesis therefore requires strong communication and co-growth between the epidermal and dermal parts.

[0007] It has long been known that transplantation of freshly isolated DP cells into adult skin can repeat this developmental process, promoting the formation of new hair follicles from undifferentiated interfollicular epidermis. When this was first discovered, it opened up the possibility that DP cells could be used to generate brand new hair follicles.

[0008] Four methods have been described that can support the expansion of DP cells in number while maintaining their hair-inductive potential to generate new hair follicles. In the first method, conditioned medium (CM) collected from mammalian epidermal cells (keratinocytes) or co-culture with keratinocytes has been shown to support both the expansion of DP cells in number and the maintenance of their hair-inductive phenotype during several culture passages. This first method is described in US5851831 and Jahoda et al. (1998, J. Invest. Dermatol. 111:767-75). The second method describes the culture of DP cells in the presence of increased levels of wnt protein or agents that mimic the effects of wnt-promoted signal transduction (see WO01 / 74164 and Kishimoto et al., 2002, Genes&Development 14:1181-85). In the second method, the wnt protein or functional fragment or analog thereof is added to the culture medium, either as a purified product or by expressing recombinant protein in producer cells and providing the wnt protein to the medium conditioned by the growth of the wnt producer cells or by co-culturing with the producer cells. The third method involves culturing hair-inducing cells in a culture medium containing prostate epithelial cells and medium conditioned by the prostate epithelial cells. The fourth method involves culturing DP cells in 3D hanging drops (see US9550976, US9109204, and Higgins et al., 2013, Proc Natl Acad Sci USA 110(49):19679-88).

[0009] However, it has also been shown that DP cells change their transcriptional profile and lose their hair-inductive potential when grown in standard 2D culture, limiting their potential to treat AGA. Higgins et al. (2013, supra) describe partial restoration of hair follicle inductive potential of DP cells in three-dimensional (3D) culture, resulting in hair follicle induction. US9109204 describes a method of collecting DP cells to promote hair follicle formation in vitro, e.g., in cut hair follicles.

[0010] Current hair loss therapies include oral and topical treatments as well as hair transplant procedures. The currently used oral and topical treatments for hair loss must be taken lifelong and are only effective during the early stages of hair loss. Only two medications, topical minoxidil and oral finasteride, are currently approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of androgenetic alopecia (AGA). Minoxidil can slow the progression of hair loss and partially restore hair, but it is expensive and effective in only a small percentage of men and women. Even those select individuals who respond need to continue the treatment to maintain the effect. Minoxidil can also cause irritation and / or allergic reactions at the application site. Finasteride is only effective in men, and only in a small percentage of men. Finasteride can also cause long-term sexual side effects such as decreased libido and erectile dysfunction. Any beneficial effects on hair growth are lost upon cessation of treatment within 6 to 12 months.

[0011] Hair loss can alternatively be treated by transplanting hair follicles from areas not expected to be affected by AGA (e.g., the front, mid-scalp and vertex areas of the scalp) into the areas where hair loss is present. However, the number of hair follicles available for transplantation is limited, and the number of hairs left in the subject after transplantation does not change, since hair is simply distributed from areas of high to low hair density. Moreover, hair transplantation is often not appropriate for men who are just beginning to be affected by AGA, since the border between affected and unaffected areas is not clearly defined. Thus, the effectiveness of hair transplantation is limited by the number of suitable patients and available donor hairs. However, despite the limitations of this procedure, more than 500,000 surgical hair restoration procedures were performed worldwide in 2016, generating a market of US$4.1 billion. Summary of the Invention [Problem to be solved by the invention]

[0012] A 2015 survey by the International Society of Hair Restoration Surgery (ISHRS) found that the most common complaints of hair transplant patients were a resultant hair density that was less than expected (57% of all complaints) or hair loss after transplant (39% of complaints). Hair transplants are considered "hair restoration" because they can only be performed when the boundaries of hair loss are precisely defined in a particular patient. As a result, roughly 80% of male hair transplant patients are over 30 years old and already suffer from severe hair loss. [Means for solving the problem]

[0013] The present invention generally relates to a method for regenerating cells of hair follicles that have become miniaturized as a result of, for example, hormonal alopecia in men or women, by introducing non-androgen-suppressed cells into the affected area to at least partially replace lost androgen-suppressed cells, thereby increasing hair shaft diameter and visible hair density, thus at least to some extent reversing the apparent hair loss.

[0014] In one aspect, the present invention is directed to a method for regenerating hair follicles, the method comprising the steps of: (1) obtaining androgen-unsuppressed cells from hair follicle tissue; (2) culturing the androgen-unsuppressed cells obtained in step (1) under conditions suitable for proliferation to generate an expanded population of androgen-unsuppressed cells; and (3) regenerating miniaturized and / or miniaturized hair follicles by transplanting the expanded population of androgen-unsuppressed cells generated in step (2) proximal to the miniaturized and / or miniaturizing hair follicle.

[0015] The androgen-unsuppressed cells may be androgen-insensitive cells, for example hair follicle tissue, hair follicle tissue of the scalp.

[0016] Alternatively, the androgen non-suppressed cells can be androgen stimulated cells, for example hair follicular tissue is beard hair follicular tissue, chest hair follicular tissue, armpit hair follicular tissue, and / or pubic hair follicular tissue.

[0017] Optionally, the androgen-unsuppressed cells in the methods of the present invention comprise or consist of dermal papilla (DP) cells. DP cells have been shown to possess particularly potent regenerative capabilities.

[0018] The androgen-unsuppressed cells transplanted in step (3) of the method of the present invention can regenerate miniaturized and / or shrinking hair follicles by reactivating and / or replacing androgen-sensitive cells in miniaturized and / or shrinking hair follicles. There may also be other mechanisms of hair regeneration.

[0019] The hair follicular tissue can be mechanically extracted, for example, using follicular unit extraction (FUE). Alternatively, the hair follicular tissue can be obtained using follicular unit transplantation (FUT).

[0020] The methods of the invention can include culturing the androgen-unsuppressed cells to expand the number of androgen-unsuppressed cells by at least about 2-20 fold, at least about 500-1000 fold, or at least 1-6 population doublings. Expanding the number of androgen-unsuppressed cells increases the number of cells available for transplantation.

[0021] Steps (1) and / or (2) of the method can include, for example, selecting and / or removing (e.g., by sorting) androgen-inhibited cells from a mixed population of cells in the hair follicle tissue using one or more biomarkers. Cell selection or sorting eliminates unwanted androgen-sensitive cells from the cell population.

[0022] In the method of the present invention, non-androgen-suppressed cells can be obtained from a subject in method step (1), cultured in method step (2), and then transplanted into the same subject in method step (3). That is, the transplanted cells are autologous. Autologous cells are more likely to be tolerated by the host tissue and / or be particularly effective in hair regeneration in certain individuals. However, the use of allogeneic cells is also contemplated in the present invention.

[0023] In another aspect, the present invention relates to a composition containing an expanded population of in vitro androgen-unsuppressed cells for regenerating hair. The androgen-unsuppressed cells can have any of the characteristics as used in the method of the present invention.

[0024] The cells of the composition may be autologous or may be allogeneic, for example related to the subject being treated with the cells.

[0025] The composition may be formulated and administered, for example, as an injection, by a physician or a non-physician medical technician.

[0026] The composition may be used in methods of treatment, for example cosmetic treatment.

[0027] The composition may be used in a method for regenerating hair growth and / or slowing hair loss.

[0028] The composition can be used in the manufacture of a medicament for regenerating hair growth and / or slowing hair loss.

[0029] The composition can be used for application to a subject having hair loss, such as male hormonal alopecia.

[0030] The compositions of the present invention can be used in systems for analyzing hair follicle cells and / or testing cosmetic drugs or pharmaceuticals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Abbreviation 3D three-dimensional 5αDHT 5-alpha-dihydrotestosterone AGA Androgenetic alopecia DHT Dihydrotestosterone DP dermal papilla EMA European Medicines Agency FDA Food and Drug Administration FUE Follicular Unit Extraction FUT Follicular Unit Transplant

[0032] Terminology Before describing the present invention in detail, it is to be understood that this invention is not limited to particular compositions or processing steps.

[0033] A cosmetic method is defined as a method for improving an individual's appearance. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature used in connection with the cell and tissue culture techniques, molecular biology, and protein and oligo- or polynucleotide chemicals and hybrids described herein are those well known and commonly used in the art. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press, Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press, and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this invention.

[0034] Standard techniques are typically used for tissue culture, including growing cells in conditions suitable for growth. Methods, reagents and conditions suitable for culturing cells obtained from hair follicle tissue are provided in the specific examples. Enzymatic reactions and purification methods can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The aforementioned techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning, Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)). The analytical chemistry, organic synthesis chemistry, and medicinal and pharmaceutical chemistry laboratory methods and techniques described herein, and the nomenclatures utilized in connection therewith, are those well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparations, formulations, preparations and delivery, and treatment of patients.

[0035] A suitable method for obtaining androgen-unsuppressed cells from tissue and transplanting a population of cells is provided in a specific embodiment. The androgen-unsuppressed cells may be androgen-insensitive cells or androgen-stimulated cells. The androgen-sensitive cells may be obtained, for example, from non-bald areas of the scalp. The androgen-stimulated cells may be obtained, for example, from beard, chest hair, armpit hair or pubic hair areas.

[0036] As utilized in accordance with the present disclosure, the following terms, unless otherwise specified, shall be understood to have the following meanings:

[0037] The term "and / or" as used herein shall be understood as a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" shall be understood as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were presented alone.

[0038] The terms "a" or "an" can refer to either one or more than one element that it modifies (e.g., "a reagent" can mean one or more than one reagent), unless the context clearly dictates otherwise.

[0039] The term "about" as used herein in connection with any and all values ​​(including the lower and upper limits of a numerical range) refers to any value having an acceptable deviation range of up to ±10% (and any value therebetween, e.g., ±0.5%, ±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%, ±4%, ±4.5%, ±5%, ±5.5%, ±6%, ±6.5%, ±7%, ±7.5%, ±8%, ±8.5%, ±9%, ±9.5%). The use of the term "about" at the beginning of a series of values ​​modifies the respective value (i.e., "about 1, 2, and 3" refers to about 1, about 2, and about 3). For example, a weight of "about 100 grams" can include weights from 90 grams to 110 grams. Additionally, when a list of values ​​is set forth herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%), the list includes all intermediate and fractional values ​​(e.g., 54%, 85.4%).

[0040] Aspects of the invention Hair exhibits repeated cycles of growth and rest. The three phases of this cycle are anagen, catagen, and telogen. In some animals, these cycles are synchronized, but each strand of hair in the human body is asynchronous and in a unique phase of the cycle. Anagen is the growth phase, which can last from several months to several years. The longer the hair remains in the anagen phase, the longer it will grow. Approximately 85% of the hair on the human scalp is in this growth phase at any one time. This growth phase is followed by its catagen phase, which lasts for about two weeks. During this period, the cellular tissue of the hair follicle shrinks to approximately 1 / 6 of its original total length. During this phase, the hair does not actively grow, but the hair shaft is still fixed in place within the skin. Following the catagen phase, the hair follicle rests in the telogen phase, remaining dormant for one to four months. At some point, a new cycle is initiated and the hair follicle tissue is completely renewed again. The hair substructure will fall out from the root and the old hair shaft will fall off. Within 2 weeks, a new hair shaft will begin to emerge as the next growth phase begins. The new hair shaft is usually of similar size and organization to the one produced in the previous cycle. In an average human lifespan, scalp hair will go through approximately 12 of these cycles. This cycle is known as androgen-dependent, and changes in androgen levels can cause noticeable changes in hair shaft diameter and total length in certain areas between one cycle and the next. For example, during puberty, changes in androgen levels can cause an increase in hair shaft diameter in certain areas. However, androgens can also cause a reduction in hair shaft diameter and a miniaturization of the hair follicle itself throughout the progressive cycle, and this miniaturization during diseases such as AGA is most noticeable on the scalp.

[0041] Thus, not all hair follicles are hormonally responsive, and AGA only affects certain androgen-sensitive hair follicles. On the scalp itself, this is most evident in men as male pattern baldness, whereby progressive temporal hair miniaturization begins at the temples and spreads posteriorly across the scalp. This miniaturization of the diameter of the hair follicle from the end to the vellus state occurs over many cycles. Moreover, during this miniaturization process, the length of time that the hair follicle remains in the anagen or growth phase of the hair follicle is greatly shortened, and balding hair follicles spend a greater proportion of their hair follicle cycle in the telogen non-growth stage, reducing both the total length and the diameter of the hair shaft.

[0042] Hair follicle size and, consequently, hair shaft diameter are determined by their mesenchymal components, and it has been shown that the miniaturization of the human hair follicle DP is a direct consequence of a decrease in the number of papilla cells in the follicle over time, which appears to occur between the anagen phase and the development of the next hair shaft.

[0043] Human DP cells from different body sites differ in their responsiveness to androgens. In particular, androgens (e.g., dihydrotestosterone [DHT] or 5α-dihydrotestosterone [5α-DHT]) stimulate the growth of hair follicles, for example, in the armpit hair, pubic hair area, and male beard, but lead to miniaturization of hair follicles in the scalp of patients suffering from AGA. Some hair follicles are insensitive to androgens and remain roughly the same size throughout life. Thus, human DP cells can be classified as androgen-suppressed (such as those affected by AGA) and androgen-unsuppressed. These androgen-unsuppressed DP cells can be further subdivided into androgen-stimulated (such as those in the male beard area) or androgen-insensitive (such as those hairs not affected by AGA).

[0044] In one aspect, the present invention is directed to a method for regenerating hair follicles, the method comprising the steps of: (1) obtaining androgen-unsuppressed cells from hair follicle tissue; (2) culturing the androgen-unsuppressed cells obtained in step (1) under conditions suitable for proliferation to generate an expanded population of androgen-unsuppressed cells; and (3) transplanting the expanded population of androgen-unsuppressed cells generated in step (2) proximal to a miniaturized and / or miniaturizing hair follicle, thereby regenerating the miniaturized and / or miniaturizing hair follicle.

[0045] The present invention can involve replacing lost androgen-sensitive DP cells in miniaturized and / or miniaturizing hair follicles with androgen-unsuppressed DP cells, such as androgen-insensitive DP cells, and also increasing the hair shaft diameter. This can be considered as essentially replacing AGA-affected hair follicles with hair follicles not affected by AGA, and changing the properties of the hair follicle itself over the course of the subsequent hair cycle.

[0046] The present invention differs from prior art disclosures in part because it relates to the use of specific androgen-unsuppressed cells to regenerate hair follicles that have become miniaturized by a reduction in the number of androgen-sensitive cells, thereby resulting in an increase in hair follicle diameter.

[0047] The androgen-unsuppressed cells may be androgen-insensitive cells. These cells may be obtained from scalp hair follicle tissue. The androgen-unsuppressed cells may be androgen-stimulated cells, and may be obtained from beard hair follicle tissue, chest hair follicle tissue, armpit hair follicle tissue, and / or pubic hair follicle tissue.

[0048] Androgen-insensitive cells can be obtained from a male subject, for example from the back of the head.

[0049] Optionally, the androgen-unsuppressed cells in the methods of the present invention comprise or consist of DP cells, which have been shown to possess particularly potent regenerative capabilities.

[0050] The androgen-unsuppressed cells transplanted in step (3) of the method of the present invention can regenerate miniaturized and / or shrinking hair follicles by reactivating and / or replacing androgen-sensitive cells in miniaturized and / or shrinking hair follicles. There may also be other mechanisms of hair regeneration.

[0051] The above-mentioned prior art methods for culturing DP cells require that DP cells maintain their ability to promote the formation of new hair follicles through the interaction of the cultured DP cells with recipient keratinocytes. However, regulating the number of DP cells in surviving miniaturized hair follicles represents an alternative strategy to prevent hair loss by repairing and regenerating these diminished hair follicles. Experimental analysis of DP cell function has shown that under appropriate conditions, transplanted mouse DP cells can "locate" to the DP region, form chimeric DPs, and affect hair shaft diameter (see McElwee et al., 2003, Invest Dermatol 121:1267-1275). It is suggested that non-androgen-suppressed cells transplanted in step (3) of the method can act in the same way.

[0052] The hair follicle tissue used in the methods of the present invention can be obtained from areas of the body known or suspected to contain androgen-unsuppressed cells, which advantageously provides an efficient collection of androgen-unsuppressed cells.

[0053] However, androgen-unsuppressed cells from hair follicle tissue are usually detected in a specific area, but in certain embodiments, a single location cannot be used to predict the androgen responsiveness of hair follicles.For example, androgen-sensitive cells can be detected in the back of the scalp of men affected by AGA, while in women, the pattern of hair loss may be more dispersed.Screening of cells obtained from hair follicle tissue of scalp for androgen sensitivity, androgen insensitivity and / or androgen stimulation may therefore include further steps in the method of the present invention.In this way, only or mainly androgen-unsuppressed cells can be cultured in step (2) of the method or transplanted in step (3) of the method.

[0054] The hair follicular tissue can be mechanically extracted, for example, using follicular unit extraction (FUE). Alternatively, the hair follicular tissue can be obtained using follicular unit transplantation (FUT).

[0055] In one embodiment of the present invention, obtaining androgen-unsuppressed cells from hair follicle tissue involves obtaining androgen-unsuppressed cells from a suspension of hair follicle tissue cells by antibody-assisted selection, which provides a marker-determined selection of androgen-unsuppressed cells.

[0056] The methods of the invention can include culturing the androgen-unsuppressed cells to expand the number of androgen-unsuppressed cells by at least about 2-20 fold, at least about 500-1000 fold, or at least 1-6 population doublings. Expanding the number of androgen-unsuppressed cells increases the number of cells available for transplantation.

[0057] Steps (1) and / or (2) of the method can include, for example, selecting and / or removing (e.g., by sorting) androgen-inhibited cells from a mixed population of cells in the hair follicle tissue using one or more biomarkers. Cell selection or sorting eliminates unwanted androgen-sensitive cells from the cell population.

[0058] In one embodiment of the present invention, the expression levels of one or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more) genes are used as androgen-insensitive biomarkers to identify and / or select androgen-unsuppressed cells. The one or more genes can be selected from the group consisting of STX17 antisense RNA1, prostaglandin I2 (prostacyclin) synthase, calmegin, glypican 6, integrin beta 8, collagen, type X, alpha 1, reelin, carbonic anhydrase XIII, DEP domain-containing MTOR interacting protein, and HAUS augmin-like complex subunit 6.

[0059] It was found that the genes listed above can be expressed at higher levels in androgen-unsuppressed DP cells, but not or only slightly expressed at lower levels in androgen-sensitive DP cells, providing efficient and accurate identification and selection of androgen-unsuppressed cells.

[0060] Additional or alternative genes whose expression or non-expression can be used as biomarkers for androgen non-suppressed or androgen suppressed cells are shown below in Example 4 (particularly in Table 1 and Lists A-D). Biomarkers can be, for example, STON1 (Stonin-1), CYB5R3 (symbolized Cytochrome B5 Reductase3) and / or SRD5A1 (symbolized Steroid 5alpha-reductase1).

[0061] The expression or non-expression of suitable control genes known in the art can be examined for comparative purposes.

[0062] Steps (1) and / or (2) of the methods of the present invention can include selecting or sorting cells for androgen-inhibited cells using a biomarker. Cell selection or sorting removes unwanted androgen-sensitive cells from the cell population.

[0063] The androgen of the method of the present invention may be dihydrotestosterone (DHT) and / or the androgen-unsuppressed cells may be human.

[0064] In the methods of the present invention, androgen-free cells can be obtained from a subject in method step (1), cultured in method step (2), and then transplanted into the same subject in method step (3). That is, the transplanted cells are autologous. Autologous cells may be more likely to be tolerated by host tissue and / or to be particularly effective in hair regeneration in certain individuals.

[0065] In another aspect, the present invention relates to a composition containing an in vitro expanded population of androgen-unsuppressed cells for regenerating hair. The androgen-unsuppressed cells can have any of the characteristics as used in the method of the present invention.

[0066] The cells of the composition may be autologous or may be allogeneic, for example related to the subject being treated with the cells.

[0067] The composition may be dispensed and administered, for example, as an injection, by a physician or a non-physician medical technician.

[0068] The composition may be used in methods of treatment, for example cosmetic treatment.

[0069] The composition may be used in a method for regenerating hair growth and / or slowing hair loss.

[0070] The composition can be used in the manufacture of a medicament for regenerating hair growth and / or slowing hair loss.

[0071] The composition can be used for application to subjects with hair loss, such as male hormonal alopecia. This use may be purely cosmetic.

[0072] The compositions of the present invention can be used in systems for analyzing hair follicle cells and / or testing cosmetic drugs or pharmaceuticals.

[0073] The entirety of each patent, patent application, publication, and document referred to in this specification is incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein. Citation of the foregoing patents, patent applications, publications, and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or dates of such publications or documents.

[0074] The technology suitably described herein as illustrative may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The terms and expressions employed are used as terms of description and not as terms of limitation, and the use of such terms and expressions does not exclude any equivalents or portions of the features shown and described, and various modifications are possible within the scope of the description set forth in the claims.

[0075] The foregoing modifications can be made without departing from the fundamental aspects of the technology. Although the technology has been described in sufficient detail with reference to one or more specific embodiments, those skilled in the art will recognize that the embodiments specifically disclosed in this application can be varied and that these modifications and improvements will still be within the scope and spirit of the technology.

[0076] Further aspects, embodiments, features and equivalents related to the invention as disclosed herein are provided in additional detail below.

[0077] Working Example The following examples, including experiments conducted and results achieved, are given to illustrate the practice of this invention. They are not intended to limit or define the entire scope of this invention.

[0078] Example 1 Obtaining hair follicle samples using follicular unit transplantation (FUT) Hair follicle cells have been obtained using the FUT surgical procedure (see Bernstein & Rassman, 2005, Dermatologic Clinics 23(3), 393-414). A small piece of tissue has been removed from an androgen-unsuppressed area of ​​the body, such as the back of the head, from which donor hair follicles can be extracted. Individual hair follicles have then been harvested from that piece. The follicles have either been frozen and stored for future use or further microdissected for culture (see also Example 3). The follicular unit is full thickness, and thus has epidermis, dermis and dermal adipose tissue.

[0079] Each follicular unit has been cut from any attached fat or connective tissue surrounding the bottom of the follicle to expose the terminal hair bulb located in the follicular substructure. Using a pair of scissors, the follicle has been cut transversely through the stroma just above the papilla to extract the terminal hair bulb. After cutting the terminal hair bulb, the dermal papilla (DP) has been extracted from the skin sheath envelope as described by Higgins et al. (2017, Br. J. Dermatol. 176(5), 1259-1269). More specifically, to extract the terminal hair bulb, the hair follicles have each been cut transversely just above the dermal papilla. The terminal hair bulb has been held in place by pressing a 27G needle against the cut apex and holding it in a Petri dish. Another 27G needle has been held in the other hand and gently pushed through the rounded base of the terminal hair bulb to invert the tissue to expose the DP. The DPs surrounded by their glassy membranes were removed using the sharp end of a needle and transferred to a 50 μl drop of collagenase in a fresh Petri dish. Each DP was similarly dissected and placed in the 50 uL drop of collagenase and incubated for 30 minutes. This ensures that the DPs adhere to the Petri dish but do not dissolve in the glassy membrane. Using 2 ml of DMEM supplemented with 10% FBS, 2 mM L-glutamine (Invitrogen, cat. no. 25030), the DPs were then carefully added to each dish, taking care not to remove the DPs, and the dishes were cultured at 37°C in a 10% CO2 incubator for 10 days without moving. The DP cells were then subsequently processed as described in Example 3.

[0080] In an alternative embodiment, cells are extracted from the follicular unit by mechanical and / or enzymatic treatment. Fibroblasts, keratinocytes and adipocytes are easily isolated by dissociating the FUT-obtained sample. Dissociation can be easily accomplished using several techniques known to those skilled in the art. Examples of such techniques include, but are not limited to, mechanical dissociation and / or treatments that can disperse the tissue into a suspension of individual cells without significant cell disruption by using digestive enzymes and / or chelating agents that weaken the connections between adjacent cells. Mechanical disruption can be accomplished by a number of methods, including, but not limited to, the use of shakers or spinners. In one embodiment, enzymatic dissociation is accomplished by splitting the tissue and treating the split tissue with trypsin. Any of a number of digestive enzymes can be used, either alone or in combination. For example, trypsin, chymotrypsin, collagenase, elastase, hyaluronidase, deoxyribonuclease, pronase and / or dispase can be used. Once the source tissue has been reduced to a suspension of individual cells, the cells are cultured in selective media to grow keratinocytes, fibroblasts, adipose-derived cells, mesenchymal cell types, or any other cell type.

[0081] Example 2 Obtaining hair follicle samples by follicular unit extraction (FUE) In FUE, each follicular unit has been individually harvested directly from an androgen-unsuppressed area of ​​the body (e.g., the back of the scalp, beard area, or other area where androgen-unsuppressed hair follicles are expected) without removing a single piece of tissue. The same procedure described in Example 1 has been used to extract DP for analysis or culture (see Example 3).

[0082] Example 3 Androgen-unsuppressed cell culture Androgen-inhibited cells have been extracted from hair follicle biopsies from non-bald (androgen-insensitive) areas obtained from FUT (as described in Example 1). Androgen-inhibited cells have been serially expanded 2-1000-fold in culture in DMEM supplemented with 10% FBS, 2 mM L-glutamine (Invitrogen, cat. no. 25030), 1% ABAM. The medium was changed every 2-3 days. Once the cultures reached a certain confluency, the cells were frozen, expanded, or used for formulation to be applied to patients. Once the cells were fully grown, they were passaged using standard cell culture techniques subcultured at a ratio of 1:2 to 1:5.

[0083] To culture the androgen-suppressed cells obtained in Example 2, the same method as described above has been used.

[0084] In another embodiment, the androgen-unsuppressed cells are expanded (at least) 2-100 fold in culture. As described by Limat et al. (1989, J. Invest. Dermatol. 92(5):758-62), keratinocytes can be expanded by 10 5 Hair follicle explants are placed on the microporous membrane of a cell culture insert carrying at its bottom a preformed feeder layer made from 100 μg / ml postmitotic human skin fibroblasts. The culture medium is 10% fetal bovine serum (FBS, Invitrogen), 10 ng / ml epidermal growth factor, 0.4 pg / ml hydrocortisone, 0.1 nM cholera toxin, 0.135 mM adenine, and final Ca 2+ The medium consists of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, cat. no. 11960) / F12 (3:1) supplemented with 2 nM triiodothyronine at a concentration of 1.5 nM. Within about 2 weeks, the keratinocytes expand and congregate. They are dissociated with trypsin / EDTA 0.1% / 0.02% and then checked for viability. They can be frozen and stored or used in formulations to treat patients. A single extended hair follicle can grow to 1-2 cm in about 2 weeks. 2The cells cover the culture surface area of ​​​​the hair follicles. These cells can be passaged in the same medium for further expansion if necessary. Fibroblasts are extracted from the explants of the hair follicles or from the suspension alone after enzymatic treatment of the hair follicles. In both cases, DMEM supplemented with 10% FBS, 2 mM L-glutamine (Invitrogen, cat.no. 25030) and 0.1 mM (final medium volume 0.7 μl / 100 ml) 2-mercaptoethanol (Sigma, cat.no. M7522) is used as the growth medium for expanding the cells. The medium is changed every 2-3 days. When the culture reaches a certain confluency, the cells are frozen, expanded or used in compositions applied to patients. The same method can be used to culture androgen-suppressed cells.

[0085] Example 4 Characterization of non-androgen-suppressed DP Samples of androgen-unsuppressed DP cells extracted as described in Example 1 and cultured as described in Example 3 have been tested for gene expression profile of androgen-unsuppressed cells, which have been compared to the gene expression profile of cultured androgen-suppressed DP cells obtained from hair follicles growing at the margins of bald areas as described in Example 2 and cultured under the same conditions as described in Example 3.

[0086] For this transcriptome analysis, RNA has been collected using RNeasy Plus Micro Kit (Qiagen). RNA has been used to synthesize first strand complementary DNA (cDNA) using Nugen Ovation V2. This has been converted to double stranded cDNA and used as a template for in vitro transcription to generate cRNA using Nugen Encore Biotin Module. cRNA has then been transcribed for hybridization and scanning using Affymetrix U133 Plus 2.0 Array (ThermoFisher Scientific Catalog no. 900466). Piecewise gene expression analysis has been performed using Transcriptome Analysis Console (TAC) software (ThermoFisher Scientific). Expression or non-expression of suitable control genes known in the art has been tested for comparison purposes.

[0087] Biomarkers (gene transcripts, including mRNA transcripts encoding non-protein components, collectively referred to herein as "genes") that exhibit the highest levels of differential expression between androgen-suppressed and androgen-unsuppressed cells are shown in Table 1 below.

[0088] [Table 1] TIFF2025000865000002.tif56124Table 1 shows the genes that were most highly expressed in one cell type ("+") compared to the other cell type ("-").

[0089] Genes that were more than 10-fold more highly expressed in androgen-unsuppressed cells compared to androgen-suppressed cells are listed below (in addition to those shown in Table 1 above). (List A)

[0090] Nuclear factor l / B, Rap guanine nucleotide exchange factor 6, CUGBP·Elav-like family member 2, receptor tyrosine kinase-like orphan receptor 1, RAB27B·member of the RAS oncogene family, metastasis suppressor 1, myelin basic protein, caspase recruitment domain family member 10, dedicator of cytokinesis 4, transcription factor 7-like 2 (T cell specific, HMG-box), DEAD (Asp-Glu-Ala-Asp) box polypeptide 52, GTPase, IMAP family member 2, spalt-like transcription factor 2, inositol 1,4,5-trisphosphate receptor type 3, family member B high mobility group AT-hook 1 with sequence homology 132, kazrin periplakin binding protein, GDNF-induced zinc finger protein 1, glutathione peroxidase 3, secretogranin II, chromosome X open reading frame 23, formin homology 2 domain-containing 3, tetraspanin 12, protein tyrosine phosphatase receptor type E, coagulation factor II (thrombin) receptor-like 2, ribosomal protein 1 (RT-PCR) Nuclease P / MRP 40 kDa subunit, leucine-rich repeat-containing 32, latexin, zinc finger protein 184, brain and acute leukemia cytoplasmic, neuronal junction molecule 1, branched-chain amino acid aminotransferase 1 cytoplasmic, calcium / calmodulin-dependent protein kinase kinase 2 beta, mitochondrial calcium uptake family member 3, homer scaffold protein 2, NADH dehydrogenase (ubiquinone) Fe-S protein 1 75 kDa (NADH-coenzyme Q reda ctase, follistatin-like 1, vitrin, lysine (K)-specific demethylase 4B, long intergenic nonprotein component coding RNA 520, nuclear factor l / B, kelch-like family member 20, claudin-1, Ras-related GTP-binding D, basonuclin 1, plexin domain-containing 1, transforming growth factor beta receptor III, adriamycin resistance-associated, DnaJ (Hsp40) homolog subfamily C member 12, claudin-like 1, and sodium channel voltage-dependent type II alpha subunit.

[0091] Genes that were more than 10-fold more highly expressed in androgen-suppressed cells compared to androgen-unsuppressed cells are shown below (in addition to those shown in Table 1 above). (List B)

[0092] AHNAK nuclear protein, serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1) member 2, lysyl oxidase, extended synaptotagmin-like protein 2, EGF-containing fibrin-like extracellular matrix protein 1, collagen type VI alpha 1, protein tyrosine phosphatase, non-receptor type 11, collagen type XII alpha 1, eukaryotic transcription elongation factor 1 delta (guanine nucleotide exchange protein), metastasis suppressor 1-like, talin 1, TSC22 domain family member β-lactamase 2, insulin-like growth factor binding protein 5, discoidin, CUB and LCCL domain-containing 2, transforming growth factor beta 2, synaptopodin 2, dehydrogenase / reductase (SDR family) member 7, consortin, connexin sorting protein, microtubule-associated monooxygenase, calponin and LIM domain-containing 2, mitochondrial ribosomal protein L41, ribosomal protein S19, nuclear casein kinase and cyclin-dependent kinase substrate 1, collagen type VI, alpha α1, FK506-binding protein 1A, cyclin K, WAS protein family member 2, tousled-like kinase 1, MDM2 proto-oncogene E3 ubiquitin protein ligase, spectrin beta non-erythroid 1, heat shock protein alpha-crystallin-related B6, RAB5A member of the RAS oncogene family, caldesmon 1, glycogen synthase kinase 3 beta, homeodomain-interacting protein kinase 3, nuclear factor l / X (CCAAT-binding transcription factor), polypyrimidine dehydrogenase lacto-binding protein 3, charged multivesicular body protein 4B, PTPRF-interacting protein-binding protein 1 (liprin beta 1), WD repeat and SOCS box-containing 1, DnaJ (Hsp40) homolog subfamily B member 4, alkB homolog 7, GTPase-activating protein (SH3 domain)-binding protein 1, microtubule-associated protein 1A, CKLF-like MARVEL transmembrane domain-containing 6, MDM2 proto-oncogene E3 ubiquitin protein ligase, t-complex 11 testis-specific-like 2, RhoGDP-dissociation inhibitor (GDI) alpha, coiled-coil containing WW domain-containing adaptor, F-box protein 32, AT-rich interacting domain 5B (MRF1-like), GrpE-like 2·mitochondrial (Escherichia coli), metastasis-associated lung adenocarcinoma transcript 1 (non-protein component coding), Aurora kinase A-interacting protein 1, ubiquitin complex enzyme E2H, AF4 / FMR2 family·member 4·QKI·KH domain-containing·RNA binding, regulator of calcineurin 1, abl-interactor 2, p21 (Cdc42 / Rac)-activating kinase 2, RAB11B·member of the RAS oncogene family, MYC-related factor X, sprouty-related EVH1 domain-containing 2, eukaryotic transcription initiation factor 4 gamma 3, ring finger protein (C3H2C3 type) 6, ribosomal protein giant P2, v-akt mouse thymoma viral oncogene homolog 2, zinc finger, DHHC type-containing 20, histone cluster 1 H1e, hyaluronan synthase 3, amyloid beta (A4) precursor-like protein 2, oxysterol-binding protein 8, heterogeneous nuclear ribonucleoprotein H1 (H), OTU deubiquitinase 4, septin 11, CD44 molecule (Indian blood group), cytoskeleton-associated protein 4, protein phosphatase Mg2 + / Mn2 + Dependence-1K, v-akt mouse thymoma viral oncogene homolog 3, neurofibromin 2 (Kokkein), ATPase-H +transport and lysosomal accessory protein 2, lysine (K)-specific demethylase 4B, SLAIN motif family member 2, transforming growth factor beta 2, zinc finger protein 460, twinfilin actin-binding protein 1, synaptosome-associated protein 23 kDa, phosphoprotein abundant in astrocytes 15, glucosaminyl (N-acetyl)transferase 4 core 2, AF4 / FMR2 family member 1, RNA polymerase II-associated protein 3, proline-rich 14, collagen type VI alpha 1, protein tyrosine phosphoprotein Phosphatase non-receptor type 11, phosphodiesterase 1C calmodulin-dependent 70 kDa, zinc finger CCHC domain-containing 7, PDZ and LIM domains 4, 5-azacytidine-inducible 2, heat shock protein alpha-crystallin-related B6, Ras homolog enriched in brain pseudogenes, ribosomal protein L27, dystroglycan 1 (dystrophin-associated glycoprotein 1), protein kinase N2, caveolin 2, DEAFI (Asp-Glu-Ala-Flis) box helicase 9, unidentified LOC105374954, tyrosine kinase tryptophan 3-monooxygenase / tryptophan 5-monooxygenase-activating protein epsilon, NCK-related protein 1, transmembrane protein 259, solute carrier family 7 (cationic amino acid transporter, y+ system) member 1, sarcoglycan beta, B double prime 1, subunit of RNA polymerase III transcription initiation factor NIB, lysine (K)-specific methyltransferase 2C, aspirin, solute carrier family 6 (neurotransmitter transporter) member 6, down-regulator of IK cytokine FILAII, transmembrane and coiled coil domain 6, TROVE domain family member 2, tumor necrosis factor receptor superfamily member 11b, centrosomal protein 89 kDa, protein tyrosine phosphatase non-receptor type 12, dynein cytoplasmic 1 heavy chain 1, annexin A2, chromobox homolog 5, CDV3 homolog (mouse), mitochondrial ribosomal protein L52, GATA zinc finger domain containing 2A, RAB7A member of the RAS oncogene family, TRAF-type zinc finger domain containing 1, transforming growth factor beta 2, H3 histone family 3A,Protein phosphatase 4, regulatory subunit 3A, protein tyrosine phosphatase non-receptor type 12, ribosomal protein L5, small nucleolar RNA FI / ACA box 66, transmembrane protein 259, iduronate 2-sulfatase, ATPase class VI type 11B, fibroblast growth factor receptor substrate 2, transcription factor AP-2 alpha (activated enhancer-binding protein 2 alpha), RNA-binding protein fox-1 homolog (C. elegans) 2, acidic nuclear phosphoprotein 32 family member A, mitochondrial ...iduronate 2-sulfatase, iduronate 2-sulfatase, iduronate 2-sulfatase, somal protein L41, interleukin 6 signal transducer, elaC ribonuclease Z2, unidentified LOC101926943, transmembrane protein 33, signal sequence receptor gamma (translocon-associated protein gamma), chloride intracellular channel 4, radixin, calreticulin, HECT domain-containing E3 ubiquitin protein ligase 1, transmembrane protein 223, large tumor suppressor kinase 1, endosulfan alpha, homeodomain-interacting protein kinase 2, protein kinase C iota, split transducin enhancer of 4, zinc finger CCCH-type antiviral 1-like, schlafen family member 5, zinc finger AN1-type domain 5, sperm specific antigen 2, DEAD (Asp-Glu-Ala-Asp) box helicase 17, dynein cytoplasmic 1 light intermediate chain 2, neuronal navigator 3, dephosphorylation-CoA kinase domain-containing, protein tyrosine phosphatase type IVA member 1, methionine adenosyltransferase II beta, protein kinase cAMP-dependent catalytic alpha, heterogeneous nuclear Ribonucleoprotein U-like 1, ras homolog family member A, TBC1 domain family member 2B, pleckstrin-phase-like domain family A member 2, vacuolar protein sorting 53 homolog (S. cerevisiae), Cbp / p300-interacting transcription activator containing Glu / Asp-rich carboxy-terminal domain 2, protein phosphatase 1 regulatory (inhibitor) subunit 14A, gamma-glutamine carboxylase, profilin 1, minichromosome maintenance 9 homologous recombination repair factor, solute carrier family 39 member 9,Topoisomerase II homolog 1 (yeast)-related protein, protein phosphatase 1 regulatory (inhibitor) subunit 2, catenin (cadherin-associated protein) beta 1, Nipped-B homolog (Drosophila), LSM12 homolog, protein phosphatase Mg2, + / Mn2 +dependent·1K, microtubule-associated protein 4, TAF9B RNA polymerase II·TATA box-binding protein (TBP)-associated factor·31 kDa, teneurin transmembrane protein 3, coiled-coil containing Myb / SANT-like DNA-binding domain-containing 4, biliverdin reductase A, WWTR1 antisense RNA 1, Yip1 domain family member 5, phosphoglycerate kinase 1, serine / threonine / tyrosine interacting protein, transmembrane protein 165, chromosome 5 open reading frame 15, ST3 beta-galactoside alpha-2,3-sialyltransferase 6, glucosamine (N-acetyl)-6-sulfatase, disc·large homolog 1 (Drosophila), TPT1 antisense RNA 1, hematological and neurological expression 1, phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2 alpha, exocyst complex component 5, nuclear paraspeckle polymerization transcript 1 (non-protein protein component coding), histone cluster 1·H2ae, GTPase-activating protein (SH3 domain)-binding protein 2, vesicle-associated membrane protein 5, EH domain-containing 2, neuron navigator 3, undefined LOC100506403, small animal-associated transcription factor 1, SPT6 homolog·histone chaperone, desumoylating isopeptidase 2, histone cluster 2·H2aa3, histone cluster 2·H2aa4, AT-rich interacting domain 5B (MRF1-like), TRAF family member-associated NFKB activator, DEAD (Asp-Glu-Ala-Asp) box helicase 3·X-linked, ecotropic virus integration site 5, F-box protein 32, exosome component 3, neudecin neurotrophic factor, mitochondrial ribosomal protein S12, translocation promoter region·nuclear basket protein, undefined LOC100506403, small animal-associated transcription factor 1, RhoGTPase-activating protein 1, pancreatic progenitor cell differentiation and growth factor, SKI-like proto-oncogene, ARP2 actin-related protein 2 homolog (yeast), CD46 molecule-complement regulatory protein, ATPase type 13A3, voltage-dependent anion channel 3, activating transcription factor 6 beta, tenascin-XB, tripeptide lipeptidyl peptidase II, limb and CNS expression 1-like, calnexin, MYC-related zinc finger protein (purine-binding transcription factor), zinc finger-MYM-type 5, endothelial PAS domain protein 1, lysyl oxidase-like 2, fumarate hydratase, 6-phosphogluconolactonase, trimethylguanosine synthase 1, glutamine-prolyl-tRNA synthase, VMA21 vacuolar H +- ATPase homolog (S. cerevisiae), ADAMTS-like 4, A-kinase (PRKA) anchor protein 13, myeloid cell leukemia 1, RAB35 member of the RAS oncogene family, CTD small phosphatase-like 2, guanylate kinase 1, nuclear receptor subfamily 2 group C member 2, protein phosphatase 1 regulatory (inhibitor) subunit 11, prostaglandin I2 (prostacyclin) receptor (IP), DEAD (Asp-Glu-Ala-Asp) box helicase 42, secretory carrier membrane protein 1, prostaglandin E receptor 3 (subtype EP3), 3-phosphoinositide-dependent critical protein kinase 1, AF4 / FMR2 family, member 4, PRKC·apoptosis·WT1, regulator, plexin A3, neudecin neurotrophic factor, discoidin domain receptor tyrosine kinase 2, KIAA0430, chromosome 16 open reading frame 72, DEAD (Asp-Glu-Ala-Asp) box helicase 3·Y-linked, basic transcription factor IIA1, transmembrane 9 superfamily member 3, sorting nexin 19, FCH domain only 2, TOX high mobility group box family member 4, NOP16 nucleolar protein, hook microtubule-tethering protein 3, zinc finger protein 460, AP2-related kinase 1, inhibitor of IKBKE1, RPA-interacting protein, zinc finger protein·X-linked·zinc finger protein·Y-linked, guanine nucleotide-binding protein (G protein), q polypeptide, Rho GTPase-activating protein 35, spectrin·beta·non-erythroid 1, FCF1 rRNA-processing protein, matrix metallopeptidase 24 (membrane inserted), tyrosine 3-monooxygenase / tryptophan 5-monooxygenase-activating protein zeta, retinoic acid receptor beta, catenin (cadherin-associated protein) alpha 1, phosphatidylinositol-4-phosphate 5-kinase type I alpha, leucine-rich repeat-containing 28, zinc finger domain 1B proximal bromo-domain, coiled-coil domain-containing 186, phosphatidylinositol-4-phosphate 5-kinase type I alpha, density regulatory protein, BUB3 mitotic checkpoint inhibitor protein, inositol monophosphatase domain-containing 1, La ribonucleoprotein domain family member 4B, poly(A) polymerase alpha, Sp3 transcription factor, activating transcription factor 1, short coiled-coil protein, serine / arginine-rich splicing factor 10, clathrin light chain B, tensin 1, metastasis-associated lung adenocarcinoma transcript 1 (non-protein coding), neutral sphingomyelinase activation-associated factor, sarcoglycan delta, early endosomal antigen 1, RAN-binding protein 1, calmodulin 1 (phosphorylase kinase delta) and calmodulin 2 (phosphorylase kinase delta),programmed cell death 1 ligand 2, tousled-like kinase 1, structural fiber of chromosome 1A, transforming growth factor beta receptor II, histamine N-methyltransferase, LYR motif-containing 2, phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2 alpha, casein kinase 1 alpha 1, CUB domain-containing protein 1, stathmin 2, SP110 nucleolar protein, thioredoxin-related transmembrane protein 1, protease serine 23, PDZ and LIM domains 5, tetratricopeptide repeat domain 37 , homeodomain-interacting protein kinase 3, centrosomal protein 89 kDa, zinc finger-BED-type containing 6, inhibin beta A, mitogen-activated protein kinase kinase kinase 2, transcription factor binding to IGHM enhancer 3, dickkopf inhibitor of WNT signaling pathway 3, fibroblast growth factor 14, histone cluster 1-H2bf, heat shock protein 90 kDa alpha (cytoplasmic)-class B member 1, ras homolog family member B, U2AF homology motif (UHM) kinase 1, calumenin, akirin 2 , transcription factor that binds IGHM enhancer 3, transmembrane and tetratricopeptide repeat-containing 3, related RAS viral (r-ras) oncogene homolog 2, ubiquitin-specific peptidase 34, growth arrest and DNA damage inducible beta, vesicle-associated membrane protein 2, solute carrier family 12 (potassium / chloride transporter) member 6, retinol dehydrogenase 10 (all transcribed), SLAIN motif family member 2, phosphatidylinositol-binding clathrin polymerization protein, solute carrier family 8 (sodium / calcium exchanger) member 1, carp coiled-coil domain-containing 88A, VPS35 retromer complex component, arginyl-tRNA synthase 2, mitochondrial, SET domain-containing 2, protein tyrosine phosphatase·receptor type·A, polymerase (RNA) II (DNA-dependent) polypeptide A·220 kDa, CD46 molecule·complement regulatory protein, zinc finger protein 460, lysine (K)-specific methyltransferase 2D, casein kinase 1·, delta, AT-rich interacting domain 1A (SWI-like), TIMP inhibitor of metallopeptidase 3, ceramide synthase 6,maternally expressed 3 (non-protein component coding), AHNAK nuclear protein 2, succinate dehydrogenase complex·subunit C, integral membrane protein·15 kDa, transmembrane and coiled-coil domains 3, RAB6A·member of RAS oncogene family, DEAD (Asp-Glu-Ala-Asp) box helicase 3·X-linked, YTH domain containing 1, S-phase kinase-associated protein 2·E3 ubiquitin protein ligase, zinc finger RNA-binding protein, wingless type MMTV integration site family · member 5B, selenoprotein T, CDK2-related · cullin domain 1, heterogeneous nuclear ribonucleoprotein D-like, TROVE domain family, member 2, Y-box binding protein 3, RING finger protein 141, uncharacterized LOC100506403 · small animal-related transcription factor 1, jumonji domain-containing 1C, nuclear factor l / X (CCAAT-binding transcription factor), ACTA2 antisense RNA1, Ras-related (RalGDS / AF-6) domain family member 3, TSC22D1 antisense RNA1, microtubule-associated protein 1A, spastic paraplegia 20 (Troyer's syndrome), serine / threonine kinase 3, SLC2A4 regulator, family containing sequence homology 171·member B, fibroblast growth factor receptor 2, histone cluster 1·H2bh, centrosomal protein 89 kDa, AF4 / FMR2 family·member 4, cortactin, transducin enhancer of split 4, family containing sequence homology 171·member B, RAB5C·member RAS oncogene family, NADFI dehydrogenase·subunit 6 (complex I), tyrosyl protein Protein sulfotransferase 2, G1-S phase transcription 1, UDP-glucose glycoprotein glucosyltransferase 2, collagen type XII alpha 1, sister chromatid attachment N-acetyltransferase 1 organization, division and polyadenylation specific factor 2, septin 9, DnaJ (Flsp40) homolog subfamily B member 5, ubiquitin-like modified activating enzyme 6, DnaJ (Flsp40) homolog subfamily C member 13, protein tyrosine phosphatase receptor type G, caldesmon 1, myosin VA,NADFI dehydrogenase (ubiquinone) Fe-S protein 1 75 kDa (NADFI-coenzyme Q reductase), zinc finger protein 146, ataxin7-like 3B, mannosidase alpha class 1A member 2, U2AF homology motif (UFIM) kinase 1, SOS Ras / Rho guanine nucleotide exchange factor 2, mitogen-activated protein kinase kinase kinase kinase 5, desumoylated isopeptidase 2, calumenin, proline-rich coiled-coil 2C, sidekick cell junction molecule 2, stress-induced phosphoprotein 1, adenomatous polyposis coli, calcium / calmodulin-dependent protein kinase kinase 2·beta, brain endothelial cell junction molecule, nuclear factor of activated T cells 5·tonicity responsiveness, tetratricopeptide repeat domain 3, RNA-binding motif protein 8A, BCL2-like 1, pre-B-cell leukemia homeobox 2, hyaluronate synthase 3, small subunit of UTP4 (SSU) processome component, heat shock protein 90 kDa beta (Grp94)·member 1, RAN-binding protein 9, neuregulin 1, dual specificity phosphatase 1, TSR3·20S rRNA accumulation homolog (S. cerevisiae), NSL1 MIS12 kinetochore complex component, histone cluster 1 H2be, homeodomain-interacting protein kinase 1, DET1 and DDB1-related 1, protein kinase DNA activation catalytic polypeptide, tripartite motif-containing 8, N(alpha)-acetyltransferase 30 NatC catalytic subunit, AT-rich interacting domain 4B (RBP1-like) RNA-binding motif protein 34, synaptopodin 2, platelet-activating factor acetylhydrolase 1b catalytic subunit 2 (30 kDa), uncharacterized LOC101243545, ELK4 ETS-domain protein (SRF accessory protein 1), chromosome 5 open reading frame 22, SAM and FID domains 1, crystallin zeta-like 1, PFID finger protein 20, ubiquitin-specific peptidase 12, PDZ and LIM domains 7 (enigma), TAF9B RNA polymerase II TATA box-binding protein (TBP)-associated factor 31 kDaPleckstrin phase-like domain family B member 2, ADAM metallopeptidase domain 9, inhibitor of T cell activation mitochondrial, MIS18 binding protein 1, YKT6 v-SNARE homolog (S. cerevisiae), RING finger protein 207, Schlafen family member 5, paired-associated homeobox 1, syntrophin beta 2 (dystrophin-associated protein A1 59 kDa basic component 2), torsinA interacting protein 2, phosphoinositide-3-kinase regulatory subunit 2 (beta), defective domain-containing 1 in DCN1 cullin neddylation 1, LRRC75A antisense RNA 1, cytoplasmic polyadenylation element binding protein 2, nuclear receptor subfamily 2 group F member 2, ribosomal protein L38, HIV-1 Tat specificity factor 1, RAB12 member of RAS oncogene family, ezrin, activator of GM2 ganglioside, pericentriolar material 1, inhibitor of cytokine signaling pathway 4, calcium / calmodulin-dependent serine protein kinase (MAGUK family), heat shock transcription factor 2, nuclear casein kinase and cyclin-dependent kinase substrate 1, transforming transcription 2 beta homolog (Drosophila), H2B histone family member S (pseudogene) histone cluster 1 H2bk histone H2B type FS-like, lysine (K)-specific demethylase 1B, Ras-related (RalGDS / AF-6) domain family (N-terminal) member 8, R-spondin 4, programmed cell death 5, itchyE3 ubiquitin protein ligase, regulation of nuclear pre-mRNA domain-containing 1A, zinc finger protein 770, galactokinase 1, protein phosphatase 3 catalytic subunit gamma isoenzyme, solute carrier family 30 (zinc transporter) member 4, MRS2 magnesium transporter, KxDL motif-containing 1, secretion-associated Ras-related GTPase 1B, zinc finger domain 1B proximal bromodomain, nestin, FK506-binding protein 1A, lectin mannose-binding 1, synaptopodin 2, laminin alpha 4, twinfilin actin-binding protein 1, twinfilin 1 pseudogene 1, ankyrin 2 neuron, cyclin-dependent kinase 6,Human immunodeficiency virus type I enhancer-binding protein 2, zinc finger-MYND-type containing 11, metastasis suppressor 1-like, cullin 4B, polymerase (DNA-dependent) eta, signaling adapter molecule (SH3 domain and ITAM motif) 2, heterogeneous nuclear ribonucleoprotein U (scaffold-binding factor A), serine / threonine kinase 4, insulin-like growth factor-binding protein 2, NOP16 nucleolar protein, peptidylarginine deiminase type II, guanine nucleotide-binding protein, protein (G protein) gamma 12, small nucleolar RNA C / D box 3A, small nucleolar RNA C / D box 3B-1, small nucleolar RNA C / D box 3B-2, small nucleolar RNA C / D box 3C, Shwachman-Bodian-Diamond syndrome, Shwachman-Bodian-Diamond syndrome pseudogene 1, Josephin domain containing 2, WD repeat domain 1, ubiquitin protein ligase E3C, coiled-coil domain containing 174, maternally expressed 3 (non-protein component coding), chromatin domain helicase DNA binding protein 9, Abhydrolase domain containing 2, leucine-rich repeat (in FLII) interacting protein 1, MET proto-oncogene receptor tyrosine kinase, neurofibromin 1, v-akt mouse thymoma virus oncogene homolog 3, lethal giant larvae homolog 1 (Drosophila), Gamma p300-binding protein gamma 1, Cbp / p300-interacting transcription activator with Glu / Asp-rich carboxy-terminal domain 2, SH3 and PX domains 2A, tropomyosin 4, homeodomain-interacting protein kinase 3, ankyrin repeat domain 19 pseudogene, aldehyde dehydrogenase 1 family member L2, binding junction molecule 3, interleukin 1 receptor type I, family containing cysteine-rich hydrophobic domain 2, sequence homology 168 member B, angiomotin-like 1, EH domain-binding protein 1, GM2 ganglioside activator, tyrosine 3-monooxygenase / tryptophan 5-monooxygenase-activating protein eta, erythrocyte membrane protein band 4.1-like 1, heterogeneous nuclear ribonucleoprotein U (scaffold-binding factor A), peroxisome biogenesis factor 13, neurofibromin 1, SH3 domain-binding glutamate-rich protein-like 3, FGFR10P N-terminal-like, G-rich RNA sequence-binding factor 1, guanine nucleotide-binding protein (G protein) alpha 13, ATPase Ca ++transport · cardiac · slow muscle 2, collagen · type I · alpha 1, alpha thalassemia / mental retardation syndrome X-linked, nuclear factor l / X (CCAAT-binding transcription factor), nuclear factor l / C (CCAAT-binding transcription factor), stromal antigen 3-like 1 (pseudogene), stromal antigen 3-like 2 (pseudogene), stromal antigen 3-like 3 (pseudogene), mannosidase · endo alpha, keratin 17 · type I, atrophin 1, RAB30 · member of the RAS oncogene family, crystallin beta B2 · crystallin beta B2 pseudogene 1, PHD finger protein 12, uncharacterized LOC100190986, KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor 1, Ral containing PH domain and SH3-binding motif 2 GEF, HECT·UBA and WWE domain-containing 1·E3 ubiquitin protein ligase, spiny homolog 3, LIM domain-containing preferred translocation partner in lipomas, TMF1-regulatory nuclear protein 1, purine-rich element-binding protein B, NADH dehydrogenase (ubiquinone) Fe-S protein 8, 23 kDa (NADH-coenzyme Q reductase), laccase (multicopy oxidoreductase) domain-containing 1, syntaxin 6, zinc finger·MYND-type containing 11, spectrin·beta·non-erythroid 1, BRICK1·SCAR / WAVE actin-nucleation complex subunit, topoisomerase II homolog 1 (yeast)-related protein, RAR-related orphan receptor A, disc·giant (Drosophila) homolog-related protein 4, F-bock and leucine-rich repeat protein 20, solute carrier family 25 (aspartate / glutamate carrier) member 13, carboxypeptidase D, AF4 / FMR2 family member 4, neuroblastoma proliferation sequence, cytochrome c oxidase oxidase subunit Vile, G elongation factor mitochondrial 2, ribulose-5-phosphate-3-epimerase-like 1, ArfGAP containing GTPase domain, ankyrin repeats and PH domain 2, ubiquitin-specific peptidase 10, dedicator of cytokinesis 9, kinesin family member 1B, lysine (K)-specific methyltransferase 2D, ATP-binding cassette subfamily D member 3, ADP-ribosyltransferase interacting protein 1, Sp1 transcription factor,Chromosome biorientation in cell division 1-like 1, ArfGAP containing FG repeats s1, general transcription factor lii, eukaryotic transcription initiation factor 4E family member 3, CASP8 and FADD-like regulator of apoptosis, leucyl-tRNA synthase, secretion-associated·Ras-related GTPase1B, cullin 5, polymerase (RNA) II (DNA-dependent) polypeptide J4·pseudogene, WNT1-induced signaling pathway protein 3, RAB3B·member of the RAS oncogene family, gamma-amyloides GABA B receptor, 2, IWS1 homolog (S. cerevisiae), interleukin-13 receptor alpha 1, PDZ and LIM domains 7 (enigma), vascular endothelial growth factor A, primase DNA polypeptide 2 (58 kDa), cardiolipin synthase 1, zinc finger protein 655, mex-3 RNA-binding family member C, gap junction protein alpha 5, casein kinase 1 alpha 1, small elongation complex ELL subunit 2 interactor, zinc finger protein 326, Ellis-van Creveld protein, integrin beta 1, mohawk homeobox essential for meiotic fission 5 homolog B, DEAD (Asp-Glu-Ala-Asp) box helicase 17, v-akt mouse thymoma viral oncogene homolog 2, sperm-associated antigen 9, PAPOLA and CPSF1 interacting factor, histone cluster 1·H2aj, heat shock transcription factor 1, leukocyte receptor cluster (LRC) member 8, MDM2 proto-oncogene·E3 ubiquitin protein ligase, mitochondrial ribosomal protein L30, tubulin·beta 2A class lla, tubulin·beta 2B class llb, Golgi-associated PDZ and coiled-coil motif-containing, DNA replication and sister chromatid cohesion 1, forkhead box O3·forkhead box O3B pseudogene, sprouty-associated·EVH1 domain-containing 1, Ral containing PH domain and SH3-binding motif 2 GEF, LSM family member 14B, dermatan sulfate epimerase-like, retinoblastoma binding protein 9, mitogen-activated protein kinase kinase kinase 7, methylenetetrahydrofolate dehydrogenase (NADP, +dependent)2·methyltetrahydrofolate cyclohydrolase, SMG7 nonsense mutant mRNA decay factor, FK506 binding protein 15, Mytilus RISC catalytic component 2, heterogeneous nuclear ribonucleoprotein U-like 2, sarcolemma-associated protein, DEAD (Asp-Glu-Ala-Asp) box helicase 17, ribosomal protein S6 kinase·70 kDa·polypeptide 1, NADH dehydrogenase (ubiquinone) 1 beta subcomplex·7·18 kDa, MLX-interacting protein, GABPB1 antisense RNA 1, SWI / SNF-related·substrate-associated ·Actin-dependent regulator of chromatin ·Subfamily a ·Member 1, zinc finger ·MYM-type 6, zinc finger and BTB domain-containing 20, capping protein (actin filament) muscle Z-line ·beta, Kruppel-like factor 12, Sestrin 3, MOB kinase activating factor 1A, KRAB box domain-containing 4 ·Zinc finger protein 674, phosphatidylinositol-4,5-bisphosphate 3-kinase ·catalytic subunit beta, N-ethylmaleimide-sensitive factor attachment protein ·gamma, mitogen-activated protein Kinase kinase kinase 7, MORN repeat-containing 2, ras homolog family member J, TRPM8 channel-associated factor 1, cAMP-regulated phosphoprotein 19 kDa, 5'-nucleotidase·ecto(CD73), transducin(beta)-like 1X-linked receptor 1, spectrin·beta·non-erythroid 1, importin8, histone cluster 1·H2bi, cofilin 1 (non-muscle), phosphocytidylyltransferase 1·choline·alpha, lysine (K)-specific methyltransferase 2A, palmdelphin, zinc finger and BTB domain-containing 24, tropomodulin 3 (ubiquitous), sushi·von Willebrand factor type A·EGF and pentraxin domain-containing 1, ubiquitin complex enzyme E2G2, acid phosphatase 1·soluble, cell division cycle 27, xylosyltransferase I, gamma-aminobutyric acid (GABA) B receptor·2, GATA zinc finger domain-containing 2B, iduronate 2-sulfatase, integrator complex subunit 1, ubiquitination factor E4B, ankyrin repeat and FYVE domain-containing 1, high mobility group box 1,TAF9B RNA polymerase II·TATA box-binding protein (TBP)-associated factor·31kDa, collagen·type VI·alpha 1, ubiquitin-specific peptidase 25, sestrin 3, F-box and WD repeat domain containing 11, histone cluster 1·H2ai, synaptosome-associated protein 23kDa, pre-mRNA processing factor 18, spermatogenesis-associated 5, huntingtin-interacting protein K·small EDRK-rich factor 2, SERF2-C15orf63 readthrough, myeloid / lymphoid or mixed lineage leukemia·translocated·1, spectrin·beta·non-erythroid 1, solute carrier family 3 (amino acid transporter heavy chain)·member 1, signal recognition particle 72kDa, ubiquitin-specific peptidase 32, sorbin and SH3 domain containing 3, thioredoxin interacting protein, HOXD antisense growth-associated long noncoding RNA, uncharacterized LOC100506403·small animal-associated transcription factor 1, ring finger protein 168, E3 ubiquitin protein ligase, RAD21 cohesin complex component, family containing sequence homology 198 member B, ubiquitin-specific peptidase 33, PR domain containing ZNF domain 2, polycomb group ring finger 5, GLI family zinc finger 4, integrin alpha 6, cytoplasmic linker-associated protein 2, guanine nucleotide-binding protein (G protein) beta polypeptide 4, glutaminase, centrosomal protein 63 kDa, voltage-dependent anion channel 1, transcription factor Dp-1, chromosome 6 open reading frame 106, lactamase beta, HERPUD family member 2, large tumor suppressor kinase 1, ezrin, SUM01 / sentrin-specific peptidase 5, chaperonin-containing TCP1 subunit 2 (beta), zinc finger protein 780A zinc finger protein 780B, coatomer protein complex subunit gamma 1, RAB23-member of the RAS oncogene family, tripartite motif-containing 8, RB-associated KRAB zinc finger, sema domain-immunoglobulin domain (Ig)-short basic domain-secreted (semaphorin) 3D, polymerase I and transcription termination factor, retinoblastoma binding protein 6, heterogeneous nuclear ribonucleoprotein H3 (2H9),mitochondrial ribosomal protein L19, sorting 1, protocadherin gamma subfamily A·11, polypyrimidine tract-binding protein 1, exosome component 3, dual specificity phosphatase 3, cyclin-dependent kinase 13, paralemmin2·PALM2-AKAP2 read-through, PX domain-containing 1, Rho guanine nucleotide exchange factor (GEF) 12, mitochondrial ribosomal protein L57, histone cluster 1, H2bd, squalene epoxidase, ubiquitin-complexing enzyme E2H, ubiquitin-specific peptidase 22, growth differentiation factor 15, prostaglandin E receptor 3 (subtype EP, 3), glycosyltransferase-like domain-containing 1, GABA(A) receptor-associated protein-like 1, phosphatidylinositol glycan anchor biosynthesis class X, CDC42 small effector 1, basic helix-loop-helix family member e41, TOR signaling pathway regulator, CCR4-NOT transcription complex subunit 4, sorting nexin 25, RING finger and CCCH-type domain 1, tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase, coiled-coil domain-containing 58, plectin, golginA8 family member A, THUMP domain-containing 3, mannosidase endo alpha, cytohesin 3, chaperonin-containing TCP1 subunit 4 (delta), and RING finger and CCCH-type domain 2.

[0093] Genes that were more highly expressed in androgen-unsuppressed cells compared to androgen-suppressed cells and were detected in both intact DP cells (obtained from hair follicles obtained in Example 2 and excised as described in Example 1) and cultured DP cells (obtained as described in Example 1) are shown below (List C).

[0094] Spalt-like transcription factor 2, interferon regulatory factor 1, PSMD5 antisense RNA 1 (head-to-head), CTTNBP2 N-terminal-like, neutral sphingomyelinase activation-associated factor, guanylate cyclase 1 soluble beta 3, trichoplain keratin filament-associated, leucine-rich repeat-containing 17, nucleolar protein 11, glutathione S-transferase mu 5, protein arginine methyltransferase 3, DEAD (Asp-Glu-Ala-Asp) box polypeptide 23, methyltransferase-like 13, alcohol dehydrogenase 5 (class III) key polypeptide, erythrocyte membrane protein band 4.1-like 5, glutathione peroxidase 3, chromatin accessibility complex 1, pumilioRNA-binding family member 3, transcription adaptor 2B, ubiquitin fold change 1, SP140 nucleolar protein-like, ribosomal RNA processing 36, BEN domain containing 3, derlin2, ArfGAP containing coiled-coil and ankyrin repeats and PH domain 3, major histocompatibility complex, class I·E, growth arrest specificity 7, transporter 2·ATP-binding cassette , subfamily B (MDR / TAP), MHC class I polypeptide-related sequence B, ribosomal protein L31, SERTA domain-containing 4, XAGE-4 protein, phosphoseric cluster sorting protein 2, coiled-coil domain-containing 51, LUC7-like 3 pre-mRNA splicing factor, pericentriolar material 1, ubiquitin-complexing enzyme E2D4 (putative), forkhead box P2, formation binding protein 4, and ubiquinol-cytochrome c reductase-binding protein.

[0095] Genes that were more highly expressed in androgen-suppressed cells compared to non-androgen-suppressed cells and were also expressed in both intact and cultured DP cells are shown below (List D).

[0096] Tachykinin 4 (hemokinin), zinc finger protein 816, glial cell line-derived neurotrophic factor, solute carrier family 5 (sodium / glucose cotransporter) member 2, TP53 target 1 (nonprotein component coding), ARPC4-TTLL3 read-through tubulin tyrosine ligase-like family member 3, discoidin CUB and LCCL domains containing 2, diacylglycerol lipase beta, breast cancer estrogen-induced apoptosis 2, serum amyloid A1, serum amyloid A2, SAA2-SAA4 read-through, untreated identified LOC101929450, transcription factor 3, RNA-binding motif protein 33, PRKC·apoptosis·WT1·regulatory factor, v-maf macrophage activating factor avian musculoaponeurotic fibrosarcoma oncogene homolog, syndecan 1, neuron navigator 1, peroxisome biogenesis factor 14·Fanconi anemia core complex-associated protein 24, zinc finger protein 528, Sin3A-related protein 18 kDa, casein kinase 1·gamma 1, unidentified LOC648987, centrosomal protein 290 kDa, exocyst complex component 3-like 1, clinovirus 1, cytoplasmic protein 290 kDa ... staphylin alpha B, autophagy-related 4D-cysteine ​​peptidase, proteasome 26S subunit non-ATPase 8, protein tyrosine phosphatase non-receptor type 23, MKL / myocardin-like 2, spastic paraplegia 11 (autosomal recessive), DENN / MADD domain-containing 5B, zinc finger protein 205, SER-TAD4 antisense RNA 1, phospholipid phosphatase-related 3, WW domain-binding protein 11, iduronidase, alpha-L-, salvador family WW domain-containing protein 1, leukocytosis alin, sialophorin, C-type lectin domain family 7 member A, ras-related C3 botulinum toxin substrate 1 (rho family, small GTP-binding protein Rad), SLC2A4 regulator, THAP domain-containing, apoptosis-related protein 2, leukosialin-sialophorin, nuclear transcription factor Y subunit gamma, latent transforming growth factor beta-binding protein 4, ubiquitin protein ligase E3 component n-recognin 3 (putative), unidentified LOC105375666, centrosomal protein 104 kDa, zinc finger protein 626,zinc finger homeobox 3, ilvB (bacterial acetolactate synthase)-like, zinc finger and BTB domain-containing 7A, trimethylguanosine synthase 1, atlastin GTPase 3, twisted gastrulation BMP signaling modulator 1, activating transcription factor 5, nuclear factor erythroid 2-like 2, C2 calcium-dependent domain-containing 4B, transforming growth factor beta regulatory factor 1, elastin, dynactin 1, 1-acylglycerol-3-phosphate O-acyltransferase 3, chloride channel CLIC-like 1, coiled-coil and ankyrin repeats and and PH domain 2-containing ArfGAP, transmembrane protein 259, RNA-binding motif protein 14, fucosidase, alpha-L-1·tissue, chromatin target of PRMT1, ELF1 homolog, elongation factor 1, TRPM8 channel-associated factor 1, NHL repeat-containing 2, integrin beta 1·integrin beta 1 pseudogene 1, tetratricopeptide repeat domain 3, inverted formin·FH2 and WH2 domain-containing, trinucleotide repeat-containing 6A, methylthio-adenosine phosphorylase, myosin·heavy chain 11·smooth muscle, ataxin1, calcium / calmodulin-dependent protein kinase II inhibitor 1, GTP-binding protein 2, ubiquitin protein ligase E3B early growth response 1, actin-related protein 2 / 3 complex subunit 5, chaperonin-containing TCP1 subunit 4(delta), Rho guanine nucleotide exchange factor (GEF) 12, PX domain-containing 1, cytoplasmic linker-associated protein 2, pre-mRNA processing factor 18, GABPB1 antisense RNA 1, LSM family member 14B, KxDL motif-containing 1, R-spondin 4, H2B histone family member S (pseudogene) histone cluster 1 H2bk histone H2B-type FS-like, NSL1 MIS12 kinetochore complex component, heat shock protein 90 kDa beta (Grp94) member 1, TSC22D1 antisense RNA 1, transcription factor binding to IGHM enhancer 3, Sp3 transcription factor, myeloid cell leukemia 1, TBC1 domain family member 2B, zinc finger AN1-type domain 5, protein tyrosine phosphatase non-receptor type 12, H3 histone family 3A, GATA zinc finger domain containing 2A,IK cytokine, down-regulator of HLAII, transmembrane and coiled-coil domain 6, protein kinase N2, ets variant 5, and collagen type VI alpha 1.

[0097] One or more genes selected from lists C and D can be used for quality control (QC) testing in cultured cells to determine whether they are androgen unsuppressed. The genes in list C can be particularly useful, for example, as they can distinguish between androgen-suppressed and androgen-unsuppressed cells, either in the intact DP before expansion in culture or in the cells after they have been cultured. Differentiated genes that express cell surface proteins can also be used as antigens for various cell separation techniques.

[0098] For the genes listed above in Lists A-D, the Affymetrix U133 Plus 2.0 Array kit uses a unique Affymetrix ID number and gene symbol.

[0099] Example 5 Identification and selection of androgen-unsuppressed cells Once the cells have expanded, and prior to clinical use in patients, the cells are evaluated for expression of biomarkers as described in Example 4. In particular, the use of the genes provided in Table 1 and / or Lists A-D allows for the discrimination between androgen-unsuppressed, hair follicle-inducing cells and androgen-sensitive cells. Expression or non-expression of suitable regulatory genes known in the art can be tested for comparison purposes.

[0100] The desired androgen-inhibited cells are selected either positively, negatively, or both. The former approach aims at the extraction of androgen-inhibited cell types from the total population, whereas the latter strategy involves the depletion of androgen-inhibited cells from the population, leaving only androgen-inhibited cells. Cells whose differentially expressed genes (e.g., as disclosed in Example 4) encode cell surface proteins (e.g., integrins) can be selectively captured by specific binding of surface antigens with either antibodies or aptamers. The captured cells are then detected with the aid of a measurable probe, e.g., fluorescent dyes or magnetic particles labeled with antibodies or aptamers. Once labeled, they can be selected either positively or negatively using techniques such as fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS).

[0101] Example 6 Preparation of cells prior to injection Once culture is complete, the cells are prepared for injection: they are harvested from the dishes by trypsinization, collected by centrifugation, and resuspended in Hypothermosol-FRS (Biolife).

[0102] Example 7 Cell Injection The in vitro expanded androgen-unsuppressed cells are counted and injected near miniaturized hairs located in bald or pre-bald areas of the scalp using a microsyringe with a 27-G needle (cells can be injected through needles as small as 30-G, but 27-G is preferred).

Claims

1. 1. A composition for use in a method for regenerating hair follicles, comprising: the composition comprises an expanded population of androgen-unsuppressed cells produced in vitro by culturing androgen-unsuppressed cells obtained from hair follicle tissue under conditions suitable for their proliferation; A composition, wherein said use comprises the step of regenerating a miniaturized hair follicle by implanting said composition proximal to said miniaturized hair follicle.

2. The composition of claim 1 , wherein the composition comprises non-androgen-suppressed cells selected from a mixed population of cells in hair follicle tissue.

3. The composition according to claim 1 or 2, wherein the androgen-unsuppressed cells are androgen-insensitive cells, for example, the hair follicle tissue is scalp hair follicle tissue.

4. 3. The composition of claim 1 or 2, wherein the androgen-unsuppressed cells are androgen-stimulated cells, for example, the hair follicle tissue is beard hair follicle tissue, chest hair follicle tissue, armpit hair follicle tissue, and / or pubic hair follicle tissue.

5. The composition of any one of claims 1 to 4, wherein the androgen-unsuppressed cells comprise or consist of dermal papilla (DP) cells.

6. The composition according to any one of claims 1 to 5, wherein the non-androgen-suppressed cells contained in the composition transplanted in the use reactivate the non-androgen-suppressed cells of the miniaturizing hair follicle and / or replace the non-androgen-suppressed cells in the miniaturizing hair follicle, thereby regenerating the miniaturizing hair follicle.

7. The composition of any one of claims 1 to 6, wherein the hair follicle tissue is obtained from an area of ​​the body known or suspected to contain androgen-unsuppressed cells.

8. The composition of any one of claims 1 to 7, wherein the hair follicle tissue has been mechanically extracted, for example using follicular unit extraction (FUE).

9. The composition according to any one of claims 1 to 8, wherein the androgen-unsuppressed cells obtained from hair follicle tissue are obtained from a suspension of hair follicle tissue cells by antibody-assisted selection.

10. 10. The composition of any one of claims 1 to 9, wherein the expanded population of androgen-unsuppressed cells is expanded in number by at least about 50-100 fold, such as at least about 500-1000 fold.

11. The composition of any one of claims 1 to 10, wherein the expression levels of one or more genes are used as androgen-insensitive biomarkers to identify and / or select the androgen-unsuppressed cells, and the one or more genes are selected from the group consisting of STX17 antisense RNA1, prostaglandin I2 (prostacyclin) synthase, calmegin, glypican 6, integrin beta 8, collagen, type X, alpha 1, reelin, carbonic anhydrase XIII, DEP domain-containing MTOR interacting protein, and HAUS augmin-like complex subunit 6.

12. The composition of claim 11 , wherein the androgen-unsuppressed cells are selected or sorted using the biomarker.

13. The composition according to any one of claims 1 to 12, wherein the androgen is dihydrotestosterone (DHT).

14. The composition according to any one of claims 1 to 13, wherein the hair follicle tissue and / or non-androgen-suppressed cells are human.

15. The composition of any one of claims 1 to 14, wherein the androgen-unsuppressed cells are obtained from a subject, cultured, and then transplanted into the same subject.

16. The composition of any one of claims 1 to 15, wherein the cells are of autologous or allogeneic origin.

17. The composition according to any one of claims 1 to 16, formulated for injection.

18. A composition according to any one of claims 1 to 17 for use in a method of treatment.

19. A composition according to any one of claims 1 to 17 for use in a method for regenerating hair growth and / or slowing hair loss.

20. Use of a composition according to any one of claims 1 to 17 in the manufacture of a medicament for regenerating hair growth and / or slowing hair loss.

21. 21. A composition for use according to claim 18 or 19, or a use according to claim 20, for application to a subject having alopecia, such as hormonal alopecia.

22. 22. A composition for use according to claim 18, 19 or 21, or a use according to claim 20 or 21, wherein said composition or use is entirely cosmetic.

23. The composition for use according to claim 18, 19, 21 or 22 or the use according to any one of claims 20 to 22, wherein the composition is administered by injection, for example by a non-physician medical technician.

24. Use of a composition according to any one of claims 1 to 17 in a system for analysing hair follicle cells and / or for testing cosmetic drugs or medicinal products.

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