Regenerative medicine and hair loss: how hair follicle culture has advanced our understanding of treatment options for androgenetic alopecia

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1 For reprint orders, please contact: Regenerative medicine and hair loss: how hair follicle culture has advanced our understanding of treatment options for androgenetic alopecia Many of the current drug therapies for androgenetic alopecia were discovered serendipitously, with hair growth observed as an off-target effect when drugs were used to treat a different disorder. Subsequently, several studies using cultured cells have enabled identification of hair growth modulators with similar properties to the currently available drugs, which may also provide clinical benefit. In situations where the current therapeutics do not work, follicular unit transplantation is an alternative surgical option. More recently, the concept of follicular cell implantation, or hair follicle neogenesis, has been attempted, exploiting the inherent properties of cultured hair follicle cells to induce de novo hair growth in balding scalp. In this review, we discuss both the advances in cell culture techniques that have led to a wider range of potential therapeutics to promote hair growth, in addition to detailing current knowledge on follicular cell implantation, and the challenges in making this approach a reality. KEYWORDS: androgenetic alopecia cell culture dermal papilla finasteride follicular cell implantation hair follicle male pattern baldness minoxidil transplantation A human hair follicle on the scalp will spend the majority of its hair growth cycle in anagen, the hair follicle growth phase [1]. During anagen, mesenchymal cells at the base of the hair follicle, in the dermal papilla, stimulate over lying epithelial cells to differentiate and form the hair shaft. Hair follicles on the scalp can spend several years in anagen, enabling growth of long hair fibers on this site [2]. At the end of anagen, the follicle transitions to a regression phase, catagen, followed by a resting phase termed telogen [3]. Catagen lasts approximately 2 weeks, during which time the hair fiber ceases to grow, forming a club hair which will later be shed from the follicle. After telogen, which lasts approximately 3 months on the human scalp, the follicle reenters anagen, and this re-entry is accompanied by the initiation of new hair fiber growth; hence, the cycle starts again [4]. Throughout an individual s lifetime, the hair follicles on the scalp go through several cycles of growth, regression and rest. There are many situations where hair growth can go awry, ranging from rare Mendelian disorders such as atrichia with papular lesions [5], through to complex diseases such as alopecia areata [6] and androgenetic alopecia [7 9]. Androgenetic alopecia is the most common form of hair loss [10], and affects around 60% of males between the ages of 30 and 50 years, and up to 17% of women under 50 years of age, increasing dramatically in prevalence in both sexes with age [11,12]. Androgenetic alopecia is commonly known as male pattern baldness, and progression is observed in a distinct pattern on the hair line and scalp vertex (Norwood Hamilton classification) [13]. Androgenetic alopecia in women is termed female pattern hair loss, and is characterized by a diffuse thinning over the crown and a widening of the hair parting (Ludwig classification) [14]. In androgenetic alopecia, miniaturization of the hair follicle is accompanied by a reduction in the size of the dermal papilla, which translates to a small unpigmented vellus hair fiber growing from the follicle, instead of the usual pigmented terminal hair fiber [15]. There is also a lag in the transition from telogen to anagen in androgenetic alopecia, so many follicles remain in a telogen state on the scalp for extended periods of time. In males, balding usually starts in the frontal region, progressing backwards towards the occipital scalp. High levels of androgen receptor, as well as the enzyme 5 a reductase have been observed in the frontal balding scalp of men with androgenetic alopecia, compared with low levels in the occipital scalp [16]. More recently, elevated levels of prostaglandin D2 synthase, and prostaglandin D2 were detected in the frontal balding scalp, when compared with occipital scalp in males with androgenetic alopecia [17]. Current treatments for hair loss are relatively limited, although one of the best available treatments is surgical relocation of hairs from the Claire A Higgins 1 & Angela M Christiano* 1,2 1 Department of Dermatology, Columbia University, New York, NY, USA 2 Department of Genetics & Development, Columbia University, New York, NY, USA *Author for correspondence: amc65@columbia.edu /RME Future Medicine Ltd Regen. Med. (2014) 9(1), ISSN

2 Higgins & Christiano occipital to the frontal scalp, known as follicular unit transplantation [18]. There are many instances, however, in which a patient is not suitable for hair follicle transplantation. This is frequently the result of having too few donor hairs for transplantation, or hair loss is not yet stable and the extent of future loss is unknown. Transplanting hairs from the occipital to the frontal scalp, only for more hair loss to occur behind the transplanted region would not be aesthetically acceptable. Moreover, women with androgenetic alopecia are often not suitable candidates for hair transplantation due to insufficient donor hair. In other cases, patients are unsuitable for hair follicle transplantation since the underlying cause of their hair loss is unknown, but is not due to androgenetic alopecia. For example, patients with alopecia areata are not usually candidates for treatment by hair follicle transplantation as there are no stable areas from which to isolate donor follicles, and follicles would be attacked again after transplant [19]. For patients in whom surgical relocation of hair follicles is not an option, there are currently two US FDA-approved drugs available for the treatment of scalp hair loss, oral finasteride and topical minoxidil, which have been shown to be beneficial, particularly in the treatment of androgenetic alopecia [20,21]. Minoxidil has been shown to have a range of efficacy depending on factors such as dosage, and extent of original hair thinning, while finasteride is predominantly effective, but only FDA approved for male patients, and not females [22 24]. Finasteride and minoxidil were both discovered to have a hair growth-promoting effect serendipitously as an unwanted side effect when the drugs were being used to treat either benign prostatic hyperplasia (in the case of finasteride) [25], or hypertension (in the case of minoxidil) [26,27]. Moreover, since their hair growth-promoting abilities were discovered as side effects, subsequent research has sought to understand their mechanism of action on the hair follicle. Hair additionally plays a central role in maintaining psychosocial wellbeing, and the loss of hair can have profound psychological consequences, particularly in women where it is most often devastating [28]. For this reason, there is an increasing interest in promoting hair restoration, whether it be through surgical regeneration means, or identification of new therapeutics to treat hair loss. Intense investigation over the past decade, in which in vitro hair follicle cell cultures have been exploited, has enabled rapid development in this area. In this review, we discuss these recent developments in the field that have arisen through the development of culture methods of hair follicles and new therapeutic approaches. Hair follicle culture methods Hair follicles can be isolated from biopsied human scalp tissue (Figure 1). This can be from a strip or punch biopsy, as long as hair follicles at the site of interest are large enough to isolate by microdissection. Human hair follicles can be isolated from the surrounding dermis and dermal adipose tissue that surrounds the miniature organ using watchmaker s forceps and scissors. In the early 1990s, Philpott and colleagues established a methodology to culture human hair follicles when removed from scalp tissue [29]. This method has now become the gold standard within the dermatological field for culture of intact human hair follicles. Follicles can be grown in a serum-free medium supplemented with insulin and hydrocortisone for approximately 7 10 days. Under normal conditions, the follicles switch from the growth phase (anagen) into the regression phase (catagen) of the hair cycle after 6 7 days. This reliable switch in cycle phase can be utilized to see if drug biologics are capable of: increasing hair growth by delaying the anagen catagen switch; or decreasing hair growth by accelerating the anagen catagen switch. More recently, intermediate hair follicles have been used in this organ culture model, as they are representative of follicles undergoing miniaturization. Subsequently, they may be more pertinent to identify drug biologics that specifically target androgenetic alopecia follicles [30]. While the hair follicle organ culture method [29] utilizes whole hair follicles, there are several research groups that have developed assays based on individual cells isolated from the hair follicle, rather than the whole organ (Figure 2). Early in the 1980s, Jahoda and Oliver first demonstrated that rat whisker dermal papillae could be isolated from the hair follicle and the cells grown in culture [31]. Messenger later showed the same properties were true for human hair follicle dermal papillae [32]. Microdissection techniques are used to remove the human dermal papilla, an onion-shaped mesenchymal cell population and the dermal sheath, a cup-like structure that surrounds the dermal papilla at the base of the hair follicle [33]. The intact papilla or sheath are separated, and placed into a dish in culture, wherein they collapse to form a dermal explant. Cells are able to migrate out from the collapsed structure, and can then be passaged and grown in culture. 102 Regen. Med. 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3 How hair follicle culture has advanced our understanding of treatment of androgenetic alopecia Sebaceous glands Hair fiber Epidermis (contains keratinocytes and melanocytes) Dermis (contains dermal fibroblasts, blood vessels and nerves) Hair follicle Dermal adipose tissue (contains intradermal adipocytes and blood vessels) Hair follicle endbulb (contains dermal papilla) Figure 1. Macroscopic image of a strip of human scalp tissue isolated from the occipital scalp. The skin is broadly divided into an epidermal layer, a dermal layer and a dermal adipose layer. Within these distinct compartments, many cell types, including keratinocytes, fibroblasts, endothelial cells, nerves and adipocytes, are housed. Within the skin, hair follicles can be found. The outer root sheath of the hair follicle is continuous with the epidermis, although the follicle protrudes down through the dermal and adipose layers of the skin. The dermal papilla is located at the base of the follicle, within the endbulb region. Signals from the dermal papilla are believed to activate overlying keratinocytes to both initiate and drive the anagen phase of the hair cycle [34]. The dermal papilla arises from the dermal condensate that is observed during follicle development, and with parallels to hair cycling, signals from this condensate to the overlying epithelium are required to initiate hair follicle development [35]. Hence, the dermal papilla is a key cellular compartment of the follicle, whose destruction results in ablation of hair growth [36]. Up until the early 1990s, it was believed that the epithelial stem cell compartment of the hair follicle was in the follicular matrix, which surrounds the dermal papilla at the base of the follicle. However, after a series of pulse-chase experiments, Cotsarelis and colleagues demonstrated that the epithelial stem cell compartment of the hair follicle resides higher up the follicle, in a region termed the hair follicle bulge [37]. Subsequently, bulge stem cells have been isolated from human hair follicles, either using microdissection, enzymatic digestion or plucking methods (bulge cells remain attached to plucked hair fibers) to isolate the cells. Bulge cells show a slow cycling nature, and enhanced growth potential [38]. After the initiating signal from the dermal papilla to initiate the hair cycle, there is cross-talk between the papilla and the bulge, or the hair germ (epithelial stem cell compartment beneath the bulge) that enables the follicle to cycle. Reciprocal signals from the bulge enable this communication; hence, the bulge plays an important role in hair growth and cycling [34]. Drug discovery using hair follicle organ cultures The hair follicle organ culture model is commonly used to determine whether small molecules or biologics may have a hair growthpromoting or a hair growth-inhibitory effect. It is a unique model in which the effect of a compound can be assessed simultaneously on the epithelial and mesenchymal compartments of the hair follicle. The human organ culture model is usually employed after the hair growth 103

4 Higgins & Christiano Hair follicle Dermal papilla Dermal sheath Outer root sheath Inner root sheath Hair shaft Scalp biopsy Organ-cultured hair follicles Cultured bulge cells Cultured dermal papilla cells Regen. Med. Future Science Group (2014) Figure 2. The various hair follicle culture systems. Intact hair follicles can be isolated from whole skin and grow as intact units in hair follicle organ culture. There are several cell layers within the follicle, including the inner root sheath and the hair fiber itself, which are relatively differentiated compared with the cells of the outer root sheath. Outer root sheath cells, which contain bulge keratinocytes, can be isolated and grown in culture. Moreover, cells from either the dermal papilla or dermal sheath can be microdissected and grown in culture. effect of a compound has been assessed on the hair cycle in mice. However, the microenvironment surrounding the hair follicle is known to have dramatic effects on hair follicle cycling [39], and this environment is not recapitulated in the ex vivo hair follicle organ culture model. If a drug or biologic is capable of inducing hair growth in the mouse hair growth model, but this effect is not translated to human hair follicle organ culture, it may provide insight into the way the drug is promoting hair growth, whether through the microenvironment or by acting directly on the follicle. Minoxidil is the most widely used treatment for androgenetic alopecia, however, there have been several conflicting studies evaluating the effect of minoxidil on hair follicle organ cultures. One particularly comprehensive study in 2004 utilized a total of 2300 human hair follicles, and concluded that minoxidil was unable to promote faster hair growth in occipital scalp follicles in the organ culture model [40]. However, more recent studies using the same model have indicated that minoxidil is capable of accelerating hair growth [41], while others have suggested that minoxidil can increase hair growth rates in approximately 20% of follicles assessed [42]. The inconsistency regarding efficacy of minoxidil is also reflected in the ambiguity surrounding its mode of action within the follicle. Moreover, clinical response to minoxidil varies depending on drug concentration and mode of delivery, and from patient to patient with approximately 60% of men showing an increased hair count with 5% formulation [43]. This variability indicates that a range of genetic or other environmental factors may contribute to the efficacy of minoxidil. Early reports indicated that minoxidil worked by increasing the blood flow surrounding the hair follicle, rather than acting directly on the hair follicle [44]. This perhaps explains the lack of concordance in minoxidil studies using the organ culture model, and highlights that this ex vivo model is not suitable for assessment of drugs that act on the follicular micro environment. Evidence now indicates that the mechanism of action of minoxidil, or minoxidil sulfate, which is a metabolite of minoxidil, is through stimulation of hair growth by opening ATPsensitive potassium channels within the dermal papilla [45], indicating that the effect is on the hair follicle itself, rather than the surrounding microenvironment. This is further supported as other potassium channel openers such as diazoxide are capable of increasing hair growth rates in the hair follicle organ culture model, indicating a similar mode of action for promoting hair growth [46]. Moreover, the effects of both minoxidil and diazoxide on hair growth can both be abrogated by coculture with a potassium channel inhibitor, tolbutamide [46]. Finasteride was also unexpectedly discovered when hair growth was reported as a side effect after its use in the treatment of benign prostate hypertrophy. Finasteride inhibits type II 104 Regen. Med. (2014) 9(1)

5 How hair follicle culture has advanced our understanding of treatment of androgenetic alopecia 5 a reductase, which in part explains its hairpromoting role, since this enzyme converts testosterone to dihydrotestosterone (DHT). DHT is a key effector in androgenetic alopecia. More recently, dutasteride has also been used in clinical trials to treat male pattern hair loss, and similarly to finasteride is a 5 a reductase inhibitor [47]. Levels of both 5 a reductase, and DHT are elevated in hair follicles in balding scalp comparative to nonbalding scalp [16,48], indicating a role for DHT in the progression of hair loss. A recent study [49] was also prompted by a serendipitous discovery of hair growth as a side effect to a drug treatment [50]. In this case it was not scalp hair, but eyelash growth that was enhanced in patients taking bimatoprost, a prostamide F2a-related analog, for treatment of glaucoma [51]. Given the recent identification of prostaglandin D2 synthase in balding scalp, this connection with hair growth is intriguing [17]. After identifying that prostanoid receptors are present within the dermal papilla and dermal sheath of the human follicles, Khidhir et al. assessed the effect of bimatoprost on human scalp hair follicles in the hair follicle organ culture model [49]. These cultures found that not only could bimatoprost significantly enhance scalp hair growth comparative to untreated controls, but that this effect could be inhibited by introduction of a prostamide F2a receptor antagonist [49]. Clinical trials evaluating bimatoprost in women with female pattern hair loss were recently completed while enrollment for a trial to assess bimatoprost in male pattern hair loss is ongoing (ClinicalTrials.gov identifier: NCT ) [201]. The results of the first trial are yet to be reported, however, a pilot study was performed with latanoprost, a prostaglandin F2a analog, whereby 50% of patients had increased hair growth [52]. These results would suggest that F2a analogs are a promising avenue to pursue for hair loss therapies. Recent evidence has pointed to an important role for hypoxia in the hair follicle, since the epithelial portion of the follicle is severely hypoxic [53]. This low oxygen state is hypothesized to be important for protection, or maintenance, of the stem cell reservoirs in the follicle [54], which are required for hair follicle regrowth. In hypoxic conditions, hypoxia inducible transcription factor is stabilized, enabling transcription of several target genes, including EPO, which prevent cells death. Not only is EPO, and its receptor EPOR, expressed within the hair follicle, but in hair follicle organ cultures that are exposed to hypoxic conditions, EPO is upregulated [55]. Interestingly, these authors found that female hair follicles grown in the presence of EPO in organ culture did not respond by growing faster than controls [55]. However, a later study by another group using male scalp hair follicles concluded that the addition of EPO to organ cultures could significantly elongate hair shafts, indicating an increased growth rate of hair follicles in the presence of EPO [56]. Within erythroid progenitor cells, vitamin D3 enhances the effect of EPO by upregulating expression of EPOR [57]. Intriguingly, low levels (10 nm) of vitamin D3 are also capable of promoting hair growth in human organ cultures, while high levels (10 µm) have an inhibitory effect on growth [58]. In vivo studies using vitamin D3 have shown that it can activate hair growth in nude mice [59]. Results are not always directly correlated between mice and humans, but if these results can be translated to humans, vitamin D3 analogs may prove beneficial in the treatment of hair loss. Finally, a last compound that has been shown to promote growth in the human hair follicle assay is spermidine. Spermidine is a compound within the polyamine pathway, known to be important in hair growth [60]. Polyamines are found at low levels in resting cells, but at high levels where there is metabolic activity or proliferation, such as in anagen hair follicles. Ramot et al. incubated organ cultured human hair follicles with spermidine, wherein they found that the polyamine was capable of increasing hair fiber elongation rates in hair follicles [61]. This is an interesting observation, as currently the only FDA-approved drug to treat hirsutism is eflornithine, which works by inhibiting the polyamine pathway, and therefore spermidine synthesis [62]. Drug discovery using hair follicle cell culture systems Although the organ culture method gives a robust assessment of whether or not a compound will have a hair-inducing effect, testing drugs or biologics on dermal papilla or bulge cells can give insightful information related to hair growth potency. This can be particularly useful if large numbers of drugs or biologics are to be tested in a high-throughput screen [63]. However, using dermal papilla cultures as a surrogate for intact hair follicle papillae is not an accurate representation since in vivo dermal papilla cells rarely divide, yet in culture they divide continuously [64,65]. More recently, spheroid cultures of dermal papilla cells have been described wherein no cell division is observed within the papilla 105

6 Higgins & Christiano spheroid [66]. Spheroid cultures are commonly used in other biological disciplines, as they provide a robust representation of intact tissue, both morphologically and biochemically [67]. Within the cancer research field, 3D culture models are extensively used over 2D models, as they exhibit behavioral characteristics of intact tumor tissue [68,69]. Screening drugs or biologics in spheroid cultures is therefore biologically appropriate, as it enables a more accurate cellular response [70]. In the future there may be a wider adoption of spheroid culture for ana lysis of hair dermal papillae, as the 3D system represents an advance over traditional 2D culture methods. As expected, one of the most common drugs that research groups have assessed using dermal papilla cultures has been minoxidil, with the theory that a balance between proliferation and apoptosis of cells in culture may translate to controlling hair follicle growth and regression in vivo. As indicated above, intact dermal papilla cells rarely divide in vivo, and therefore an assessment of proliferation is not necessarily the best readout for indicating whether a drug will promote hair growth. When used in dermal papilla cultures, minoxidil is capable of not only increasing proliferation, but there is also increased phosphorylation of ERK and AKT (which both mediate cell survival) and an increase in the BCL-2:BAX ratio (indicating reduced apoptosis) [71]. Minoxidil has also been shown to increase levels of VEGF [72] or adenosine [45] in dermal papilla cell cultures, while a recent study indicates that minoxidil can also activate the b-catenin pathway in cultured cells [73]. These studies again highlight the variable understanding that we have of minoxidil action within the follicle [74]. There have been several other studies that have examined the effect of active compounds on phosphorylation of ERK and AKT, as well as the BCL-2:BAX ratio in human dermal papilla cells, since an increase in phosphorylation and the ratio is believed to represent anagen [75]. Another predicted route of action for minoxidil in dermal papilla cells is through adenosine activation [45]. Minoxidil is predicted to activate a signaling cascade through adenosine receptors, resulting in the production of adenosine. Furthermore, adenosine activation in hair follicle dermal papilla cells can stimulate phosphorylation of ERK and AKT, as well as inducing expression of several growth factors, including FGF7 [76], which has previously been implicated in hair growth activation [34], and can increase hair growth rates in the human organ culture model [76]. Transcriptional activation of b-catenin has also been demonstrated in dermal papilla cells after adenosine activation [77], perhaps explaining the activation of this pathway after treatment with minoxidil [73]. Although adenosine was not evaluated in the human organ culture model, studies have shown that it can increase hair growth in organ cultures of rodent whisker follicles [77]. Moreover, adenosine has been used in clinical trials to treat androgenetic alopecia, and is able to result in increased hair growth in both men and women after topical treatment [78]. Regeneration of hair follicles using hair follicle cell cultures The notion of using hair follicle dermal papilla cells for regeneration of hair follicle structures comes from the demonstration more than 30 years ago that cultured rodent dermal papilla cells are capable of inducing growth of de novo follicles in recipient epithelium [79]. The dermal papilla has long been known to have a hairinducing capacity, and since the direct demonstration of this in rodent tissues in the 1980s, there has been intense interest in showing the same principle holds true for cultured human dermal papilla cells [80 82]. The concept behind using human hair follicle cells to promote hair growth is referred to as hair follicle neogenesis. Neogenesis simply refers to de novo follicle growth, which is uncommon in adults except for circumstances described in mice and rabbits after wounding [83,84]. We are referring specifically to neogenesis after follicular cell implantation. The proposed model is to isolate hair follicles from the occipital scalp of patients with androgenetic alopecia. Dermal papilla, or dermal sheath cells, can then be expanded in culture, and injected back into the frontal or balding scalp where they will either: induce entirely new follicles to grow; or alternatively, augment existing dermal papilla in miniaturized hair follicles, to enable thickening of existing hair fibers. Compellingly, dermal cells (both papilla and sheath) are capable of inducing hair growth when they are transplanted as intact units into recipient epithelium [81,82,85]. However, the path to human hair follicle induction is not without obstacles, not least because human dermal papilla cells behave very differently than their rodent counterparts, and appear to lose their inductive potential very quickly once grown in culture [86]. One theory explaining why hair follicle dermal cells lose their inductive potential is that 106 Regen. Med. (2014) 9(1)

7 How hair follicle culture has advanced our understanding of treatment of androgenetic alopecia when they are removed from the proximity of the follicle epithelium and placed in culture, they subsequently lose their inductive cues from the overlying epithelium. In light of this, Teumer and colleagues used a supplemented keratinocyte-conditioned medium to grow human dermal papilla cells, and were able to demonstrate an improvement in human dermal papilla inductivity when the cells were challenged to induce hair growth in recipient mouse neonatal epidermal tissue [87]. Additionally, Inoue et al. grew human dermal papilla cells in a vitamin D3 analog, selected after screening keratinocyte-conditioned medium for factors of interest, and demonstrated that this analog alone could increase inductivity in papilla cells placed against recipient rodent epithelium to test inductive potency [88]. Despite both these advances, these results have not been translated to humans, that is, human dermal papilla cells grown in these conditions have not yet been shown to be capable of inducing hair growth in recipient human tissues. Human dermal cells tend to lose their inductive potential immediately upon growth in culture. Comparatively, rodent cells are inductive early in primary culture, but quickly lose their inductive potential at higher passages [89,90]. Thus, studies that maintain rodent dermal papilla activity for longer in culture may provide an insight into factors that will promote human dermal cell inductivity, at least for a short period in culture. Members of the Wnt, BMP and FGF families have been shown to be capable of prolonging the hair-inducing effect of cultured mouse dermal papilla cells [91 93]. Restoring the Wnt, BMP and FGF influence in cultured human dermal papilla cells can also partially restore key intact dermal papilla signature genes, whose expression is usually absent in cultured human papilla cells [94]. Using a different approach, several groups have begun using spheroid cultures of dermal papilla cells, rather than regular 2D culture methods. Spheroid cultures of dermal papilla more closely resemble intact papillae, both morpho logically and with regard to their transcriptional signature [66]. Growth of mouse dermal papilla cells in spheroid culture can extend their inductive phenotype by several passages compared with monolayer cultures [92]. Moreover, human dermal papilla cells grown as spheroids, and combined with mouse epithelial cells in the patch assay, are capable of inducing hair growth while monolayer cultures lack this capacity [95]. This advance is of great interest, as it is an intrinsic modification of the cells, rather than the introduction of an external factor, that is conferring an inductive effect upon the papilla cells. Interestingly, an increase in the size of human papilla spheroids resulted in an increased number of hairs, but not thicker hair fibers [96]. While the size or type of the dermal papilla in mice can determine the thickness or type of hair fiber (awl/auchene, guard or zigzag) [97,98], the recipient epithelial tissue still determines the origin of the hair [99,100], and whether it is a human or a mouse fiber. More recently, we established a human-tohuman surgical assay to allow us to assess the potential of human dermal papilla spheroids to induce human hair fiber growth. We inserted spheroids between human glabrous skin and demonstrated that spheroid culture can restore the inductive properties of human dermal papilla cells, enabling them to induce an human hair follicle, unlike their monolayer counterparts [86]. This proof-of-concept study is a significant advance for human hair follicle neogenesis after follicular cell transplantation. Conclusion Isolation of hair follicles, and subsequently isolation and culture of dermal papilla cells was first demonstrated over 30 years ago [31], when perhaps the significance of this observation on hair loss treatments was not obvious. After identifying the hair growth-inducing potential of rodent dermal papilla cells, many groups from around the world have investigated and exploited human cells in an effort to develop treatments for androgenetic alopecia. In this review, we summarized the advances that have been made using both intact follicle organ cultures and cultured dermal papilla cells in identifying new treatments to promote hair growth. More recently, in a shift from traditional studies that use cell cultures to gain insight, many groups have begun to exploit the inherent properties present within hair follicle dermal cells to attempt to induce new hair follicles in recipient tissues. Future perspective There is a robust effort by pharmaceutical and cosmetic companies to utilize hair follicle cell culture to find new treatments that will have a hair growth-promoting effect. For follicular cell implantation, there are currently some Phase I/II trials in progress that are assessing the ability of either dermal papilla or sheath cells to promote hair growth in patients with androgenetic alopecia. Furthermore, we predict the development 107

8 Higgins & Christiano of growing hair follicles in bio engineered skin, which would bring rapid advance to the skin graft field, with the ability to generate a functional skin complete with appendages. Financial & competing interests disclosure The authors are grateful for support from the Dermatology Foundation (Career Development Award to CA Higgins) and NYSTAR and NYSTEM (to AM Christiano). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. Executive summary Hair follicle culture methods Whole hair follicles can be isolated from the scalp and used to assess hair growth in an organ culture model. Dermal papilla and sheath cells can be microdissected from follicles, and the cells can be grown in culture for use in subsequent studies. Hair follicle bulge keratinocytes can be isolated from the follicle and grown in culture. Drug discovery using hair follicle organ cultures Minoxidil exerts its effect on the hair follicle by opening adenosine-sensitive potassium channels in the dermal papilla. Finasteride acts on the hair follicle by inhibiting the conversion of testosterone to dihydrotestosterone. The glaucoma drug bimatoprost can also increase length of human scalp hairs in hair follicle organ cultures. Hypoxic conditions in the hair follicle can increase growth rates of hair fibers. Drug discovery using hair follicle cell culture systems Minoxidil may act on dermal papilla cells by phosphorylating Erk and Akt, and by increasing the Bcl-2:Bax ratio. Minoxidil may also upregulate adenosine, which in turn is capable of inducing several hair growth-related genes, such as FGF7, and was successful as a hair growth inducer in a recent clinical trial. Regeneration of hair follicles using hair follicle cell cultures Follicular cell implantation is based upon the observation that human dermal papilla cells are capable of inducing hair growth in recipient epithelium. Restoring the epithelial influence, via the introduction of defined factors to the culture medium, may increase the inductive potential of human papilla cells. Restoring the intrinsic signaling network via 3D culture appears to have an induction-promoting effect on both mouse and human dermal papilla cells. References Papers of special note have been highlighted as: of interest of considerable interest 1 Chase HB. Growth of the hair. Physiol. Rev. 34(1), (1954). 2 Kligman AM. The human hair cycle. J. Invest. Dermatol. 33, (1959). 3 Price ML, Griffiths WA. 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Androgenetic alopecia: identification of four genetic risk loci and evidence for the contribution of WNT signaling to its etiology. J. Invest. Dermatol. 133(6), (2013). 10 Springer K, Brown M, Stulberg DL. Common hair loss disorders. Am. Fam. Physician 68(1), (2003). 11 Krupa Shankar D, Chakravarthi M, Shilpakar R. Male androgenetic alopecia: population-based study in 1,005 subjects. Int. J. Trichol. 1(2), (2009). 12 Birch MP, Lalla SC, Messenger AG. Female pattern hair loss. Clin. Exp. Dermatol. 27(5), (2002). 13 Norwood OT. Male pattern baldness. classification and incidence. South Med. J. 68(11), (1975). 14 Ludwig E. Classification of the types of androgenetic alopecia (common baldness) occurring in the female sex. Br. J. Dermatol. 97(3), (1977). 15 Whiting DA. Possible mechanisms of miniaturization during androgenetic alopecia or pattern hair loss. J. Am. Acad. Dermatol. 45(Suppl. 3), S81 S86 (2001). 16 Sawaya ME, Price VH. 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