Human hair growth enhancement in vitro by green tea epigallocatechin-3-gallate (EGCG)

Green tea is a popular worldwide beverage, and its potential beneficial effects such as anti-cancer and anti-oxidant properties are believed to be mediated by epigallocatechin-3-gallate (EGCG), a major constituent of polyphenols. Recently, it was reported that EGCG might be useful in the prevention or treatment of androgenetic alopecia by selectively inhibiting 5alpha-reductase activity. However, no report has been issued to date on the effect of EGCG on human hair growth. This study was undertaken to measure the effect of EGCG on hair growth in vitro and to investigate its effect on human dermal papilla cells (DPCs) in vivo and in vitro. EGCG promoted hair growth in hair follicles ex vivo culture and the proliferation of cultured DPCs. The growth stimulation of DPCs by EGCG in vitro may be mediated through the upregulations of phosphorylated Erk and Akt and by an increase in the ratio of Bcl-2/Bax ratio. Similar results were also obtained in in vivo dermal papillae of human scalps. Thus, we suggest that EGCG stimulates human hair growth through these dual proliferative and anti-apoptotic effects on DPCs.     

6-Gingerol Inhibits Hair Shaft Growth in Cultured Human Hair Follicles and Modulates Hair Growth in Mice

Ginger (Zingiber officinale) has been traditionally used to check hair loss and stimulate hair growth in East Asia. Several companies produce shampoo containing an extract of ginger claimed to have anti-hair loss and hair growth promotion properties. However, there is no scientific evidence to back up these claims. This study was undertaken to measure 6-gingerol, the main active component of ginger, on hair shaft elongation in vitro and hair growth in vivo, and to investigate its effect on human dermal papilla cells (DPCs) in vivo and in vitro. 6-Gingerol suppressed hair growth in hair follicles in culture and the proliferation of cultured DPCs. The growth inhibition of DPCs by 6-gingerol in vitro may reflect a decrease in the Bcl-2/Bax ratio. Similar results were obtained in vivo. The results of this study showed that 6-gingerol does not have the ability to promote hair growth, on the contrary, can suppress human hair growth via its inhibitory and pro-apoptotic effects on DPCs in vitro, and can cause prolongation of telogen phase in vivo. Thus, 6-gingerol rather than being a hair growth stimulating drug, it is a potential hair growth suppressive drug; i.e. for hair removal.     

Fibroblast growth factor 5 inhibits hair growth by blocking dermal papilla cell activation.

Fibroblast growth factor (FGF) 5 inhibits hair growth and induces catagen in mouse hair follicles, in vivo. Given that FGF-5 receptor (FGFR1) is expressed in dermal papilla cells (DPCs), which are known to stimulate outer root sheath cell (ORSC) proliferation, we hypothesized that FGF-5 attenuates DPC-mediated ORSC proliferation. In the present study, DPCs and ORSCs were isolated from rat vibrissae, after which the effects of FGF-5 on proliferation of ORSCs cultured in DPC-conditioned medium were assessed. We first confirmed that FGFR1 was expressed in cultured DPCs and detected FGFR2-4 as well. ORSC proliferation was increased approximately twofold when the cells were cultured in DPC-conditioned medium, and the effect was unaltered by FGF-5. In addition, FGF-5 did not directly inhibit ORSC proliferation; indeed, it actually promoted proliferation of both DPCs and ORSCs. When DPCs were first activated by exposure to FGF-1 and FGF-2, which are expressed in hair follicles during anagen, ORSC proliferation observed in the resultant conditioned medium was substantially greater than in medium conditioned by unstimulated DPCs. The FGF-1-induced enhancement was reversed by FGF-5, diminishing ORSC proliferation to control levels. By contrast, the enhancement of DPC-mediated ORSC proliferation by FGF-2 was not suppressed by FGF-5. Proliferation of ORSCs did not depend on DPC proliferation, nor did FGF-1 directly promote ORSC proliferation. Dermal papillae thus appear to require activation before they will efficiently stimulate hair growth, and FGF-5 appears to inhibit hair growth and induce catagen by blocking that activation.     

Hair multiplication with dermal papilla like tissue containing human dermal papilla cells

Alopecia is not a critical disease; however it is a disease that can affect the quality of life. Many remedies have been developed to cure alopecia, but only two have been approved by the FDA. Due to the steadily increasing number of young alopecia patients, the need for new therapies for curing alopecia is very high. Recent studies on cell therapy have reported using technique to treat various diseases. We introduce upgraded hair cell therapy which tested hair structure inducing activity with bioartificial dermal papilla tissue. Hair follicles contain two types of stem cells: Outer root sheath cells (ORSCs) derived epithelial cells, and dermal cells (DPCs). In this study, we reconstructed DP-like tissues (DPLTs) using cultured dermal papilla cells (DPCs) from human hair follicles. The DPLTs were produced special media (Dermal Papilla Forming Media: DPFM) conditions in vitro, which can induce epithelial stands from implanted healthy hair without DP. We tested in vivo hair-inducing with a modified hair sandwich model. Two to three weeks DPLT injection into the mouse scalp skin, we observed new hair in the injected site and detected injected human cells from DPLTs and Outer Root Sheath Cells (ORSCs) in the new hair via human Alu-DNA-specific probe. In the future, reconstructed DPLTs may be used in in vitro studies of hair development and the morphogenesis mechanism, as well as in vitro studies of the efficacy and toxicity of drugs for baldness. These tissues will be used as an alternative medicine product for hair transplantation     

黄芪、何首乌、女贞子、菟丝子混合提取物对体外培养毛囊生长的影响及药理作用研究

目的:通过体内外实验探讨黄芪、何首乌、女贞子、菟丝子混合中药提取物对毛囊增殖的影响作用以及其作用机理。方法:通过体外培养的C57BL/6小鼠毛囊器官模型观察不同浓度中药提取物对毛囊生长的影响;采用MTT法测定不同浓度中药提取物对毛乳头增殖的影响;蛋白免疫印迹法(Western Blot)和ELISA检测中药提取物对毛乳头细胞分泌肝细胞生长因子(HGF)的影响。结果:中药提取物能够刺激体外培养的小鼠毛囊的生长,800μg/mL浓度的促进作用最强;160μg/mL中药提取物对毛乳头细胞的增殖作用最强,与米诺地尔、齐墩果酸阳性对照存在显著性差异(P0.05)。而且,药提取物促进了毛乳头细胞分泌HGF。结论:黄芪、何首乌、女贞子、菟丝子混合中药提取物在促进毛发生长中起到重要作用,促进毛乳头细胞增殖和分泌HGF是促进毛囊生长的可能性药理机制。     

Comparative secretome analysis of human follicular dermal papilla cells and fibroblasts using shotgun proteomics

The dermal papilla cells (DPCs) of hair follicles are known to secrete paracrine factors for follicular cells. Shotgun proteomic analysis was performed to compare the expression profiles of the secretomes of human DPCs and dermal fibroblasts (DFs). In this study, the proteins secreted by DPCs and matched DFs were analyzed by 1DE/LTQ FTICR MS/MS, semi-quantitatively determined using emPAI mole percent values and then characterized using protein interaction network analysis. Among the 1,271 and 1,188 proteins identified in DFs and DPCs, respectively, 1,529 were further analyzed using the Ingenuity Pathway Analysis tool. We identified 28 DPC-specific extracellular matrix proteins including transporters (ECM1, A2M), enzymes (LOX, PON2), and peptidases (C3, C1R). The biochemically- validated DPC-specific proteins included thrombospondin 1 (THBS1), an insulin-like growth factor binding protein3 (IGFBP3), and, of particular interest, an integrin beta1 subunit (ITGB1) as a key network core protein. Using the shotgun proteomic technique and network analysis, we selected ITGB1, IGFBP3, and THBS1 as being possible hair-growth modulating protein biomarkers.     

Induction of transforming growth factor-beta 1 by androgen is mediated by reactive oxygen species in hair follicle dermal papilla cells

The progression of androgenetic alopecia is closely related to androgen-inducible transforming growth factor (TGF)-β1 secretion by hair follicle dermal papilla cells (DPCs) in bald scalp. Physiological levels of androgen exposure were reported to increase reactive oxygen species (ROS) generation. In this study, rat vibrissae dermal papilla cells (DP-6) transfected with androgen receptor showed increased ROS production following androgen treatment. We confirmed that TGF-β1 secretion is increased by androgen treatment in DP-6, whereas androgeninducible TGF-β1 was significantly suppressed by the ROSscavenger, N-acetyl cysteine. Therefore, we suggest that induction of TGF-β1 by androgen is mediated by ROS in hair follicle DPCs. [BMB Reports 2013; 46(9): 460-464]     

Expression of basement membrane components through morphological changes in the hair growth cycle

The amount and distribution of fibronectin associated with hair follicles was found to vary during the hair growth cycle in the rat. Immunocytochemical staining of follicles in mid-late anagen (the growth stage) revealed the presence of fibronectin in the dermal papilla matrix, in the basement membrane separating this from the epithelial cells of the hair bulb, and in the basement membrane and connective tissue sheath which underly the cells of the outer root sheath. Early in catagen, the transitional stage, staining of the dermal papilla matrix disappeared. Fibronectin persisted in the basement membrane and connective tissue sheath, which undergo corrugation and apparent thickening in catagen. After follicle shortening, the telogen (resting) stage is reached, at which point fibronectin staining was found to be minimal, being restricted to the basement membrane around the secondary germ. The onset of anagen, involving cell division and follicle elongation, was associated with a great increase in the amount of fibronectin in this zone and in and around the dermal papilla. Analysis of entry into anagen by [3H]thymidine incorporation and autoradiography revealed that growth could be detected before the increase in fibronectin expression. However, growing cells, even in a suprabasal position, always had some fibronectin at their surface. Immunoelectron microscopy of early anagen follicles confirmed the light microscopic findings and also showed that fibronectin was present in small vesicles close to the surface of dermal papilla and some epithelial cells. Increased deposition of laminin and type IV collagen in early anagen follicles was also noted, emphasizing the importance of basement membrane components during morphogenetic events in vivo.     

Valproic acid induces hair regeneration in murine model and activates alkaline phosphatase activity in human dermal papilla cells

BACKGROUND: Alopecia is the common hair loss problem that can affect many people. However, current therapies for treatment of alopecia are limited by low efficacy and potentially undesirable side effects. We have identified a new function for valproic acid (VPA), a GSK3β inhibitor that activates the Wnt/β-catenin pathway, to promote hair re-growth in vitro and in vivo. METHODOLOGY/ PRINCIPAL FINDINGS: Topical application of VPA to male C3H mice critically stimulated hair re-growth and induced terminally differentiated epidermal markers such as filaggrin and loricrin, and the dermal papilla marker alkaline phosphatase (ALP). VPA induced ALP in human dermal papilla cells by up-regulating the Wnt/β-catenin pathway, whereas minoxidil (MNX), a drug commonly used to treat alopecia, did not significantly affect the Wnt/β-catenin pathway. VPA analogs and other GSK3β inhibitors that activate the Wnt/β-catenin pathway such as 4-phenyl butyric acid, LiCl, and BeCl(2) also exhibited hair growth-promoting activities in vivo. Importantly, VPA, but not MNX, successfully stimulate hair growth in the wounds of C3H mice. CONCLUSIONS/ SIGNIFICANCE: Our findings indicate that small molecules that activate the Wnt/β-catenin pathway, such as VPA, can potentially be developed as drugs to stimulate hair re-growth.     

Hair cycle-dependent changes of alkaline phosphatase activity in the mesenchyme and epithelium in mouse vibrissal follicles

Alkaline phosphatase (ALP) activity was detected in the restricted mesenchymal and epithelial regions in mouse vibrissal follicles. Its localization and strength dramatically changed during the hair cycle. Activity in the dermal papilla (DP) was moderate in very early anagen, reached a maximal level in early anagen, decreased at the proximal region of DP after mid anagen, and was kept at a low level during catagen. The bulbar dermal sheath showed intense ALP activity only in early anagen. Although most bulbar epithelium did not show ALP activity, germinative epidermal cells that were adjacent to the ALP-negative DP cells became ALP-positive in mid anagen and rearranged in a single layer so as to encapsulate the DP in mid catagen. During catagen, the outermost layer of bulbar epithelium became ALP-positive, which could be follicular epithelial precursors migrating from the bulge. Before the initiation of hair formation, ALP activity in the bulbar epithelium rapidly decreased and that in DP increased. These dynamic changes of ALP expression might be related to DP's functions in hair induction and also to reconstruction of the bulbar structure during the hair cycle.     

The hair follicle: a paradoxical androgen target organ

Androgens are the main regulator of normal human hair growth. After puberty, they promote transformation of vellus follicles, producing tiny, unpigmented hairs, to terminal ones, forming larger pigmented hairs, in many areas, e.g. the axilla. However, they have no apparent effect on the eyelashes, but can cause the opposite transformation on the scalp leading to the replacement of terminal hairs by vellus ones and the gradual onset of androgenetic alopecia. This paradox appears to be an unique hormonal effect. Hair follicles are mainly epithelial tissues, continuous with the epidermis, which project into the dermis. A mesenchyme-derived dermal papilla enclosed within the hair bulb at the base controls many aspects of follicle function. In the current hypothesis for androgen regulation, the dermal papilla is also considered the main site of androgen action with androgens from the blood binding to receptors in dermal papilla cells of androgen-sensitive follicles and causing an alteration of their production of paracrine factors for target cells e.g. keratinocytes. Studies of cultured dermal papilla cells from sites with different responses to androgens in vivo have confirmed the paradoxical responses. All dermal papilla cells from androgen-sensitive sites contain low capacity, high affinity androgen receptors. However, only some cells formed 5alpha-dihydrotestosterone, e.g. beard but not axillary cells, in line with hair growth in 5alpha-reductase deficiency. Incubation with androgens also stimulated the mitogenic capacity of beard cell media, but inhibited that produced by scalp cells. This suggests that the paradoxical differences are due to differential gene expression within hair follicles, presumably caused during embryogenesis.     

Different patterns of 5alpha-reductase expression, cellular distribution, and testosterone metabolism in human follicular dermal papilla cells

Androgens regulate hair growth, and 5α-reductase (5αR) plays a pivotal role in the action of androgens on target organs. To clarify the molecular mechanisms responsible for controlling hair growth, the present study presents evidence that the human follicular dermal papilla cells (DPCs) from either beard (bDPCs) or scalp hair (sDPCs) possess endogenous 5αR activity. Real-time RT-PCR revealed that the highest level of 5αR1 mRNA was found in bDPCs, followed by sDPCs, and a low but detectable level of 5αR1 mRNA was observed in fibroblasts. Minimally detectable levels of 5αR2 mRNA were found in all three cell types. A weak band at 26 kDa corresponding to the human 5αR1 protein was detected by Western blot in both DPCs, but not in fibroblasts. Immuonofluorescence analysis confirmed that 5αR1 was localized to the cytoplasm rather than in the nuclei in both DPCs Furthermore, a 5αR assay using [¹⁴C]testosterone labeling in intact cells revealed that testosterone was transformed primarily into androstenedione, and in small amounts, into DHT. Our results demonstrate that the 5αR activities of either bDPCs or sDPCs are stronger than that of dermal fibroblasts, despite the fact that the major steroidogenic activity is attributed to 17β-HSD rather than 5αR among the three cell types. The 5αR1 inhibitor MK386 exhibited a more potent inhibitory effect on 5αR activity than finasteride (5αR2 inhibitor) in bDPCs.     

Bone morphogenetic proteins 4 and 2/7 induce osteogenic differentiation of mouse skin derived fibroblast and dermal papilla cells

Heterotopic ossification is a pathological condition in which bone forms outside the skeletal system. It can also occur in skin, which is the case in some genetic disorders. In addition to precursor cells and the appropriate tissue environment, heterotopic ossification requires inductive signals such as bone morphogenetic proteins (BMP). BMPs are growth and differentiation factors that have the ability to induce cartilage and bone formation in ectopic sites. The objective of this study is to explore the effect of the BMP-4 homodimer and BMP-2/7 heterodimer on the osteogenic differentiation of primary mouse skin fibroblasts and hair follicle dermal papilla (DP) cells. Osteogenic differentiation was induced by osteogenic induction medium (OS) containing 10 nM dexamethasone. The effect of BMP-4 and BMP-2/7 was studied using alkaline phosphatase (ALP) and calcium assays after 1.5, 3 and 5 weeks of differentiation. Fibroblasts and DP cells were able to differentiate into osteoblast-like matrix mineralizing cells. The first visible sign of differentiation was the change of morphology from rounded to more spindle-shaped cells. BMP-4 and BMP-2/7 exposure elevated ALP activity and calcium production significantly more than OS alone. The osteogenic response to BMP-4 and BMP-2/7 was similar in fibroblasts, whereas, in DP cells, BMP-2/7 was more potent than BMP-4. OS alone could not induce osteogenic differentiation in DP cells. Clear and consistent results show that dermal fibroblasts and stem cells from the dermal papilla were capable of osteogenic differentiation. The BMP-2/7 heterodimer was significantly more effective on hair follicular dermal stem cell differentiation.     

Molecular biology of hair morphogenesis: development and cycling

In mammals, hair follicles produce hairs that fulfill a number of functions including thermoregulation, collecting sensory information, protection against environmental trauma, social communication, and mimicry. Hair follicles develop as a result of epithelial-mesenchymal interactions between epidermal keratinocytes committed to hair-specific differentiation and cluster of dermal fibroblasts that form follicular papilla. During postnatal life, hair follicles show patterns of cyclic activity with periods of active growth and hair production (anagen), apoptosis-driven involution (catagen), and relative resting (telogen). During last decade, substantial progress has been achieved in delineating molecular mechanisms that control hair follicle development and cyclic activity. In this review, we summarize the data demonstrating that regulation of hair follicle development in the embryo and control of hair follicle growth during postnatal life are highly conserved and both require involvement of similar molecular mechanisms. Since many of the molecules that control hair follicle development and cycling are also involved in regulating morphogenesis and postnatal biology of other ectodermal derivatives, such as teeth, feathers, and mammary glands, basic principles and molecular mechanisms that govern hair follicle development and growth may also be applicable for other developmental systems.     

Restoration of hair growth by surgical implantation of follicular dermal sheath.

The capacity of lower follicle dermal sheath to restore hair growth was tested by removing the lower halves of follicles, and then immediately implanting material containing dermal sheath cells from these bases, into the remaining upper epidermal follicle cavity. Over 60% of recipient follicles produced stout emergent vibrissa fibres and some operations resulted in multiple hair production from a single follicle. Histological examination revealed new dermal papillae within large bulb structures which were sited below the level of amputation--a feature that indicated that the new dermal papilla was derived from implanted material. For many follicles, the failure to produce emergent fibres could be accounted for after histological examination. These results provide clear evidence that lower follicle dermal sheath cells are capable of replacing those of the dermal papilla and it shows that they can do so in the context of the upper follicle. However, because elements of lower follicle epidermis were present in the implant material, the interactive sequence of events cannot be established. Dermal sheath cells have immense potential for papilla cell replacement: questions remain as to whether the distinction between sheath and papilla cells is one of context, or whether the transition requires specific external influences.     

Localization of TIMP in cycling mouse hair

TIMP (tissue inhibitor of metalloproteinase) is a glycoprotein inhibitor of metalloproteinases that we hypothesize to be involved in the tissue remodeling that occurs during each hair growth cycle. We examined this hypothesis by studying the expression of TIMP at selected times during a single hair cycle using TIMP-lacZ transgenic mice to localize TIMP gene activity in the hair follicle. TIMP gene induction was visualized by staining mouse back skin for beta-galactosidase (beta-gal) activity. Paraffin sections were analyzed for the localization of TIMP expression. TIMP gene activation appears in hair follicles only during the mid-anagen (the growing stage of the hair cycle) primarily in Henle's layer of the inner root sheath. Some expression of TIMP is also seen in a few connective tissue cells, in the sebaceous gland and in cells at the proximity of the dermal papilla cells in catagen (regressing) and telogen (resting) follicles. These results are consistent with a role for TIMP in cyclic remodeling of connective tissue in hair follicles.     

Optimization of the reconstruction of dermal papilla like tissues employing umbilical cord mesenchymal stem cells

Alopecia is not life threatening, but patients who undergo alopecia often experience severe mental stress. In addition, the number of individuals afflicted by alopecia has been increasing steadily. The most effective treatment of alopecia developed to date is auto hair transplantation. To overcome the limitations associated with current therapies for the treatment of alopecia, many researchers have attempted to revive hair follicles by in vitro culture of hair follicle cells and subsequent implantation in the treatment area. Previously, we demonstrated that umbilical cord-derived mesenchymal stem cells (UC-MSCs) could be isolated and expanded successfully from the Wharton’s Jelly. Cultureexpanded UC-MSCs formed aggregates similar to native dermal papilla (DP) in special media (DPFM) and reconstructed dermal papilla like tissues (DPLTs) could induce new hair follicles. The purpose of the present study was to optimize the reconstruction of DPLTs. As in the case of MSCs, when compared to differentiated cells, DPLTs require an additional step to induce differentiation into dermal papilla cells. However, it is necessary to use hepatocyte growth factor (HGF) in the differentiation step, which is relatively expensive. To reduce the expenses associated with cell therapy using MSCs, it is necessary to optimize this differentiation step. To accomplish this, we evaluated the effects of cell inoculation density and growth factors during differentiation.     

Androgen Receptor Accelerates Premature Senescence of Human Dermal Papilla Cells in Association with DNA Damage

The dermal papilla, located in the hair follicle, expresses androgen receptor and plays an important role in hair growth. Androgen/Androgen receptor actions have been implicated in the pathogenesis of androgenetic alopecia, but the exact mechanism is not well known. Recent studies suggest that balding dermal papilla cells exhibit premature senescence, upregulation of p16^INK4a, and nuclear expression of DNA damage markers. To investigate whether androgen/AR signaling influences the premature senescence of dermal papilla cells, we first compared frontal scalp dermal papilla cells of androgenetic alopecia patients with matched normal controls and observed that premature senescence is more prominent in the dermal papilla cells of androgenetic alopecia patients. Exposure of androgen induced premature senescence in dermal papilla cells from non-balding frontal and transitional zone of balding scalp follicles but not in beard follicles. Overexpression of the AR promoted androgen-induced premature senescence in association with p16^INK4a upregulation, whereas knockdown of the androgen receptor diminished the effects of androgen. An analysis of γ-H2AX expression in response to androgen/androgen receptor signaling suggested that DNA damage contributes to androgen/androgen receptor-accelerated premature senescence. These results define androgen/androgen receptor signaling as an accelerator of premature senescence in dermal papilla cells and suggest that the androgen/androgen receptor-mediated DNA damage-p16^INK4a axis is a potential therapeutic target in the treatment of androgenetic alopecia.