Organ-cultured Human Hair Follicles Research Articles

Revealing novel insights on how oral supplementation with collagen peptides may prevent hair loss: Lessons from the human hair follicle organ culture

Dietary supplementation with bioactive collagen peptides (CPs) may be a helpful adjuvant strategy in reducing the excessive hair shedding and thinning associated with aging or patterned hair loss. However, the underlying biological effects of CPs on hair follicle (HF) function have not yet been characterized. Excessive hair loss occurs when HFs exit the growth phase (anagen) prematurely due to, among other factors, impaired epithelial HF stem cell (eHFSC) activities. The aims of this study were to investigate the response of human HFs to peptides from the two most popular CP sources, i.e. marine and bovine collagens, which were previously in vitro-digested to mimic gastrointestinal breakdown, in one of the most clinically-relevant ex vivo HF models. Both types of collagens reduced the proliferation of pluripotent K15+ eHFSCs and enhanced the generation of K19+ and/or CD34+ stem cell progenies. In addition, bovine CPs significantly increased K15+ cells in the bulge and marine CPs significantly maintained HFs longer in anagen. Our results suggest that both marine and bovine CPs may help to prevent hair loss and maintain healthy hair by preserving eHFSCs and/or improving the generation of SC progenies. This data invites the further exploration of other CPs for the prevention of excessive hair shedding.

650 Controlling mTORC1 activity as a novel therapeutic strategy for managing human hair growth and pigmentation

The mechanistic Target of Rapamycin Complex 1 (mTORC1) exerts multiple physiological functions, including nutrient sensing and the control of metabolism, proliferation, senescence and Wnt signaling. Yet, the role of mTORC1 activity in human hair follicle (HF) biology remains unknown. Since patients with a loss-of-function mutation of the key endogenous inhibitor of mTORC1 activity, tuberous sclerosis complex (TSC), can show poliosis, we probed the hypothesis that mTORC1 regulates human HF pigmentation, using HF organ culture.

Induction of hair growth in hair follicle cells and organ cultures upon treatment with 30 kHz frequency inaudible sound via cell proliferation and antiapoptotic effects.

Androgenic alopecia is a hair loss disease mediated by dihydrotestosterone (DHT) and is currently treated using minoxidil, finasteride, or low-level laser therapy. However, these treatments have side-effects, indicating the need for an alternative treatment. In the present study, it was demonstrated that inaudible sound at 30 kHz significantly induced proliferative and anti-apoptotic effects in human dermal papilla cells (hDPCs) and outer root sheath keratinocytes. Cell viability assay, ELISA, reverse transcription quantitative PCR and TUNEL assays were performed to evaluate the effect of inaudible sound. Inaudible sound was also demonstrated to significantly inhibit the hair loss signals induced by DHT treatment in hDPCs. Furthermore, inaudible sound significantly induced hair follicle (HF) elongation and hair matrix keratinocyte proliferation in human HF organ culture. Overall, the results suggested that inaudible sound may be effective in treating hair loss and could be used to develop a new hair loss treatment approach.

Human epithelial stem cell survival within their niche requires "tonic" cannabinoid receptor 1-signalling-Lessons from the hair follicle.

The endocannabinoid system (ECS) regulates multiple aspects of human epithelial physiology, including inhibition/stimulation of keratinocyte proliferation/apoptosis, respectively. Yet, how the ECS impacts on human adult epithelial stem cell (eSC) functions remains unknown. Scalp hair follicles (HFs) offer a clinically relevant, prototypic model system for studying this directly within the native human stem cell niche. Here, we show in organ-cultured human HFs that, unexpectedly, selective activation of cannabinoid receptor-1 (CB1)-mediated signalling via the MAPK (MEK/Erk 1/2) and Akt pathways significantly increases the number and proliferation of cytokeratin CK15+ or CK19+ human HF bulge eSCs in situ, and enhances CK15 promoter activity in situ. In striking contrast, CB1-stimulation promotes apoptosis in the differentiated progeny of these eSCs (CK6+ HF keratinocytes). Instead, intrafollicular CB1 gene knockdown or CB1 antagonist treatment significantly reduces human HF eSCs numbers and stimulates their apoptosis, while CB1 knockout mice exhibit a reduced bulge eSCs pool in vivo. This identifies "tonic" CB1 signalling as a required survival stimulus for adult human HF eSCs within their niche. This novel concept must be taken into account whenever the human ECS is targeted therapeutically.

Polygonum multiflorum extract support hair growth by elongating anagen phase and abrogating the effect of androgen in cultured human dermal papilla cells

BackgroundDermal papilla cells (DPCs) play a key role in hair growth among the various cell types in hair follicles. Especially, DPCs determine the fate of hair follicle such as anagen to telogen transition and play a pivotal role in androgenic alopecia (AGA). This study was performed to elucidate the hair growth promoting effects of Polygonum multiflorum extract (PM extract) in cultured human DPCs and its underlying mechanisms.MethodsThe effects of PM extract on cultured DPCs were investigated. Cell viability and mitochondrial activity were measured by CCK-8 and JC-1 analysis, respectively. Western blotting, dot blotting, ELISA analysis, immunocytochemistry and real-time PCR analysis were also performed to elucidate the changes in protein and mRNA levels induced by PM extract. 3D cultured DPC spheroids were constructed for mimicking the in vivo DPs. The hair growth stimulatory effect of PM extract was evaluated using human hair follicle organ culture model.ResultsPM extract increased the viability and mitochondrial activity in cultured human DPCs in a dose dependent manner. The expression of Bcl2, an anti-apoptotic protein expressed dominantly in anagen was significantly increased and that of BAD, a pro-apoptotic protein expressed in early catagen was decreased by PM extract in cultured DPCs and/or 3D DPC spheroid culture. PM extract also decreased the expression of catagen inducing protein, Dkk-1. Growth factors including IGFBP2, PDGF and VEGF were increased by PM extract, revealed by dot blot protein analysis. We also have found that PM extract could reverse the androgenic effects of dihydrotestosterone (DHT), the most potent androgen. Finally, PM extract prolonged the anagen of human hair follicles by inhibiting catagen entry in human hair follicle organ culture model.ConclusionOur data strongly suggest that PM extract could promote hair growth by elongating the anagen and/or delaying the catagen induction of hair follicles through activation of DPCs.

CDK4/6 inhibition mitigates stem cell damage in a novel model for taxane‐induced alopecia

Taxanes are a leading cause of severe and often permanent chemotherapy‐induced alopecia. As the underlying pathobiology of taxane chemotherapy‐induced alopecia remains poorly understood, we investigated how paclitaxel and docetaxel damage human scalp hair follicles in a clinically relevant ex vivo organ culture model. Paclitaxel and docetaxel induced massive mitotic defects and apoptosis in transit amplifying hair matrix keratinocytes and within epithelial stem/progenitor cell‐rich outer root sheath compartments, including within Keratin 15+ cell populations, thus implicating direct damage to stem/progenitor cells as an explanation for the severity and permanence of taxane chemotherapy‐induced alopecia. Moreover, by administering the CDK4/6 inhibitor palbociclib, we show that transit amplifying and stem/progenitor cells can be protected from paclitaxel cytotoxicity through G1 arrest, without premature catagen induction and additional hair follicle damage. Thus, the current study elucidates the pathobiology of taxane chemotherapy‐induced alopecia, highlights the paramount importance of epithelial stem/progenitor cell‐protective therapy in taxane‐based oncotherapy, and provides preclinical proof‐of‐principle in a healthy human (mini‐) organ that G1 arrest therapy can limit taxane‐induced tissue damage.

Human Perifollicular Macrophages Undergo Apoptosis, Express Wnt Ligands, and Switch their Polarization during Catagen

Human Perifollicular Macrophages Undergo Apoptosis, Express Wnt Ligands, and Switch their Polarization during Catagen

Expression Pattern and Role of Klotho in Human Hair Follicles.

BackgroundKlotho protein plays a pivotal role in aging regulation. However, it is unclear whether klotho is expressed in human hair follicles and is correlated with hair growth.ObjectiveThe purpose of this study was to determine the expression pattern and role of klotho in human hair follicles.MethodsWe examined the klotho expression patterns in human hair follicles from young and aged donors. Furthermore, we examined the functional roles of klotho on human hair growth using klotho siRNA and klotho recombinant protein.ResultsInterestingly, klotho was expressed in human hair follicles at both gene and protein levels. In hair follicles, prominent klotho expression was mainly observed in the outermost regions of the outer root sheath and hair bulb matrix cells. Quantification of klotho protein expression in young and aged donors showed that klotho expression decreased with aging. In human hair follicle organ culture, klotho silencing promoted premature catagen induction and inhibited human hair growth. Otherwise, klotho protein prolonged human hair growth.ConclusionThese results indicate that klotho might be an important regulatory factor for human hair growth and hair cycle change.

Nature-derived lignan compound VB-1 exerts hair growth-promoting effects by augmenting Wnt/β-catenin signaling in human dermal papilla cells.

BackgroundVitexin is a kind of lignan compound which has been shown to possess a variety of pharmacological effects, such as anti-inflammatory, anti-oxidative and anti-cancer activities. However the effect of vitexin on hair regeneration has not been elaborated.MethodsThe proliferation of human dermal papilla cells (hDPCs) was examined by cell counting and continuous cell culture after vitexin compound 1 (VB-1) was treated. The expression of lef1, wnt5a, bmp2, bmp4, alpl and vcan was examined by RT-PCR. The expression of dkk1, tgf-β1, active-β-Catenin, and AXIN2 was examined by RT-PCR or immunoblotting. Hair shaft growth was measured in the absence or presence of VB-1.ResultsWe demonstrated that VB-1 significantly promotes the proliferation of hDPCs in a concentration-dependent manner within a certain concentration range. Among the hair growth-related genes investigated, dkk1 was clearly down-regulated in hDPCs treated with VB-1. The increased active β-Catenin and decreased AXIN2 protein levels suggest that VB-1 facilitates Wnt/β-catenin signaling in hDPCs in vitro. The expression of DP signature genes was also upregulated after VB-1 treatment. Our study further indicated that VB-1 promotes human hair follicle (HF) growth by HF organ culture assay.DiscussionVB-1 may exert hair growth-promoting effects via augmenting Wnt/β-catenin signaling in hDPCs.

Activation of Wnt/β-catenin signaling is involved in hair growth-promoting effect of 655-nm red light and LED in in vitro culture model.

Activation of the Wnt/β-catenin signaling pathway plays an important role in hair follicle morphogenesis and hair growth. Recently, low-level laser therapy (LLLT) was evaluated for stimulating hair growth in numerous clinical studies, in which 655-nm red light was found to be most effective and practical for stimulating hair growth. We evaluated whether 655-nm red light + light-emitting diode (LED) could promote human hair growth by activating Wnt/β-catenin signaling. An in vitro culture of human hair follicles (HFs) was irradiated with different intensities of 655-nm red light + LED, 21h7 (an inhibitor of β-catenin), or both. Immunofluorescence staining was performed to assess the expression of β-catenin, GSK3β, p-GSK3β, and Lef1 in the Wnt/β-catenin signaling. The 655-nm red light + LED not only enhanced hair shaft elongation, but also reduced catagen transition in human hair follicle organ culture, with the greatest effectiveness observed at 5min (0.839J/cm2). Additionally, 655-nm red light + LED enhanced the expression of β-catenin, p-GSK3β, and Lef1, signaling molecules of the Wnt/β-catenin pathway, in the hair matrix. Activation of Wnt/β-catenin signaling is involved in hair growth-promoting effect of 655-nm red light and LED in vitro and therefore may serve as an alternative therapeutic option for alopecia.

β-Catenin is involved in oleanolic acid-dependent promotion of proliferation in human hair matrix cells in an in vitro organ culture model

Oleanolic acid (OA), a pentacyclic triterpenoid compound which can be found in >1600 plants, has been shown to promote hair growth. To study the mechanisms of OA on hair growth, we investigated hair follicle (HF) growth on four different concentration OA using human hair follicle organ culture model. We found that HFs treated with 1 or 10μg/mL OA showed statistically enhanced elongation of the hair shaft and anagen-like stage. Moreover, higher positive rate of Ki-67, a matrix cellular marker of proliferation, was detected in the same groups treated with 1 or 10μg/mL than those treated with vehicle. We further demonstrated that β-catenin, a key Wnt signaling transducer, was highly expressed in the OA treated groups using immunofluorescence stain assay. These results suggest that OA may promote human hair growth by stimulating hair matrix cell proliferation through the Wnt/β-catenin pathway.

Human hair follicle organ culture: theory, application and perspectives.

For almost a quarter of a century, ex vivo studies of human scalp hair follicles (HFs) have permitted major advances in hair research, spanning diverse fields such as chronobiology, endocrinology, immunology, metabolism, mitochondrial biology, neurobiology, pharmacology, pigmentation and stem cell biology. Despite this, a comprehensive methodological guide to serum-free human HF organ culture (HFOC) that facilitates the selection and analysis of standard HF biological parameters and points out both research opportunities and pitfalls to newcomers to the field is still lacking. The current methods review aims to close an important gap in the literature and attempts to promote standardisation of human HFOC. We provide basic information outlining the establishment of HFOC through to detailed descriptions of the analysis of standard read-out parameters alongside practical examples. The guide closes by pointing out how serum-free HFOC can be utilised optimally to obtain previously inaccessible insights into human HF biology and pathology that are of interest to experimental dermatologists, geneticists, developmental biologists and (neuro-) endocrinologists alike and by highlighting novel applications of the model, including gene silencing and gene expression profiling of defined, laser capture-microdissected HF compartments.

Protective effect of Korean Red Ginseng against chemotherapeutic drug-induced premature catagen development assessed with human hair follicle organ culture model

BackgroundChemotherapy-induced alopecia (CIA) is one of the most distressing side effects for patients undergoing chemotherapy. This study evaluated the protective effect of Korean Red Ginseng (KRG) on CIA in a well-established in vitro human hair follicle organ culture model as it occurs in vivo. MethodsWe examined whether KRG can prevent premature hair follicle dystrophy in a human hair follicle organ culture model during treatment with a key cyclophosphamide metabolite, 4-hydroperoxycyclophosphamide (4-HC). Results4-HC inhibited human hair growth, induced premature catagen development, and inhibited proliferation and stimulated apoptosis of hair matrix keratinocytes. In addition, 4-HC increased p53 and Bax protein expression and decreased Bcl2 protein expression. Pretreatment with KRG protected against 4-HC-induced hair growth inhibition and premature catagen development. KRG also suppressed 4-HC-induced inhibition of matrix keratinocyte proliferation and stimulation of matrix keratinocyte apoptosis. Moreover, KRG restored 4-HC-induced p53 and Bax/Bcl2 expression. ConclusionOverall, our results indicate that KRG may protect against 4-HC-induced premature catagen development through modulation of p53 and Bax/Bcl2 expression.

Mimicking Hair Disorders by Genetic Manipulation of Organ-Cultured Human Hair Follicles

Human hair follicles can be dissected out of scalp skin and cultured in vitro in defined growth medium. Hair follicle organ cultures have previously been used to investigate the molecular and cellular mechanisms through which various factors regulate the maintenance and cycling of adult hair follicles. In this issue, Samuelov et al. transfected organ-cultured human hair follicles with siRNA nucleotides and suppressed the expression of the endogenous P-cadherin gene in follicular keratinocytes. Knocking down the expression of P-cadherin in hair follicles in vitro recapitulated the hair follicle phenotype observed in patients with hypotrichosis with juvenile macular dystrophy (HJMD) and enabled the authors to establish a cause-effect relationship between loss of P-cadherin and suppression of the canonical Wnt signaling pathway and upregulation of TGFβ2 during development of the hair abnormalities observed in HJMD patients.

Utilizing the hair follicle to dissect the regulation and autocrine/paracrine activities of prolactin in humans

to the editor: Ferraris et al. ([4][1]) have recently reported an increase in anterior pituitary cell proliferation in Δ1-9-G129R-hPRL (competitive prolactin receptor antagonist) -expressing transgenic mice compared to wild-type controls. In addition, they reported increased proliferation/decreased

Endocannabinoids limit excessive mast cell maturation and activation in human skin

Mast cells (MCs) crucially contribute to many inflammatory diseases. However, the physiological controls preventing excessive activities of MCs in human skin are incompletely understood. Since endocannabinoids are important neuroendocrine MC modifiers, we investigated how stimulation/inhibition of cannabinoid 1 (CB1) receptors affect the biology of human skin MCs in situ. This was investigated in the MC-rich connective tissue sheath of organ-cultured human scalp hair follicles by quantitative (immuno)histomorphometry, ultrastructural, and quantitative PCR techniques with the use of CB1 agonists or antagonists, CB1 knockdown, or CB1 knockout mice. Kit+ MCs within the connective tissue sheath of human hair follicles express functional CB1 receptors, whose pharmacological blockade or gene silencing significantly stimulated both the degranulation and the maturation of MCs from resident progenitor cells in situ (ie, enhanced the number of tryptase+, FcεRIα, or chymase+ connective tissue sheath-MCs). This was, at least in part, stem cell factor-dependent. CB1 agonists counteracted the MC-activating effects of classical MC secretagogues. Similar phenomena were observed in CB1 knockout mice, attesting to the invivo relevance of this novel MC-inhibitory mechanism. By using human hair follicle organ culture as an unconventional, but clinically relevant model system for studying the biology of MCs in situ, we show that normal skin MCs are tightly controlled by the endocannabinoid system. This limits excessive activation and maturation of MCs from resident progenitors via "tonic" CB1 stimulation by locally synthesized endocannabinoids. The excessive numbers and activation of MCs in allergic and other chronic inflammatory skin diseases may partially arise from resident intracutaneous MC progenitors, for example, because of insufficient CB1 stimulation. Therefore, CB1 stimulation is a promising strategy for the future management of allergy and MC-dependent skin diseases.

Thyrotropin-Releasing Hormone Selectively Stimulates Human Hair Follicle Pigmentation

In amphibians, thyrotropin-releasing hormone (TRH) stimulates skin melanophores by inducing secretion of α-melanocyte-stimulating hormone in the pituitary gland. However, it is unknown whether this tripeptide neurohormone exerts any direct effects on pigment cells, namely, on human melanocytes, under physiological conditions. Therefore, we have investigated whether TRH stimulates pigment production in organ-cultured human hair follicles (HFs), the epithelium of which expresses both TRH and its receptor, and/or in full-thickness human skin in situ. TRH stimulated melanin synthesis, tyrosinase transcription and activity, melanosome formation, melanocyte dendricity, gp100 immunoreactivity, and microphthalmia-associated transcription factor expression in human HFs in a pituitary gland-independent manner. TRH also stimulated proliferation, gp100 expression, tyrosinase activity, and dendricity of isolated human HF melanocytes. However, intraepidermal melanogenesis was unaffected. As TRH upregulated the intrafollicular production of "pituitary" neurohormones (proopiomelanocortin transcription and ACTH immunoreactivity) and as agouti-signaling protein counteracted TRH-induced HF pigmentation, these pigmentary TRH effects may be mediated in part by locally generated melanocortins and/or by MC-1 signaling. Our study introduces TRH as a novel, potent, selective, and evolutionarily highly conserved neuroendocrine factor controlling human pigmentation in situ. This physiologically relevant and melanocyte sub-population-specific neuroendocrine control of human pigmentation deserves clinical exploration, e.g., for preventing or reversing hair graying.

A simple assay for the study of human hair follicle damage induced by ionizing irradiation

Due to its rapidly proliferating matrix keratinocytes, the hair follicle is highly sensitive to ionizing irradiation (IR)-induced skin damage and thus an instructive and clinically relevant model organ for investigating the effects of IR on rapidly dividing epithelial-mesenchymal interaction systems. Here, we have assessed the impact of IR on organ-cultured human scalp hair follicles. We show that IR significantly inhibits the proliferation and induces apoptosis of hair follicle matrix keratinocytes, disrupts normal hair follicle pigmentation, and upregulates a number of quantitative toxicity and viability markers (oxidative stress indicators, DNA oxidative damage, LDH release). This introduces human hair follicle organ culture as an excellent novel research tool for radiobiology and invites exploitation as a preclinical assay system for testing candidate radioprotectants.

Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture

The organ culture of human scalp hair follicles (HFs) is the best currently available assay for hair research in the human system. In order to determine the hair growth-modulatory effects of agents in this assay, one critical read-out parameter is the assessment of whether the test agent has prolonged anagen duration or induced catagen in vitro. However, objective criteria to distinguish between anagen VI HFs and early catagen in human HF organ culture, two hair cycle stages with a deceptively similar morphology, remain to be established. Here, we develop, document and test an objective classification system that allows to distinguish between anagen VI and early catagen in organ-cultured human HFs, using both qualitative and quantitative parameters that can be generated by light microscopy or immunofluorescence. Seven qualitative classification criteria are defined that are based on assessing the morphology of the hair matrix, the dermal papilla and the distribution of pigmentary markers (melanin, gp100). These are complemented by ten quantitative parameters. We have tested this classification system by employing the clinically used topical hair growth inhibitor, eflornithine, and show that eflornithine indeed produces the expected premature catagen induction, as identified by the novel classification criteria reported here. Therefore, this classification system offers a standardized, objective and reproducible new experimental method to reliably distinguish between human anagen VI and early catagen HFs in organ culture.

Prolactin—a novel neuroendocrine regulator of human keratin expressionin situ

The controls of human keratin expression in situ remain to be fully elucidated. Here, we have investigated the effects of the neurohormone prolactin (PRL) on keratin expression in a physiologically and clinically relevant test system: organ-cultured normal human hair follicles (HFs). Not only do HFs express a wide range of keratins, but they are also a source and target of PRL. Microarray analysis revealed that PRL differentially regulated a defined subset of keratins and keratin-associated proteins. Quantitative immunohistomorphometry and quantitative PCR confirmed that PRL up-regulated expression of keratins K5 and K14 and the epithelial stem cell-associated keratins K15 and K19 in organ-cultured HFs and/or isolated HF keratinocytes. PRL also up-regulated K15 promoter activity and K15 protein expression in situ, whereas it inhibited K6 and K31 expression. These regulatory effects were reversed by a pure competitive PRL receptor antagonist. Antagonist alone also modulated keratin expression, suggesting that "tonic stimulation" by endogenous PRL is required for normal expression levels of selected keratins. Therefore, our study identifies PRL as a major, clinically relevant, novel neuroendocrine regulator of both human keratin expression and human epithelial stem cell biology in situ.