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Background: Hair loss is linked to dysfunction of the growth (anagen), regression (catagen) and rest (telogen) phases of the hair follicle (HF) cycle. Aims: To evaluate the effects of a Silybum marianum extract (SME), manganese PCA (MnPCA), and a Lespedeza capitata extract (LCE) on markers of hair growth and anchorage in human follicle dermal papilla cells (HFDPCs), and to investigate the ability of a topical serum containing these active ingredients to improve HF growth in an ex vivo human scalp skin model. Methods: In HFDPCs, we assessed receptor tyrosine kinase phosphorylation and Wnt/β‐catenin pathway activation; quantified versican, vascular endothelial growth factor (VEGF) and Dickkopf‐1 (DDK1) secretion; and evaluated 5α‐reductase (5αR) activity. Using scalp skin biopsies from two female donors, we measured hair shaft elongation, analyzed hair matrix keratinocyte proliferation and apoptosis, and determined HF cycle stage and score. Results: Compared to untreated HFDPCs, SME upregulated phosphorylation of growth factor receptors (EGFR:1.9 × and PDGFR: 2.8 ×) and their downstream effectors (ERK, GSK3, Akt, and STAT: 1.2–2.0 ×); MnPCA enhanced versican (33.0 ×) and VEGF (3.3 ×) secretion, and stimulated the Wnt/β‐catenin pathway (+80%); and LCE reduced DKK1 secretion (−72%) and 5αR activity (dihydrotestosterone/testosterone ratio: −60%). Compared to untreated scalp skin biopsies, the serum enhanced hair shaft elongation (+102%), and significantly prolonged the anagen phase by improving hair cycle scores and stimulating hair matrix keratinocyte proliferation (+58%). Conclusions: SME, MnPCA, and LCE displayed complementary anti‐hair loss properties. The serum combining these active ingredients may be useful in hair loss treatment. [ABSTRACT FROM AUTHOR] more...

Abstract Background Stress is an important cause of skin disease, including hair loss. The hormonal response to stress is due to the HPA axis, which comprises hormones such as corticotropin releasing factor (CRF), adrenocorticotropic hormone (ACTH), and cortisol. Many reports have shown that CRF, a crucial stress hormone, inhibits hair growth and induces hair loss. However, the underlying mechanisms are still unclear. The aim of this study was to examine the effect of CRF on human dermal papilla cells (DPCs) as well as hair follicles and to investigate whether the HPA axis was established in cultured human DPCs. Results CRF inhibited hair shaft elongation and induced early catagen transition in human hair follicles. Hair follicle cells, both human DPCs and human ORSCs, expressed CRF and its receptors and responded to CRF. CRF inhibited the proliferation of human DPCs through cell cycle arrest at G2/M phase and induced the accumulation of reactive oxygen species (ROS). Anagen-related cytokine levels were downregulated in CRF-treated human DPCs. Interestingly, increases in proopiomelanocortin (POMC), ACTH, and cortisol were induced by CRF in human DPCs, and antagonists for the CRF receptor blocked the effects of this hormone. Conclusion The results of this study showed that stress can cause hair loss by acting through stress hormones. Additionally, these results suggested that a fully functional HPA axis exists in human DPCs and that CRF directly affects human DPCs as well as human hair follicles under stress conditions. more...

RNA synthesis can be detected by 5-ethynyl uridine (EU) incorporation and click chemistry. Despite identifying a fundamental functional process, this technique has yet to be widely applied to complex human tissue systems. By incorporating EU into human hair follicle (HF) organs cultured ex vivo, nascent RNA synthesis was detected in situ. EU differentially incorporated across the HF epithelium. Interestingly, RNA synthesis did not correlate with protein synthesis, proliferation or epithelial progenitor cell marker expression. By treating human HFs with the cytotoxic cell cycle inhibitor (R)-CR8, which inhibits transcriptional regulators CDK7 and CDK9, it was further shown that this technique can be used to sensitively detect changes in global RNA synthesis in situ. Together, this work delineates new insights into nascent RNA synthesis within a human (mini)- organ and describes a novel read-out parameter that will enrich future ex vivo human tissue research studies. more...

Background: Stress is an important cause of skin disease, including hair loss. The hormonal response to stress is due to the HPA axis, which comprises hormones such as corticotropin releasing factor (CRF), adrenocorticotropic hormone (ACTH), and cortisol. Many reports have shown that CRF, a crucial stress hormone, inhibits hair growth and induces hair loss. However, the underlying mechanisms are still unclear. The aim of this study was to examine the effect of CRF on human dermal papilla cells (DPCs) as well as hair follicles and to investigate whether the HPA axis was established in cultured human DPCs. Results: CRF inhibited hair shaft elongation and induced early catagen transition in human hair follicles. Hair follicle cells, both human DPCs and human ORSCs, expressed CRF and its receptors and responded to CRF. CRF inhibited the proliferation of human DPCs through cell cycle arrest at G2/M phase and induced the accumulation of reactive oxygen species (ROS). Anagen-related cytokine levels were downregulated in CRF-treated human DPCs. Interestingly, increases in proopiomelanocortin (POMC), ACTH, and cortisol were induced by CRF in human DPCs, and antagonists for the CRF receptor blocked the effects of this hormone. Conclusion: The results of this study showed that stress can cause hair loss by acting through stress hormones. Additionally, these results suggested that a fully functional HPA axis exists in human DPCs and that CRF directly affects human DPCs as well as human hair follicles under stress conditions. [ABSTRACT FROM AUTHOR] more...

Our previous studies showed that nestin-expressing hair follicle-associated-pluripotent (HAP) stem cells, which reside in the bulge area of the hair follicle, could restore injured nerve and spinal cord and differentiate into cardiac muscle cells. Here we transplanted mouse green fluorescent protein (GFP)-expressing HAP stem-cell colonies enclosed on polyvinylidene fluoride membranes (PFM) into the severed thoracic spinal cord of nude mice. After seven weeks of implantation, we found the differentiation of HAP stem cells into neurons and glial cells. Our results also showed that PFM-captured GFP-expressing HAP stem-cell colonies assisted complete reattachment of the thoracic spinal cord. Furthermore, our quantitative motor function analysis with the Basso Mouse Scale for Locomotion (BMS) score demonstrated a significant improvement in the implanted mice compared to non-implanted mice with a severed spinal cord. Our study also showed that it is easy to obtain HAP stem cells, they do not develop teratomas, and do not loose differentiation ability when cryopreserved. Collectively our results suggest that HAP stem cells could be a better source compared to induced pluripotent stem cells (iPS) or embryonic stem (ES) cells for regenerative medicine, specifically for spinal cord repair. [ABSTRACT FROM AUTHOR] more...

Human hair follicle (HF) growth and hair shaft formation require terminal differentiation-associated cell cycle arrest of highly proliferative matrix keratinocytes. However, the regulation of this complex event remains unknown. CIP/KIP family member proteins (p21 CIP1 , p27 KIP1 and p57 KIP2 ) regulate cell cycle progression/arrest, endoreplication, differentiation and apoptosis. Since they have not yet been adequately characterized in the human HF, we asked whether and where CIP/KIP proteins localise in the human hair matrix and pre-cortex in relation to cell cycle activity and HF-specific epithelial cell differentiation that is marked by keratin 85 (K85) protein expression. K85 expression coincided with loss or reduction in cell cycle activity markers, including in situ DNA synthesis (EdU incorporation), Ki-67, phospho-histone H3 and cyclins A and B1, affirming a post-mitotic state of pre-cortical HF keratinocytes. Expression of CIP/KIP proteins was found abundantly within the proliferative hair matrix, concomitant with a role in cell cycle checkpoint control. p21 CIP1 , p27 KIP1 and cyclin E persisted within post-mitotic keratinocytes of the pre-cortex, whereas p57 KIP2 protein decreased but became nuclear. These data imply a supportive role for CIP/KIP proteins in maintaining proliferative arrest, differentiation and anti-apoptotic pathways, promoting continuous hair bulb growth and hair shaft formation in anagen VI. Moreover, post-mitotic hair matrix regions contained cells with enlarged nuclei, and DNA in situ hybridisation showed cells that were >2N in the pre-cortex. This suggests that CIP/KIP proteins might counterbalance cyclin E to control further rounds of DNA replication in a cell population that has a propensity to become tetraploid. These data shed new light on the in situ -biography of human hair matrix keratinocytes on their path of active cell cycling, arrest and terminal differentiation, and showcase the human HF as an excellent, clinically relevant model system for cell cycle physiology research of human epithelial cells within their natural tissue habitat. [ABSTRACT FROM AUTHOR] more...

The aim of the present study was to investigate whether feeder layers composed of human hair follicle-derived mesenchymal stem cells (hHFDCs) are able to support human embryonic stem cells (hESCs). hHFDCs and mouse embryonic fibroblasts (MEFs) were isolated and cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 and low-glucose DMEM, respectively. hHFDCs were passaged three times and subsequently characterized. hHFDCs and MEFs were mitotically inactivated with mitomycin C for 3 h prior to co-culture with H9-hESCs. hESCs were initially established on a mouse feeder layer, subsequently transferred onto a human feeder layer and split every 5 days. Cell morphology, expression of specific 'undifferentiation' markers and growth factors, and the differentiation capacity of hESCs grown on the hHFDC feeder layer were analyzed. hHFDCs are adherent to plastic, possess the classic mesenchymal stem cell phenotype [they express cluster of differentiation (CD)90, CD73 and CD105] and are able to differentiate into adipocytes, chondroblasts and osteocytes, indicating that these cells are multipotent. Population-doubling time analysis revealed that hHFDCs rapidly proliferate over 34.5 h. As a feeder layer, hHFDC behaved similarly to MEF in maintaining the morphology of hESCs. The results of alkaline phosphatase activity, reverse transcription-quantitative polymerase chain reaction analysis of the expression of pluripotency transcription factors [octamer-binding transcription factor 4 (Oct4), Nanog and sex determining region Y-box 2], and immunofluorescence assays of markers (stage-specific embryonic antigen-4 and Oct4) in hESCs co-cultured over hHFDC, indicated that the undifferentiated state of hESCs was preserved. No change in the level of growth factor transcripts (bone morphogenetic protein 4, fibroblast growth factor-2, vascular endothelial growth factor, Pigment epithelium-derived factor and transforming growth factor-ß1) was detected for either feeder layer prior to or following inactivation. Similar phenotypes of embryoid body formation, size and morphology were observed in the hHFDC and MEF feeders. In conclusion, hHFDC maintained hESCs in an undifferentiated state comparable to MEF in standard conditions, which may be an important finding regarding the establishment of stem cell-based translational applications. [ABSTRACT FROM AUTHOR] more...

The cell cycle is of major importance to human hair follicle ( HF) biology. Not only is continuously active cell cycling required to facilitate healthy hair growth in anagen VI HFs, but perturbations in the cell cycle are likely to be of significance in HF pathology (i.e. in scarring, non-scarring, chemotherapy-induced and androgenic alopecias). However, cell cycle dynamics of the human hair follicle ( HF) are poorly understood in contrast to what is known in mouse. The current Methods Review aims at helping to close this gap by presenting a primer that introduces immunohistological/immunofluorescent techniques to study the cell cycle in the human HF. Moreover, this primer encourages the exploitation of the human HF as a powerful and clinically relevant tool to investigate mammalian cell cycle biology in situ. To achieve this, we describe methods to study markers of general 'proliferation' (nuclei count, Ki-67 expression), apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labelling, cleaved caspase 3), mitosis (phospho-histone H3, ' pS780'), DNA synthesis (5-ethynyl-2′-deoxyuridine) and cell cycle regulation (cyclins) in the human HF. In addition, we provide specific examples of dual immunolabelling for instructive cell cycle analyses and for investigating the cell cycle behaviour of specific HF keratinocyte subpopulations, such as keratin 15+ stem/progenitor cells. [ABSTRACT FROM AUTHOR] more...

The complex mechanical properties of biomaterials such as hair, horn, skin, or bone are determined by the architecture of the underlying fibrous bionetworks. Although much is known about the influence of the cytoskeleton on the mechanics of isolated cells, this has been less studied in tridimensional tissues. We used the hair follicle as a model to link changes in the keratin network composition and architecture to the mechanical properties of the nascent hair. We show using atomic force microscopy that the soft keratinocyte matrix at the base of the follicle stiffens by a factor of ∼360, from 30 kPa to 11 MPa along the first millimeter of the follicle. The early mechanical stiffening is concomitant to an increase in diameter of the keratin macrofibrils, their continuous compaction, and increasingly parallel orientation. The related stiffening of the material follows a power law, typical of the mechanics of nonthermal bending-dominated fiber networks. In addition, we used X-ray diffraction to monitor changes in the (supra)molecular organization within the keratin fibers. At later keratinization stages, the inner mechanical properties of the macrofibrils dominate the stiffening due to the progressive setting up of the cystine network. Our findings corroborate existing models on the sequence of biological and structural events during hair keratinization. [ABSTRACT FROM AUTHOR] more...

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 serumfree 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. [ABSTRACT FROM AUTHOR] more...

7-Phloroeckol, phloroglucinol derivative isolated from marine brown algae, has anti-oxidative, anti-inflammatory responses and MMP inhibitory activities. In this study, we evaluated the hair growth-promoting effects of 7-phloroeckol in human hair follicles. To investigate cell viability of human dermal papilla cells (DPCs) and outer root sheath (ORS) cells in the presence or absence of 7-phloroeckol treatment, MTT assay was employed. Moreover, gene expression and protein concentration of insulin-like growth factor (IGF)-1 was measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. 7-Phloroeckol induced an increase in proliferation of DPCs and ORS cells. In addition, hair shaft growth was measured using the hair-follicle organ culture system. 7-Phloroeckol resulted in elongation of the hair shaft in cultured human hair follicles. 7-Phloroeckol induced an IGF-1 mRNA expression and protein concentration in DPCs and conditioned media, respectively. These results suggest that 7-phloroeckol promotes hair growth through stimulation of DPCs and ORS cells. [ABSTRACT FROM AUTHOR] more...

Reprogramming of somatic cells into inducible pluripotent stem cells (iPSCs) provides an alternative to using embryonic stem cells (ESCs). Mesenchymal stem cells derived from human hair follicles (hHF-MSCs) are easily accessible, reproducible by direct plucking of human hairs. Whether these hHF-MSCs can be reprogrammed has not been previously reported. Here we report the generation of iPSCs from hHF-MSCs obtained by plucking several hairs. hHF-MSCs were isolated from hair follicle tissues and their mesenchymal nature confirmed by detecting cell surface antigens and multilineage differentiation potential towards adipocytes and osteoblasts. They were then reprogrammed into iPSCs by lentiviral transduction with Oct4, Sox2, c-Myc and Klf4. hHF-MSC-derived iPSCs appeared indistinguishable from human embryonic stem cells (hESCs) in colony morphology, expression of alkaline phosphotase, and expression of specific hESCs surface markers, SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, Nanog, Oct4, E-Cadherin and endogenous pluripotent genes. When injected into immunocompromised mice, hHF-MSC-derived iPSCs formed teratomas containing representatives of all three germ layers. This is the first study to report reprogramming of hHF-MSCs into iPSCs. [ABSTRACT FROM AUTHOR] more...

During the morphogenesis of hair follicles, the invasive migration of basal keratinocytes resembles cell’s dissemination of tissue remodeling. The urokinase-type plasminogen activator receptor (uPAR) appears to be a key molecule in the metastasis. In order to elucidate the relationship between uPAR and the invasion of the human hair follicle, immunohistochemistry, RT-PCR, plasmids transfection, and western blot were used. The results showed that uPAR was expressed in the outermost epithelial cells of the hair follicle and the basal keratinocytes of epidermis, and that the expression decreased with the development of the hair follicle. The cells of the outer root sheath (ORS) and interfollicle epidermis, which overexpressed uPAR, acquired increased invasiveness; however, they showed decreased invasion with overexpression of the urokinase-type plasminogen activator amino terminal fragment (uPA ATF), which inhibited the combination of uPAR and uPA competitively, and the cell invasive migration with overexpressed uPAR was required activated extracellular signal-regulated kinases (ERK). These results implied that overexpression of uPAR promote the invasive migration of hair follicle into the dermis in uPA-dependent and independent manner during human prenatal development. [ABSTRACT FROM AUTHOR] more...

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. [ABSTRACT FROM AUTHOR] more...

The article reports on the study done on the effects of α-melanocyte-stimulating hormone-related tripeptide K(D)PT on the hair follicles of human scalp under proinflammatory conditions. It mentions that the method is advantageous to these patients because it will mediate immune privilege (IP) and tolerance induction in the brain and eye. Moreover, it states that it also re-establishes major histocompatibility complex (MHC) class I-dependent IP in the human anagen of hair bulbs. more...

Background Keratin family proteins are generally accepted as being restricted to epithelial cells. However, several studies have challenged this paradigm by reporting, for example, that melanoma cells can express keratins and that normal human epidermal melanocytes, which derive from the neural crest, express keratin 16 (K16) in situ. Objectives We wished to confirm or refute that K16 and/or its intermediate filament partner, keratin 6 (K6), are expressed in normal human epidermal and/or hair follicle melanocytes in situ. Methods Cryosections of normal human scalp skin were subjected to highly sensitive double immunohistochemistry with specific antibodies against K16 or K6 and against the melanocyte-specific marker NKI/beteb (gp100). Immunoreactivity (IR) was visualized by conventional light microscopy and confocal fluorescence microscopy. Results Despite the use of different, high-sensitivity immunostaining methods, stringent positive and negative controls, and monospecific, well-characterized antikeratin antibodies, we could detect neither K16 nor K6 IR within intraepidermal or intrafollicular pigment cells of normal human scalp skin. Instead, NKI/beteb+ cells were found to be intimately embedded in foci of K16+ and/or K6+ keratinocytes, which might create the illusion of keratin expression by these cells. Conclusions Human epidermal or hair follicle melanocytes do not express K16 and/or K6 while residing in their natural habitat. [ABSTRACT FROM AUTHOR] more...

Background Hair follicle (HF) ostia represent a potential port of microbial entry into the skin. However, they rarely show clinical signs of infection. This suggests the presence of local, efficient, antimicrobial defence systems, which may include antimicrobial peptides (AMPs). Objectives We determined the presence and distribution of the major AMPs, RNase 7 and psoriasin (S100A7), in human scalp HFs. We investigated whether HF production of these AMPs was induced by prototypic microbial products and proinflammatory cytokines, i.e. interferon (IFN)-γ. Finally, we examined whether the classical pathways for AMP induction, such as toll-like receptor (TLR)4 and TLR5 expression, are present in human HFs and up-regulated after stimulation with bacterium-associated ligands. Methods Cryosections from fresh or organ-cultured full-thickness normal human scalp skin treated with lipopolysaccharide (LPS), flagellin, protein A, lipoteichoic acid (LTA) or IFN-γ were stained for psoriasin and RNase 7 immunoreactivity (IR) as well as for TLR4 and TLR5. In addition, outer root sheath cell culture and semiquantitative analysis of mRNA expression levels of RNase 7 and psoriasin were performed. Results Specific RNase 7 IR was present throughout the entire HF outer root sheath in situ and in cell culture, whereas psoriasin IR was present only in the most distal compartment and not detectable in cultured ORS cells. Upon treatment with Gram-positive (LTA, protein A) or Gram-negative bacterial (LPS, flagellin) cell wall components, or with the cytokine IFN-γ, the IR of both psoriasin and RNase 7 was modified. TLR4 and TLR5 IR was detected in the normal HF epithelium and were upregulated after treatment with their respective ligand. The mRNA analysis confirmed the immunohistochemistry results. Conclusions This pilot study suggests that normal human scalp HF epithelium possesses a functional antimicrobial defence system, which includes the AMPs RNase 7 and psoriasin, and TLRs, and that these are induced by classical microbial products. [ABSTRACT FROM AUTHOR] more...

Background α-Melanocyte stimulating hormone (α-MSH) is a well-tolerated immunomodulator with cytoprotective and anti-inflammatory effects that is known to stimulate melanogenesis and proliferation of follicular melanocytes. As human hair follicles (HFs) locally synthesize α-MSH, pharmacologically more easily handled α-MSH-related tripeptides, such as K(D)PT, may imitate this endogenous regulation, and may show a favourable side-effect profile on clinical use. Objectives To investigate the effect of the synthetic, α-MSH-related peptide K(D)PT [which is identical to interleukin (IL)-1β193–195] on melanogenesis in human anagen HFs, under normal and proinflammatory growth conditions. Methods Normal human anagen VI scalp HFs were microdissected and organ cultured with different concentrations of K(D)PT with or without coadministration of a proinflammatory, catagen-inducing stimulus, interferon (INF)-γ. Masson–Fontana histochemistry and NKI/beteb immunohistochemistry were employed to assess changes in the degree of human HF pigmentation and melanocyte dendricity. Results As confirmed by quantitative (immuno-)histomorphometry, compared with controls, K(D)PT alone did not affect human HF pigmentation in organ culture. However, in the presence of a strong, prototypic proinflammatory stimulus (IFN-γ), K(D)PT significantly stimulated HF melanin content and melanocyte dendrite formation in situ. Conclusions The IL-1β- and α-MSH-related tripeptide, K(D)PT, displays interesting hair pigmentation-stimulatory activities under proinflammatory conditions. These might become exploitable for innovative antigreying strategies, notably in postinflammatory poliosis (regrowth of white hair, e.g. during recovery from alopecia areata), where no effective clinical therapy is yet available. [ABSTRACT FROM AUTHOR] more...

Since the discovery of epithelial hair follicle stem cells (eHFSCs) in the bulge of human hair follicles (HFs) an important quest has started: to define useful markers. In the current study, we contribute to this by critically evaluating corresponding published immunoreactivity (IR) patterns, and by attempting to identify markers for the in situ identification of human eHFSCs and their niche. For this, human scalp skin cryosections of at least five different individuals were examined, employing standard immunohistology as well as increased sensitivity methods. Defined reference areas were compared by quantitative immunohistochemistry for the relative intensity of their specific IR. According to our experience, the most useful positive markers for human bulge cells turned out to be cytokeratin 15, cytokeratin 19 and CD200, but were not exclusive, while β1 integrin and Lhx2 IR were not upregulated by human bulge keratinocytes. Absent IR for CD34, connexin43 and nestin on human bulge cells may be exploited as negative markers. α6 integrin, fibronectin, nidogen, fibrillin-1 and latent transforming growth factor (TGF)-beta-binding protein-1 were expressed throughout the connective tissue sheath of human HFs. On the other hand, tenascin-C was upregulated in the bulge and may thus constitute a component of the bulge stem cell niche of human HFs. These immunophenotyping results shed further light on the in situ expression patterns of claimed follicular ‘stem cell markers’ and suggest that not a single marker alone but only the use of a limited corresponding panel of positive and negative markers may offer a reasonable and pragmatic compromise for identifying human bulge stem cells in situ. [ABSTRACT FROM AUTHOR] more...

Abstract: Integrins are transmembrane adhesion proteins that convey critical topobiological information and exert crucial signalling functions. In skin and hair follicle biology, β1 integrins and their ligands are of particular interest. It is not yet known whether β1 integrins play any role in the regulation of human hair growth and the expression pattern of β1 integrin in the human pilosebaceous unit remains ill-defined. Here, we show that pilosebaceous immunoreactivity for β1 integrin is most prominent in the outermost layer of the outer root sheath and the surrounding connective tissue sheath of human scalp hair follicles in situ and in vitro. Sites of β1 integrin immunoreactivity co-express fibronectin and tenascin-C. Contrary to previous reports, β1 integrin immunoreactivity in situ was not significantly upregulated in the human bulge region. Functionally, two β1 integrin-activating antibodies (12G10, TS2/16) and ligand-mimicking RGD peptides promoted the growth of microdissected, organ-cultured human scalp hair follicles in vitro and inhibited spontaneous hair follicle regression. This supports the concept that β1 integrin-mediated signalling is also important in human hair growth control. The physiologically relevant organ culture assay employed here is a potential research tool for exploring whether targeted stimulation of β1 integrin-mediated signalling is a suitable candidate for human hair loss management. [Copyright &y& Elsevier] more...

Recent articles on hair follicle stem cells have summarized the current state of knowledge of what has been termed the hair follicle ‘bulge’. During the course of immunohistological studies aimed at characterizing the expression of selected extracellular matrix proteins in the – as yet insufficiently characterized – niche of human bulge hair follicle stem cells, we have recently come across a largely forgotten, peculiar epithelial protrusion of the outer root sheath, which was visible in only a minority of all examined hair follicles. The morphology and immunoreactivity patterns of this structure, the ‘follicular trochanter’, are described herein. [ABSTRACT FROM AUTHOR] more...

We previously showed that the homeodomain protein HOXC13 is involved in the expression control of the early human hair keratin genes hHa5 and hHa2, which contain specific HOXC13 binding sites in their proximal promoters. Hox specificity is generally thought to be enhanced by the interaction with members of the TALE superclass of homeodomain proteins Pbx, Meis, and Prep. Using reverse transcription PCR with total human hair follicle RNA, we demonstrated transcripts of the major TALE proteins PBX1-4, MEIS1, 2 and PREP1, 2 in the human hair follicle. In view of the presence of MEIS/PREP responsive elements in close vicinity to the HOXC13 binding sites of the hHa5 and hHa2 promoters, we determined the expression sites of these TALE proteins in the human hair follicle. We found that MEIS1, MEIS2, PREP1 and PREP2 were differentially expressed in the three layers of the inner root sheath. In addition, MEIS2 and PREP1 exhibited expression in the mid-to upper hair cortex, with PREP1 being also expressed in the dermal papilla and the connective tissue sheath of the hair follicle. In virtually all cases, the expression of these TALE proteins was exclusively cytoplasmic. Considering that in contrast, HOXC13 is expressed in the nuclei of matrix, precortex and lower cuticle cells of the hair follicle, our data suggest that despite the presence of MEIS/PREP binding sites in the hHa5 and hHa2 promoters, the HOXC13-controlled activation of these genes in the hair follicle does not seem to involve these TALE proteins as cofactors. [ABSTRACT FROM AUTHOR] more...

Neurotrophin (NT)-3 and its high-affinity receptor tyrosine kinase C (Trk C) are essential for nervous system development. These members of the NT family are also involved in murine hair morphogenesis and cycling. However, their role in human hair follicle (HF) biology remains to be elucidated. To explore the role of NTs in human skin and HF biology. The immunoreactivity (IR) of NT-3 and Trk C was studied in human scalp skin and HFs by immunofluorescent and light microscopic immunohistology. Skin biopsies were obtained from normal human scalp containing mainly anagen VI HFs from women (age 53–57 years) undergoing elective plastic surgery. Both NT-3 and Trk C showed prominent, yet distinct, IR patterns in human scalp anagen HFs (anagen VI), whereas they were weakly expressed in catagen and increased again in telogen HFs. Within HF compartments, NT-3 IR was prominent in the outer root sheath, inner root sheath, dermal papilla and connective tissue sheath. Trk C IR was prominent in all HF epithelial and mesenchymal compartments. Outside the HF, both NT-3 and Trk C showed prominent IR in the epidermis, sebaceous glands and sweat glands. These observations provide the first indication that NT-3 and Trk C are expressed in human scalp skin and HFs, and suggest that Trk C-mediated signalling is involved not only in murine but also in human HF biology. They may be useful in determining therapeutic strategies for the treatment of hair cycle and skin-related disorders. [ABSTRACT FROM AUTHOR] more...

Abstract: The keratin family includes epithelial (soft) keratins and hair (hard) keratins, and can be divided into acidic type I and basic to neutral type II subfamilies. Recently, nine type I and six type II hair keratin genes have been characterized through the screening of a human PAC library. The expression of these genes in the hair follicle was determined in vivo and a combined catalog of acidic and basic hair keratins was established. In this study, we investigated the expression and localization of most of the human hair keratin members of both types in human hair grown in vitro . We show that in vitro growth of hair follicles for 10 days in complete William's E culture medium did not alter the expression pattern of hair keratins. Similarly to the in vivo situation, each hair keratin was localized in precise and discrete compartments of the follicle, ranging from the matrix to the upper cortex and/or the hair cuticle. This study shows that the increase in length of in vitro grown follicles was accompanied by the proper hair shaft keratinization process. It also shows that hair follicle integrity was maintained in vitro , both in terms of gross morphology and molecular organization despite the complexity of the keratin expression pattern. [ABSTRACT FROM AUTHOR] more...

In skin and hair research drug targeting to the hair follicle is of great interest. Therefore the influence of permeant lipophilicity and vehicle composition on local accumulation has been examined using confocal laser scanning microscopy (CLSM). Formulations saturated with either Oregon Green® 488, Bodipy® FL C5 or Bodipy® 564/570 C5 were prepared. The dyes were applied in citric acid buffer, 8% (w/v) surfactants in citric acid buffer or 8% (w/v) surfactants/20% (w/v) propylene glycol in citric acid buffer. Flow-through diffusion experiments were performed with fresh human scalp skin, after which the skin was imaged using CLSM. Diffusion studies showed for Oregon Green® 488 (low lipophilicity) a higher flux when applied in citric acid buffer compared to surfactants. In contrast the fluxes of the more lipophilic dyes (Bodipy® FL C5 and Bodipy® 564/570 C5) are highest when applied in surfactants/propylene glycol. CLSM studies revealed that follicular accumulation increased with (i) a lipophilic dye and (ii) application of lipophilic dyes in surfactants–propylene glycol. Therefore we conclude that targeting to the hair follicle can be increased by the use of lipophilic drugs in combination with surfactant solutions and propylene glycol. [Copyright &y& Elsevier] more...

Ultrastructural changes of the connective tissue sheath (CTS), including the hyaline membrane, of human hair follicles during the hair cycle, were studied in normal scalp skin specimens. In early anagen, the CTS was composed of a thin basal lamina and surrounding collagen tissue. The collagen tissue gradually thickened during the development of the hair and hair follicle. In mature anagen hair follicles, the collagen tissue was separated into three layers. The inner collagen layer, just outside the basal lamina, was thin and composed of collagen fibres running longitudinally parallel to the hair axis. The middle collagen layer was very thick with its collagen fibres running transversely against the hair axis and surrounding the inner hair tissue. Many fibroblasts were present among the collagen fibres in the middle layer, whereas the inner layer contained almost none. In the outer collagen layer, collagen fibres ran in various directions parallel to the outer surface of the outer root sheath cells. In late anagen, the basal lamina became very thick. In catagen, the basal lamine and the inner collagen layer became corrugated and showed oedematous change and degeneration. Surrounding fibroblasts showed active production of new collagen fibres, which seemed to fill the spaces left by the retraction of the hair follicle and hyaline membrane. These ultrastructural changes of the CTS show that there may be dynamic metabolic changes of the connective tissue around human hair follicles during the hair cycle. [ABSTRACT FROM AUTHOR] more...

Whole human scalp hair follicles were cultured. The follicles were dissected from skin pieces of normal scalp and put into 1.5 ml of incubation medium in a closed 5 ml glass tube under an atmosphere of 95% O and 5% CO. The tube was rolled at 15 rpm at 36‡C. Remarkable hair growth was noticed for 7 to 8 days. Hair root sheaths also grew with the hair shafts. The structure of the hair bulbs was well maintained for at least 6 days, and then the hair matrix cells started to degenerate. Fetal calf serum was not essential for hair growth in vitro, but increased the growth rate slowly. Testosterone and oestrogen inhibited hair growth in vitro to a similar extent. The minimum effective doses of both hormones to suppress hair growth were around 5 ng/ml, which corresponds well to the normal plasma level of testosterone in adult males in vivo, suggesting that scalp hair growth may be critically controlled by testosterone in adult males. [ABSTRACT FROM AUTHOR] more...

Reactive oxygen species (ROS) may severely affect the biochemical viability of most cells. However, ROS may act also as key second messengers regulating important physiological functions in eukaryotic organisms. Of special interest is the potential role of ROS in the regulation of stem cell function and tissue homeostasis and regeneration in adult mammalian tissues. In this context, the hair follicle constitutes an excellent experimental model to study this aspect of ROS biology.Here we present a robust protocol to promote a sustained growth of ex vivo cultured human hair follicles based on the induction of a transient/modulable production of nonlethal endogenous ROS levels in the tissue through a protoporphyrin IX-dependent photodynamic procedure. The light-switchable ROS production activates hair follicle stem cell niches, induces cell proliferation, and maintains the growth/anagen phase for long time. This approach constitutes a complementary experimental tool to study the physiological roles of ROS in human tissues. more...

Hair follicles are easily accessible skin appendages that protect against cold and potential injuries. Hair follicles contain various pools of stem cells, such as epithelial, melanocyte, and mesenchymal stem cells (MSCs) that continuously self-renew, differentiate, regulate hair growth, and maintain skin homeostasis. Recently, MSCs derived from the dermal papilla or dermal sheath of the human hair follicle have received attention because of their accessibility and broad differentiation potential. In this review, we describe the applications of human hair follicle-derived MSCs (hHF-MSCs) in tissue engineering and regenerative medicine. We have described protocols for isolating hHF-MSCs from human hair follicles and their culture condition in detail. We also summarize strategies for maintaining hHF-MSCs in a highly proliferative but undifferentiated state after repeated in vitro passages, including supplementation of growth factors, 3D suspension culture technology, and 3D aggregates of MSCs. In addition, we report the potential of hHF-MSCs in obtaining induced smooth muscle cells and tissue-engineered blood vessels, regenerated hair follicles, induced red blood cells, and induced pluripotent stem cells. In summary, the abundance, convenient accessibility, and broad differentiation potential make hHF-MSCs an ideal seed cell source of regenerative medical and cell therapy., Competing Interests: Conflict-of-interest statement: Liu JY reports grants from the China National Natural Science Foundation, grants from the Joint Construction Project between Jilin Province and Provincial Colleges, grants from the Jilin Province Science and Technology Development Plan, grants from the Innovative Special Industry Fund Project in Jilin Province, grants from the Open Research Project of the State Key Laboratory of Industrial Control Technology, Zhejiang University, China, during the conduct of the study., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.) more...

Glycosaminoglycans (GAGs) and associated proteoglycans have important functions in homeostatic maintenance and regenerative processes (e.g., wound repair) of the skin. However, little is known about the role of these molecules in the regulation of the hair follicle cycle. Here we report that growing human hair follicles ex vivo in a defined GAG hydrogel mimicking the dermal matrix strongly promotes sustained cell survival and maintenance of a highly proliferative phenotype in the hair bulb and suprabulbar regions. This significant effect is associated with the activation of WNT/β-catenin signaling targets (CCDN1, AXIN2) and with the expression of stem cell markers (CK15, CD34) and growth factors implicated in the telogen/anagen transition (TGFβ2, FGF10). As a whole, these results point to the dermal GAG matrix as an important component in the regulation of the human hair follicle growth cycle, and to GAG-based hydrogels as potentially relevant modulators of this process both in vitro and in vivo. [ABSTRACT FROM AUTHOR] more...

The article focuses on a study related to the use of spinning-disk microscopy as a complementary approach for studying epithelial precursor cells of human hair follicles (hHFs). Topics discussed include benefits of using laser scanning confocal microscopy for imaging of thick samples in three-dimensional as compared to widefield microscopy, recording immunofluorescence to avoid any cell population loss, and options provided by the approach such as spectral analysis and photon counting. more...

The culture of microdissected hair follicles (HFs) and scalp skin enriched in terminal HFs are the best currently available preclinical assays for studying hair and skin biology/pathology in the human system. While microdissected HF organ culture only allows the testing of compounds added into the culture medium, mimicking a systemic application, the scalp skin organ culture also is suitable to test topical and intradermal applications. Here, we describe different methods for isolation of human scalp HFs, the procedures for culturing the scalp skin and microdissected HFs and we also outline different delivery techniques (e.g., topical, systemic) to test active and control substances. more...

Background: Klotho 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., Objective: The purpose of this study was to determine the expression pattern and role of klotho in human hair follicles., Methods: We 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., Results: Interestingly, 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., Conclusion: These results indicate that klotho might be an important regulatory factor for human hair growth and hair cycle change., Competing Interests: CONFLICTS OF INTEREST: The authors have nothing to disclose., (Copyright © 2019 The Korean Dermatological Association and The Korean Society for Investigative Dermatology.) more...