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Клетки дермальной папиллы волосяного фолликула человека в условиях культивирования

Журнальные статьи

1. Ahn S.-Y. et al. Effect of IGF-I on Hair Growth Is Related to the Anti-Apoptotic Effect of IGF-I and Up-Regulation of PDGF-A and PDGF-B // Ann. Dermatol. 2012. Vol. 24, № 1. P. 26–31.

Background: Insulin-like growth factor-I (IGF-I) shares a high degree of structural and functional homology with insulin and is a potent mitogen supporting cell growth and survival in many kinds of the tissues and cells. It also plays a role in some differentiation and anti-apoptotic functions. In previous reports, it has been shown that IGF-I stimulates hair follicle (HF) growth, maintains the anagen stage, and postpones the catagen stage. Objective: The exact mechanism of the effect of IGF-I on HF growth is not yet established. Therefore, we investigated the relationships between IGF-I and various other factors (i.e. apoptosis related molecules, pro-inflammatory cytokines, other growth factors, etc.) in the control of HF growth. Methods: The effect of IGF-I on human hair growth was measured using an organ culture model of human HFs and compared with a control group that did not receive IGF-I. We also measured mRNA expression of factors related to hair growth and apoptosis (which was determined by reverse transcription polymerase chain reaction (RT-PCR). RT-PCR was done on days 2, 4, 6, and 8 of organ culture. Results: In organ cultured human hair follicles, IGF-I had a positive effect on the rate of linear hair growth. IGF-I maintained the anagen phase. IGF-I increased the expression of platelet-derived growth factor (PDGF)-A, PDGF-B and the expression ratio of Bcl-2/Bax. Conclusion: The effect of IGF-I on hair growth appears to be related to the upregulation of PDGF-A and PDGF-B and to the anti-apoptotic effect of IGF-I. (Ann Dermatol 24(1) 26 similar to 31, 2012)

https://doi.org/10.5021/ad.2012.24.1.26

2. Aljuffali I.A. et al. Squarticles as a Lipid Nanocarrier for Delivering Diphencyprone and Minoxidil to Hair Follicles and Human Dermal Papilla Cells // AAPS J. 2014. Vol. 16, № 1. P. 140–150.

Delivery of diphencyprone (DPCP) and minoxidil to hair follicles and related cells is important in the treatment of alopecia. Here we report the development of "squarticles," nanoparticles formed from sebum-derived lipids such as squalene and fatty esters, for use in achieving targeted drug delivery to the follicles. Two different nanosystems, nanostructured lipid carriers (NLC) and nanoemulsions (NE), were prepared. The physicochemical properties of squarticles, including size, zeta potential, drug encapsulation efficiency, and drug release, were examined. Squarticles were compared to a free control solution with respect to skin absorption, follicular accumulation, and dermal papilla cell targeting. The particle size of the NLC type was 177 nm; that of the NE type was 194 nm. Approximately 80% of DPCP and 60% of minoxidil were entrapped into squarticles. An improved drug deposition in the skin was observed in the in vitro absorption test. Compared to the free control, the squarticles reduced minoxidil penetration through the skin. This may indicate a minimized absorption into systemic circulation. Follicular uptake by squarticles was 2- and 7-fold higher for DPCP and minoxidil respectively compared to the free control. Fluorescence and confocal images of the skin confirmed a great accumulation of squarticles in the follicles and the deeper skin strata. Vascular endothelial growth factor expression in dermal papilla cells was significantly upregulated after the loading of minoxidil into the squarticles. In vitro papilla cell viability and in vivo skin irritancy tests in nude mice suggested a good tolerability of squarticles to skin. Squarticles provide a promising nanocarrier for topical delivery of DPCP and minoxidil.

https://doi.org/10.1208/s12248-013-9550-y

3. Al-Nuaimi Y. et al. A Meeting of Two Chronobiological Systems: Circadian Proteins Period1 and BMAL1 Modulate the Human Hair Cycle Clock // J. Invest. Dermatol. 2014. Vol. 134, № 3. P. 610–619.

The hair follicle (HF) is a continuously remodeled mini organ that cycles between growth (anagen), regression (catagen), and relative quiescence (telogen). As the anagen-to-catagen transformation of microdissected human scalp HFs can be observed in organ culture, it permits the study of the unknown controls of autonomous, rhythmic tissue remodeling of the HF, which intersects developmental, chronobiological, and growth-regulatory mechanisms. The hypothesis that the peripheral clock system is involved in hair cycle control, i.e., the anagen-to-catagen transformation, was tested. Here we show that in the absence of central clock influences, isolated, organ-cultured human HFs show circadian changes in the gene and protein expression of core clock genes (CLOCK, BMAL1, and Period1) and clock-controlled genes (c-Myc, NR1D1, and CDKN1A), with Period1 expression being hair cycle dependent. Knockdown of either BMAL1 or Period1 in human anagen HFs significantly prolonged anagen. This provides evidence that peripheral core clock genes modulate human HF cycling and are an integral component of the human hair cycle clock. Specifically, our study identifies BMAL1 and Period1 as potential therapeutic targets for modulating human hair growth.

https://doi.org/10.1038/jid.2013.366

4. Amoh Y. et al. Nestin-positive hair follicle pluripotent stem cells can promote regeneration of impinged peripheral nerve injury // J. Dermatol. 2012. Vol. 39, № 1. P. 33–38.

Nestin-positive, keratin 15 (K15)-negative multipotent hair follicle stem cells are located above the hair follicle bulge. We have termed this location the hair follicle pluripotent stem cell area. We have previously shown that transplantation of nestin-expressing hair follicle stem cells can regenerate peripheral nerve and spinal cord injuries. In the present study, we regenerated the impinged sciatic nerve by transplanting hair follicle pluripotent stem cells. Human hair follicle stem cells were transplanted around the impinged sciatic nerve of ICR nude (nu/nu) mice. The hair follicle stem cells were transplanted between impinged sciatic nerve fragments of the mouse where they differentiated into glial fibrillary acidic protein-positive Schwann cells and promoted the recovery of pre-existing axons. The regenerated sciatic nerve functionally recovered. These multipotent hair follicle stem cells thereby provide a potential accessible, autologous source of stem cells for regeneration therapy of nerves degenerated by compression between bony or other hard surfaces.

https://doi.org/10.1111/j.1346-8138.2011.01413.x

5. Bassino E. et al. Effects of the biomimetic peptide Sh-Polypeptide 9 (CG-VEGF) on cocultures of human hair follicle dermal papilla cells and microvascular endothelial cells // Exp. Dermatol. 2016. Vol. 25, № 3. P. 237–239.

https://doi.org/10.1111/exd.12906

6. Bassino E. et al. Paracrine crosstalk between human hair follicle dermal papilla cells and microvascular endothelial cells // Exp. Dermatol. 2015. Vol. 24, № 5. P. 388–390.

Human follicle dermal papilla cells (FDPC) are a specialized population of mesenchymal cells located in the skin. They regulate hair follicle (HF) development and growth, and represent a reservoir of multipotent stem cells. Growing evidence supports the hypothesis that HF cycling is associated with vascular remodeling. Follicular keratinocytes release vascular endothelial growth factor (VEGF) that sustains perifollicular angiogenesis leading to an increase of follicle and hair size. Furthermore, several human diseases characterized by hair loss, including Androgenetic Alopecia, exhibit alterations of skin vasculature. However, the molecular mechanisms underlying HF vascularization remain largely unknown. In vitro coculture approaches can be successfully employed to greatly improve our knowledge and shed more light on this issue. Here we used Transwell-based co-cultures to show that FDPC promote survival, proliferation and tubulogenesis of human microvascular endothelial cells (HMVEC) more efficiently than fibroblasts. Accordingly, FDPC enhance the endothelial release of VEGF and IGF-1, two well-known proangiogenic growth factors. Collectively, our data suggest a key role of papilla cells in vascular remodeling of the hair follicle.

https://doi.org/10.1111/exd.12670

7. Bertolini M. et al. Vasoactive intestinal peptide, whose receptor-mediated signalling may be defective in alopecia areata, provides protection from hair follicle immune privilege collapse // Br. J. Dermatol. 2016. Vol. 175, № 3. P. 531–541.

BackgroundAlopecia areata (AA) is an autoimmune disorder whose pathogenesis involves the collapse of the relative immune privilege (IP) of the hair follicle (HF). Given that vasoactive intestinal peptide (VIP) is an immunoinhibitory neuropeptide released by perifollicular sensory nerve fibres, which play a role in IP maintenance, it may modulate human HF-IP and thus be therapeutically relevant for AA. ObjectivesTo answer the following questions: Do human HFs express VIP receptors, and does their stimulation protect from or restore experimentally induced HF-IP collapse? Is VIP signalling defective in AA HFs? MethodsFirstly, VIP and VIP receptor (VPAC1, VPAC2) expression in human scalp HFs and AA skin was assessed. In HF organ culture, we then explored whether VIP treatment can restore and/or protect from interferon--induced HF-IP collapse, assessing the expression of the key IP markers by quantitative (immuno-)histomorphometry. ResultsHere we provide the first evidence that VIP receptors are expressed in the epithelium of healthy human HFs at the gene and protein level. Furthermore, VIP receptor protein expression, but not VIP+ nerve fibres, is significantly downregulated in lesional hair bulbs of patients with AA, suggesting defects in VIP receptor-mediated signalling. Moreover, we show that VIP protects the HF from experimentally induced IP collapse invitro, but does not fully restore it once collapsed. ConclusionsThese pilot data suggest that insufficient VIP receptor-mediated signalling may contribute to impairing HF-IP in patients with AA, and that VIP is a promising candidate HF-IP guardian' that may be therapeutically exploited to inhibit the progression of AA lesions.

https://doi.org/10.1111/bjd.14645

8. Bhogal R.K. et al. Protease activity, localization and inhibition in the human hair follicle // Int. J. Cosmetic Sci. 2014. Vol. 36, № 1. P. 46–53.

ObjectiveIn humans, the process of hair shedding, referred to as exogen, is believed to occur independently of the other hair cycle phases. Although the actual mechanisms involved in hair shedding are not fully known, it has been hypothesized that the processes leading to the final step of hair shedding may be driven by proteases and/or protease inhibitor activity. In this study, we investigated the presence of proteases and protease activity in naturally shed human hairs and assessed enzyme inhibition activity of test materials. MethodsWe measured enzyme activity using a fluorescence-based assay and protein localization by indirect immunohistochemistry (IHC). We also developed an ex vivo skin model for measuring the force required to pull hair fibres from skin. ResultsOur data demonstrate the presence of protease activity in the tissue material surrounding club roots. We also demonstrated the localization of specific serine protease protein expression in human hair follicle by IHC. These data provide evidence demonstrating the presence of proteases around the hair club roots, which may play a role during exogen. We further tested the hypothesis that a novel protease inhibitor system (combination of Trichogen((R)) and climbazole) could inhibit protease activity in hair fibre club root extracts collected from a range of ethnic groups (UK, Brazil, China, first-generation Mexicans in the USA, Thailand and Turkey) in both males and females. Furthermore, we demonstrated that this combination is capable of increasing the force required to remove hair in an ex vivo skin model system. ConclusionThese studies indicate the presence of proteolytic activity in the tissue surrounding the human hair club root and show that it is possible to inhibit this activity with a combination of Trichogen((R)) and climbazole. This technology may have potential to reduce excessive hair shedding.

https://doi.org/10.1111/ics.12091

9. Boehm M. et al. alpha-Melanocyte-stimulating hormone: a protective peptide against chemotherapy-induced hair follicle damage? // Br. J. Dermatol. 2014. Vol. 170, № 4. P. 956–960.

Background Effective, safe and well-tolerated therapeutic and/or preventive regimens for chemotherapy-induced alopecia (CIA) still remain to be developed. Because alpha-melanocyte-stimulating hormone (alpha-MSH) exerts a number of cytoprotective effects and is well tolerated, we hypothesized that it may be a candidate CIA-protective agent. Objectives To explore, using a human in vitro model for chemotherapy-induced hair follicle (HF) dystrophy that employs the key cyclophosphamide metabolite (4-hydroperoxy- cyclophosphamide, 4-HC), whether alpha-MSH protects from 4-HC-induced HF dystrophy. Methods Microdissected human scalp HFs from four individuals were treated with 4-HC, alpha-MSH and 4-HC plus alpha-MSH. Changes in HF cycling, melanin distribution and hair matrix keratinocyte proliferation/apoptosis were examined by quantitative (immune-) morphometry. Expression of the cytoprotective enzyme haem oxygenase-1 (HO-1) was determined by real-time reverse transcriptase-polymerase chain reaction in HF of two individuals. Results In 50% of the individuals alpha-MSH reduced melanin clumping as an early sign of 4-HC-induced disruption of follicular pigmentation. a-MSH reduced 4- HC-induced apoptosis in the HFs of one female patient. These protective effects of alpha-MSH were not associated with changes in 4-HC-induced catagen induction. alpha-MSH and 4-HC both increased HO-1 mRNA expression, while the combination of both agents had additive effects on HO-1 transcription. Conclusions Exogenous alpha-MSH exerts moderate HF-protective effects against 4-HC-induced human scalp HF damage and upregulates the intrafollicular expression of a key cytoprotective enzyme. However, as substantial interindividual response variations were found, further studies are needed to probe alpha-MSH as a candidate CIA-protective agent.

https://doi.org/10.1111/bjd.12759

10. Buffoli B. et al. The human hair: from anatomy to physiology // Int. J. Dermatol. 2014. Vol. 53, № 3. P. 331–341.

Background Hair is a unique character of mammals and has several functions, from protection of the skin to sexual and social communication. In literature, there are various studies about hair that take into consideration different aspects within many fields of science, including biology, dermatology, cosmetics, forensic sciences, and medicine. Methods We carried out a search of studies published in PubMed up to 2013. Results In this review, we summarized the principal anatomical and physiological aspects of the different types of human hair, and we considered the clinical significance of the different structures and the distribution of the hair in the human body. Conclusion This review could be the basis for improvement and progression in the field of hair research.

https://doi.org/10.1111/ijd.12362

11. Buscone S. et al. A New Path in Defining Light Parameters for Hair Growth: Discovery and Modulation of Photoreceptors in Human Hair Follicle // Lasers Surg. Med. 2017. Vol. 49, № 7. P. 705–718.

Background and Objective: Though devices for hair growth based on low levels of light have shown encouraging results, further improvements of their efficacy is impeded by a lack of knowledge on the exact molecular targets that mediate physiological response in skin and hair follicle. The aim of this study was to investigate the expression of selected light-sensitive receptors in the human hair follicle and to study the impact of UV-free blue light on hair growth ex vivo. Material and Methods: The expression of Opsin receptors in human skin and hair follicles has been characterized using RT-qPCR and immunofluorescence approaches. The functional significance of Opsin 3 was assessed by silencing its expression in the hair follicle cells followed by a transcriptomic profiling. Proprietary LED-based devices emitting two discrete visible wavelengths were used to access the effects of selected optical parameters on hair growth ex vivo and outer root sheath cells in vitro. Results: The expression of OPN2 (Rhodopsin) and OPN3 (Panopsin, Encephalopsin) was detected in the distinct compartments of skin and anagen hair follicle. Treatmentwith 3.2 J/cm(2) of blue light with 453 nm central wavelength significantly prolonged anagen phase in hair follicles ex vivo that was correlated with sustained proliferation in the light-treated samples. In contrast, hair follicle treatment with 3.2 J/cm(2) of 689nmlight (red light) did not significantly affect hair growth ex vivo. Silencing of OPN3 in the hair follicle outer root sheath cells resulted in the altered expression of genes involved in the control of proliferation and apoptosis, and abrogated stimulatory effects of blue light (3.2 J/cm(2); 453nm) on proliferation in the outer root sheath cells. Conclusions: We provide the first evidence that (i) OPN2 and OPN3 are expressed in human hair follicle, and (ii) A 453nm blue light at low radiant exposure exerts a positive effect on hair growth ex vivo, potentially via interaction with OPN3. (C) 2017 Wiley Periodicals, Inc.

https://doi.org/10.1002/lsm.22673

12. Cerqueira M.T. et al. Human Adipose Stem Cells Cell Sheet Constructs Impact Epidermal Morphogenesis in Full-Thickness Excisional Wounds // Biomacromolecules. 2013. Vol. 14, № 11. P. 3997–4008.

Among the wide range of strategies to target skin repair/regeneration, tissue engineering (TE) with stem cells at the forefront, remains as the most promising route. Cell sheet (CS) engineering is herein proposed, taking advantage of particular cell-cell and cell-extracellular matrix (ECM) interactions and subsequent cellular milieu, to create 3D TE constructs to promote full-thickness skin wound regeneration. Human adipose derived stem cells (hASCs) CS were obtained within five days using both thermoresponsive and standard cell culture surfaces. hASCs-based constructs were then built by superimposing three CS and transplanted into full-thickness excisional mice skin wounds with delayed healing. Constructs obtained using thermoresponsive surfaces were more stable than the ones from standard cell culture surfaces due to the natural adhesive character of the respective CS. Both CS-generating strategies lead to prolonged hASCs engraftment, although no transdifferentiation phenomena were observed. Moreover, our findings suggest that the transplanted hASCs might be promoting neotissue vascularization and extensively influencing epidermal morphogenesis, mainly through paracrine actions with the resident cells. The thicker epidermis, with a higher degree of maturation characterized by the presence of rete ridges-like structures, as well as a significant number of hair follicles observed after transplantation of the constructs combining the CS obtained from the thermoresponsive surfaces, reinforced the assumptions of the influence of the transplanted hASCs and the importance of the higher stability of these constructs promoted by cohesive cell-cell and cell-ECM interactions. Overall, this study confirmed the potential of hASCs CS-based constructs to treat full-thickness excisional skin wounds and that their fabrication conditions impact different aspects of skin regeneration, such as neovascularisation, but mainly epidermal morphogenesis.

https://doi.org/10.1021/bm4011062

13. Danby F.W. et al. Preliminary findings suggesthidradenitis suppurativa may be due to defective follicular support // Br. J. Dermatol. 2013. Vol. 168, № 5. P. 1034–1039.

Background The initial pathology in hidradenitis suppurativa (HS)/acne inversa takes place in the folliculopilosebaceous unit (FPSU) and its surrounding tissue. The process involves follicular hyperkeratosis, inflammation and perifolliculitis. Identification of the exact origin of inflammation may shed new light on the pathogenesis and aetiology of the disease. Objectives To study the morphology of the basement membrane zone (BMZ) in patients with HS. Methods In total, 65 operative specimens from 20 patients diagnosed with HS were cut stepwise. Within each specimen, the focus was set on heavily involved HS regions (centre) and clinically uninvolved regions (border). All specimens were stained with periodic acidSchiff (PAS) to visualize the epithelial support structures of the FPSU (i.e. the BMZ), the sinus tracts (STs) and the interfollicular basement membrane (BM). The intensity of BMZ PAS staining was graded from 0 to 4+. Results Compared with the axillary skin of human controls, the sebofollicular junction in patients with HS was found to be almost devoid of PAS-positive material (grade 0/1+) in both the border and centre lesions of HS, whereas STs and BMs showed uniformly grade 23+ positivity irrespective of any inflammation present. The distribution of inflammatory cells around the sebofollicular junction occurred predominantly in areas of BMZ thinning. Conclusions The BMZ PAS positivity of clinically uninvolved FPSUs of patients with HS appears to be wispy or not present at all. It is speculated that this may explain the apparent fragility of the sebofollicular junction. There is an increased concentration of inflammatory cells adjacent to these areas, while inflammatory cells are scarce in areas where the PAS-positive material is intact. It is hypothesized that the PAS gap identifies (i) areas susceptible to leakage, trauma and rupture, leading to release of materials that trigger inflammatory mediators, and (ii) the seeding of the dermis with free-living stem cells generating benign but invasive epithelialized sinuses, spreading horizontally in and below the dermis.

https://doi.org/10.1111/bjd.12233

14. Dimitrov A. et al. Spinning-disk microscopy: a complementary approach to study epithelial precursor cells in dissected human hair follicles // Exp. Dermatol. 2016. Vol. 25, № 8. P. 644–645.

https://doi.org/10.1111/exd.13048

15. Fedorovska M.I., Polovko N.P., Antymis O.V. THE STUDY OF THE FOLLICLE STIMULATING ACTION OF THE CREAM WITH PLANT SUBSTANCES // Клінічна фармація. 2017. № 3. С. 35-40.

Одним из важных подходов к терапии АА является применение сосудорасширяющих средств природного и синтетического происхождения. Цель исследования. Изучение фолликулостимулирующей и вазодилатирующей активности крема с экстрактом пальмы сабаль и настойкой софоры японской. Материалы и методы. Фармакологические исследования проводили на взрослых крысах-самцах, в которых стимулировали облысение путем перорального введения (5 мг/кг) борной кислоты в течение 14 суток. Разработанный крем и препарат сравнения (Аллотон спрей) наносили на выстриженный участок спины в течение следующих 14 суток, при этом проводили контроль длины шерсти. В конце сравнивали массу выстриженной новой шерсти и микроскопически определяли процент дистрофических волосков. Рандомизированно осуществляли биопсию кожи и в полученных образцах методами гистологического анализа оценивали сосудистое русло, плотность волосяных фолликул. Результаты. Как показало исследование, разработанный крем по сравнению с группой контроля (нелеченые животные) ускорял восстановление роста шерсти крыс, улучшал ее качество за счет увеличения массы и уменьшения процента дистрофических волос. Применение разработанного средства стимулировало расширение кровеносных сосудов подсосочковой и дермальной сетей. Усиление кровоснабжения сопровождалось ростом количества мастоцитов за счет их дегрануляции. Наблюдалось увеличение толщины эпидермиса, увеличение количества волосяных фолликулов (в стадии анагена их находилось 89 %) и их активная регенерация. Выводы. Таким образом, разработанный крем с экстрактом пальмы сабаль и настойкой софоры японской при местном применении оказывает сосудорасширяющее и фолликулостимулирующего действие.

https://doi.org/10.24959/cphj.17.1429

16. Fernandes B. et al. Improved Poly (D,L-lactide) nanoparticles-based formulation for hair follicle targeting // Int. J. Cosmetic Sci. 2015. Vol. 37, № 3. P. 282–290.

Synopsis ObjectiveHair follicles are widely recognized as the preferential target and site of accumulation for nanoparticles after topical application. This feature is of particular importance for hair cosmetics, having the potential to refine the treatment of several hair follicle-related disorders. The aim of this work was to improve the preparation of Poly (D,L-lactide) (PLA) nanoparticles for in vivo follicular target and drug delivery. MethodsEnvisaging a future industrial scale-up of the process, nanoprecipitation method was used to prepare PLA nanoparticles: the effect of several processing parameters on their properties was examined and the yield of nanoparticles formation determined. Encapsulation efficiencies and in vitro release profiles of lipophilic and hydrophilic model compounds were also assessed. In vitro cytotoxicity and ex vivo penetration studies were performed on a reference skin cell line (NCTC2455, human skin keratinocytes) and porcine skin, respectively. ResultsUsing acetone:ethanol (50:50, v/v) as the solvent phase, 0.6% (w/w) of Pluronic((R)) F68 as a surfactant agent and agitation to mix the solvent and non-solvent phases, a monodispersed population of non-cytotoxic spherical nanoparticles of approximately 150nm was obtained. The yield of nanoparticles for this formulation was roughly 90%. After encapsulation of model compounds, no significant changes were found in the properties of particles and the entrapment efficiencies were above 80%. The release kinetics of dyes from PLA nanoparticles indicate an anomalous transport mechanism (diffusion and polymer degradation) for Nile Red (lipophilic) and a Fickian diffusion of first order for fluorescein 5(6)-isothiocyanate (hydrophilic). Ex vivo skin penetration studies confirmed the presence of nanoparticles along the entire follicular ducts. ConclusionsThe optimized method allows the preparation of ideal PLA nanoparticles-based formulations for hair follicle targeting. PLA nanoparticles can effectively transport and release lipophilic and hydrophilic compounds into the hair follicles, and the yields obtained are acceptable for industrial purposes. Resume ObjectifLes follicules pileux sont largement reconnus comme la cible preferentielle et le site de l'accumulation des nanoparticules apres application topique. Cette caracteristique est particulierement importante pour les produits cosmetiques pour les cheveux, ayant la possibilite d'affiner le traitement de plusieurs troubles des follicules de cheveux. Le but de ce travail etait d'ameliorer la preparation de nanoparticules poly (D,L-lactide) (PLA) pour une administration folliculaire in vivo ciblee de drogues. MethodesEn envisageant un avenir a l'echelle industrielle du procede, une methode de nanoprecipitation a ete utilise pour preparer des nanoparticules de PLA: l'effet de plusieurs parametres de traitement sur leurs proprietes a ete examine et le rendement de la formation des nanoparticules a ete determine. Les efficacites d'encapsulation et de profils de liberation in vitro de composes modeles lipophiles et hydrophiles ont egalement ete evaluees. La cytotoxicite in vitro et ex vivo des etudes de penetration a ete effectuee sur une lignee de cellules de peau de reference (NCTC2455, des keratinocytes de peau humaine) et la peau de porc, respectivement. ResultatsEn utilisant l'acetone:ethanol (50:50, v/v) comme phase solvant, 0,6% (p/p) de Pluronic (R) F68 a titre d'agent tensioactif et l'agitation pour melanger les phases de solvant et de non-solvant, une population monodispersee des nanoparticules spheriques non cytotoxiques d'environ 150 nm a ete obtenue. Le rendement de nanoparticules pour cette formulation etait d'environ 90%. Apres encapsulation de composes modeles, aucune modification significative n'a ete observee dans les proprietes des particules et les efficacites de piegeage ont ete superieures a 80%. La cinetique de liberation de colorants de nanoparticules de PLA indique un mecanisme de tr

https://doi.org/10.1111/ics.12197

17. Fernandez Flores A. et al. Expression of connexin 43 in the human hair follicle: emphasis on the connexin 43 protein levels in the bulge and through the keratinization process // J. Cutan. Pathol. 2018. Vol. 45, № 1. P. 8–15.

Background: Gap junctions form communication compartments between cells. These channels assemble from connexin subunits. Objective: To investigate the immunoexpression of connexin 43 (Cx43) in adult human hair follicles. Methods: Cases were retrospectively obtained from our archives. Results: We identified immunoexpression of Cx43 in the matrix, the papilla, the outer root sheath, the bulge, the medulla, the cortex, the shaft and the secretory part of the sebaceous gland. There was very low expression (VLE) of Cx43 in the perifollicular sheath, the mantle and the arrector pili muscle. The internal root sheath showed high-density expression in the bulb. Such expression abruptly decreased at different points in each of its layers at the point of keratinization. The isthmus showed Cx43-positive staining in the middle layers and all along, whereas there was VLE in the two outermost layers. The infundibulum showed expression all along the middle layers, whereas it showed VLE in the 2 outermost layers and in the 2 or 3 innermost layers. Conclusions: The bulge contains Cx43. Our results suggest that keratinization in the hair follicle is closely related to the decrease in Cx43 expression.

https://doi.org/10.1111/cup.13050

18. Fischer T.W. et al. Differential effects of caffeine on hair shaft elongation, matrix and outer root sheath keratinocyte proliferation, and transforming growth factor-beta 2/insulin-like growth factor-1-mediated regulation of the hair cycle in male and female human hair follicles in vitro // Br. J. Dermatol. 2014. Vol. 171, № 5. P. 1031–1043.

Background Caffeine reportedly counteracts the suppression of hair shaft production by testosterone in organ-cultured male human hair follicles (HFs). Objectives We aimed to investigate the impact of caffeine (i) on additional key hair growth parameters, (ii) on major hair growth regulatory factors and (iii) on male vs. female HFs in the presence of testosterone. Methods Microdissected male and female human scalp HFs were treated in serum-free organ culture for 120 h with testosterone alone (0.5 mu g mL(-1)) or in combination with caffeine (0.005-0.0005%). The following effects on hair shaft elongation were evaluated by quantitative (immuno)histomorphometry: HF cycling (anagen-catagen transition); hair matrix keratinocyte proliferation; expression of a key catagen inducer, transforming growth factor (TGF)-beta 2; and expression of the anagen-prolonging insulin-like growth factor (IGF)-1. Caffeine effects were further investigated in human outer root sheath keratinocytes (ORSKs). Results Caffeine enhanced hair shaft elongation, prolonged anagen duration and stimulated hair matrix keratinocyte proliferation. Female HFs showed higher sensitivity to caffeine than male HFs. Caffeine counteracted testosterone-enhanced TGF-beta 2 protein expression in male HFs. In female HFs, testosterone failed to induce TGF-beta 2 expression, while caffeine reduced it. In male and female HFs, caffeine enhanced IGF-1 protein expression. In ORSKs, caffeine stimulated cell proliferation, inhibited apoptosis/necrosis, and upregulated IGF-1 gene expression and protein secretion, while TGF-beta 2 protein secretion was downregulated. Conclusions This study reveals new growth-promoting effects of caffeine on human hair follicles in subjects of both sexes at different levels (molecular, cellular and organ).

https://doi.org/10.1111/bjd.13114

19. Gay D.L. et al. CD133 Expression Correlates with Membrane Beta-Catenin and E-Cadherin Loss from Human Hair Follicle Placodes during Morphogenesis // J. Invest. Dermatol. 2015. Vol. 135, № 1. P. 45–55.

Genetic studies suggest that the major events of human hair follicle development are similar to those in mice, but detailed analyses of this process are lacking. In mice, hair follicle placode "budding" is initiated by invagination of Wnt-induced epithelium into the underlying mesenchyme. Modification of adherens junctions (AJs) is clearly required for budding. Snail-mediated downregulation of AJ component E-cadherin is important for placode budding in mice. Beta-catenin, another AJ component, has been more difficult to study owing to its essential functions in Wnt signaling, a prerequisite for hair follicle placode induction. Here, we show that a subset of human invaginating hair placode cells expresses the stem cell marker CD133 during early morphogenesis. CD133 associates with membrane beta-catenin in early placodes, and its continued expression correlates with loss of beta-catenin and E-cadherin from the cell membrane at a time when E-cadherin transcriptional repressors Snail and Slug are not implicated. Stabilization of CD133 via anti-CD133 antibody treatment of human fetal scalp explants depresses beta-catenin and E-cadherin membrane localization. We discuss this unique correlation and suggest a hypothetical model whereby CD133 promotes morphogenesis in early hair follicle placodes through the localized removal of membrane beta-catenin proteins and subsequent AJ dissolution.

https://doi.org/10.1038/jid.2014.292

20. Gerhards N.M. et al. Stem Cell-Associated Marker Expression in Canine Hair Follicles // J. Histochem. Cytochem. 2016. Vol. 64, № 3. P. 190–204.

Functional hair follicle (HF) stem cells (SCs) are crucial to maintain the constant recurring growth of hair. In mice and humans, SC subpopulations with different biomarker expression profiles have been identified in discrete anatomic compartments of the HF. The rare studies investigating canine HF SCs have shown similarities in biomarker expression profiles to that of mouse and human SCs. The aim of our study was to broaden the current repertoire of SC-associated markers and their expression patterns in the dog. We combined analyses on the expression levels of CD34, K15, Sox9, CD200, Nestin, LGR5 and LGR6 in canine skin using RT-qPCR, the corresponding proteins in dog skin lysates, and their expression patterns in canine HFs using immunohistochemistry. Using validated antibodies, we were able to define the location of CD34, Sox9, Keratin15, LGR5 and Nestin in canine HFs and confirm that all tested biomarkers are expressed in canine skin. Our results show similarities between the expression profile of canine, human and mouse HF SC markers. This repertoire of biomarkers will allow us to conduct functional studies and investigate alterations in the canine SC compartment of different diseases, like alopecia or skin cancer with the possibility to extend relevant findings to human patients.

https://doi.org/10.1369/0022155415627679

21. Gho C.G. et al. Isolation, expansion and neural differentiation of stem cells from human plucked hair: a further step towards autologous nerve recovery // Cytotechnology. 2016. Vol. 68, № 5. P. 1849–1858.

Stem cells from the adult hair follicle bulge can differentiate into neurons and glia, which is advantageous for the development of an autologous cell-based therapy for neurological diseases. Consequently, bulge stem cells from plucked hair may increase opportunities for personalized neuroregenerative therapy. Hairs were plucked from the scalps of healthy donors, and the bulges were cultured without prior tissue treatment. Shortly after outgrowth from the bulge, cellular protein expression was established immunohistochemically. The doubling time was calculated upon expansion, and the viability of expanded, cryopreserved cells was assessed after shear stress. The neuroglial differentiation potential was assessed from cryopreserved cells. Shortly after outgrowth, the cells were immunopositive for nestin, SLUG, AP-2 alpha and SOX9, and negative for SOX10. Each bulge yielded approximately 1 x 10(4) cells after three passages. Doubling time was 3.3 (+/- 1.5) days. Cellular viability did not differ significantly from control cells after shear stress. The cells expressed class III beta-tubulin (TUBB3) and synapsin-1 after 3 weeks of neuronal differentiation. Glial differentiation yielded KROX20- and MPZ-immunopositive cells after 2 weeks. We demonstrated that human hair follicle bulge-derived stem cells can be cultivated easily, expanded efficiently and kept frozen until needed. After cryopreservation, the cells were viable and displayed both neuronal and glial differentiation potential.

https://doi.org/10.1007/s10616-015-9938-x

22. Guiraud B. et al. Characterization of a human epidermis model reconstructed from hair follicle keratinocytes and comparison with two commercially models and native skin // Int. J. Cosmetic Sci. 2014. Vol. 36, № 5. P. 485–493.

Synopsis ObjectiveOuter root sheath (ORS) cells of human hair follicles are a readily available, non-invasive source of keratinocytes for epidermis reconstruction. The aim of this study was to characterize a model of epidermis reconstructed from ORS cells (ORS-derived model) and to evaluate its reproducibility, in comparison with native human skin and two marketed reconstructed skin models (model A, Episkin((R)) and model B, Skinethic((R))). MethodsCell morphology and tissue architecture of the three models were analysed histologically and proliferation and differentiation marker expression by immunohistochemistry and mRNA quantification. ResultsAll models displayed the same general epidermal architecture as native epidermis, but with a thicker stratum corneum in models A and B. Compared with native epidermis, Ki67 was correctly localized in epidermal basal cells in all models, as K10 in suprabasal layers. In all skin models, transglutaminase 1 (TGM1) was prematurely expressed in suprabasal layers. However, this expression was only observed from the upper stratum spinosum in the ORS-derived model. In this model, filaggrin and loricrin were correctly located in the stratum granulosum. Filaggrin, involucrin, loricrin and TGM1 mRNAs (markers of keratinocyte terminal differentiation) were transcriptionally expressed in all models. In the ORS-derived model, transcriptional expression level was similar to that of native skin. ConclusionORS cell-based reconstructed epidermis is a valid and reproducible model for human epidermis and it may be used to evaluate the effects of active substances and cosmetic formulations. Resume ObjectifLes cellules ORS de la gaine epitheliale externe des follicules pileux humains sont une source facilement disponible, non invasive de keratinocytes pour la reconstruction des epidermes. L'objectif de la presente etude est de caracteriser un modele d'epiderme reconstitue realise a partir des cellules ORS (modele derive d'ORS), et d'evaluer sa reproductibilite, par rapport a la peau humaine native et a deux modeles commercialises d'epidermes reconstruits (modele A, Episkin ((R)) et le modele B, Skinethic ((R))). MethodesLa morphologie cellulaire et l'architecture des tissus des 3 modeles ont ete analyses sur le plan histologique et l'expression des marqueurs de proliferation et de differenciation ont ete analyses par immunohistochimie et quantification d'ARNm. ResultatsTous les modeles ont une architecture epidermique generale similaire a celle de l'epiderme natif, mais avec un stratum corneum plus epais pour les modeles A et B. Par comparaison avec l'epiderme natif, Ki67 est correctement localise dans les cellules basales epidermiques dans tous les modeles, de meme que K10 dans les couches suprabasales. Dans tous les modeles, la transglutaminase 1 (TGM1) est prematurement exprimee dans les couches suprabasales. Toutefois, cette expression a ete observee uniquement a partir de la couche epineuse superieure dans le modele derive d'ORS. Dans ce modele, la filaggrine et la loricrine sont correctement situees au niveau de la couche granuleuse. Les ARNm de la filaggrine, l'involucrine, la loricrine et la TGM1 (marqueurs de la differenciation terminale des keratinocytes) sont exprimes dans tous les modeles. Dans le modele derive d'ORS, le niveau d'expression transcriptionnel est semblable a celui de la peau native. ConclusionCe modele reconstruit est un modele d'epiderme humain reproductible et interessant pour l'evaluation de principes actifs et des produits cosmetiques.

https://doi.org/10.1111/ics.12150

23. Haslam I.S. et al. Oxidative Damage Control in a Human (Mini-) Organ: Nrf2 Activation Protects against Oxidative Stress-Induced Hair Growth Inhibition // J. Invest. Dermatol. 2017. Vol. 137, № 2. P. 295–304.

The in situ control of redox insult in human organs is of major clinical relevance, yet remains incompletely understood. Activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the "master regulator" of genes controlling cellular redox homeostasis, is advocated as a therapeutic strategy for diseases with severely impaired redox balance. It remains to be shown whether this strategy is effective in human organs, rather than only in isolated human cell types. We have therefore explored the role of Nrf2 in a uniquely accessible human (mini-) organ: scalp hair follicles. Microarray and qRT-PCR analysis of human hair follicles after Nrf2 activation using sulforaphane identified the modulation of phase II metabolism, reactive oxygen species clearance, the pentose phosphate pathway, and glutathione homeostasis. Nrf2 knockdown (small interfering RNA) in cultured human hair follicles confirmed the regulation of key Nrf2 target genes (i.e., heme oxygenase-1, NAD(P)H dehydrogenase, quinone 1, glutathione reductase, glutamate-cysteine ligase catalytic subunit, ABCC1, peroxiredoxin 1). Importantly, Nrf2 activation significantly reduced reactive oxygen species levels and associated lipid peroxidation. Nrf2 preactivation reduced premature catagen and hair growth inhibition induced by oxidative stress (H2O2 or menadione), significantly ameliorated the H2O2-dependent increase in matrix keratinocyte apoptosis and reversed the reactive oxygen species-induced reduction in hair matrix proliferation. This study thus provides direct evidence for the crucial role of Nrf2 in protecting human organ function (i.e., scalp hair follicles) against redox insult.

https://doi.org/10.1016/j.jid.2016.08.035

24. Hill R.P. et al. Human hair follicle dermal sheath and papilla cells support keratinocyte growth in monolayer coculture // Exp. Dermatol. 2013. Vol. 22, № 3. P. 236–238.

Traditional skin grafting techniques are effective but limited methods of skin replacement. Autologous transplantation of rapidly cultured keratinocytes is successful for epidermal regeneration, but the current gold-standard technique requires mouse fibroblast feeders and serum-rich media, with serum-free systems and dermal fibroblast (DF) feeders performing relatively poorly. Here, we investigated the capacity of human hair follicle dermal cells to act as alternative supports for keratinocyte growth. Dermal papilla (DP) dermal sheath (DS), DF and 3T3 cells were used as inactivated feeder cells for human keratinocyte coculture. Under conditions favouring dermal cells, proliferation of keratinocytes in the presence of either DS or DP cells was significantly enhanced compared with DF cells, at levels comparable to keratinocytes cultured under gold-standard conditions. Secreted protein acidic and rich in cysteine (SPARC) expression increased DS and DP cells relative to DFs; however, further experiments did not demonstrate a role in keratinocyte support.

https://doi.org/10.1111/exd.12107

25. Hill R.P., Haycock J.W., Jahoda C.A.B. Human hair follicle dermal cells and skin fibroblasts show differential activation of NF-kappa B in response to pro-inflammatory challenge // Exp. Dermatol. 2012. Vol. 21, № 2. P. 158–160.

The underlying mechanism of immune privilege in hair follicle cell dermal papilla (DP) and sheath (DS) populations is not well understood, and the responsiveness of hair follicle dermal cells to pro-inflammatory challenge presently remains unknown. In this work, we describe acute NF-?B activation in human DS, DP and dermal fibroblast (DF) cells challenged with TNF-alpha and IL1-beta. In contrast, the DS and DP cells revealed an unexpected tolerance to bacterial LPS challenge relative to DF cells. Understanding follicle cell responses to typical pro-inflammatory stimuli is critical for diseases where collapse of hair follicle immune privilege is observed, and to further applications in autologous stem cell/wound healing therapeutics.

https://doi.org/10.1111/j.1600-0625.2011.01401.x

26. Hochfeld L.M. et al. Expression profiling and bioinformatic analyses suggest new target genes and pathways for human hair follicle related microRNAs // BMC Dermatol. 2017. Vol. 17. P. 3.

Background: Human hair follicle (HF) cycling is characterised by the tight orchestration and regulation of signalling cascades. Research shows that micro(mi)RNAs are potent regulators of these pathways. However, knowledge of the expression of miRNAs and their target genes and pathways in the human HF is limited. The objective of this study was to improve understanding of the role of miRNAs and their regulatory interactions in the human HF. Methods: Expression levels of ten candidate miRNAs with reported functions in hair biology were assessed in HFs from 25 healthy male donors. MiRNA expression levels were correlated with mRNA-expression levels from the same samples. Identified target genes were tested for enrichment in biological pathways and accumulation in protein-protein interaction (PPI) networks. Results: Expression in the human HF was confirmed for seven of the ten candidate miRNAs, and numerous target genes for miR-24, miR-31, and miR-106a were identified. While the latter include several genes with known functions in hair biology (e.g., ITGB1, SOX9), the majority have not been previously implicated (e.g., PHF1). Target genes were enriched in pathways of interest to hair biology, such as integrin and GnRH signalling, and the respective gene products showed accumulation in PPIs. Conclusions: Further investigation of miRNA expression in the human HF, and the identification of novel miRNA target genes and pathways via the systematic integration of miRNA and mRNA expression data, may facilitate the delineation of tissue-specific regulatory interactions, and improve our understanding of both normal hair growth and the pathobiology of hair loss disorders.

https://doi.org/10.1186/s12895-017-0054-9

27. Holub B.S. et al. The neuropeptide galanin is a novel inhibitor of human hair growth // Br. J. Dermatol. 2012. Vol. 167, № 1. P. 10–16.

Background Galanin is a trophic factor of the central and peripheral nervous system that shows widespread distribution in human skin. However, the exact localization and the role of galanin in the hair follicle (HF) remain to be clarified. Objectives To characterize galanin expression in human scalp HFs and to examine the effects of galanin on normal human scalp HF growth in organ culture. Methods Immunohistochemistry was performed on cryosections of human female scalp skin. Anagen HFs were microdissected and cultured up to 9 days and treated with 100 nmol L-1 galanin. Staining for Ki-67, TUNEL and Masson-Fontana were used to analyse proliferation, apoptosis and hair cycle staging of the HFs. Functional effects of galanin were tested in serum-free HF organ culture. Results Galanin-like immunoreactivity was detected in the outer root sheath (ORS) and inner root sheath. Additionally, galanin mRNA was detected in ORS keratinocytes and all HF samples tested. Galanin receptor transcripts (GalR2, GalR3) were also detected in selected samples. Galanin reduced proliferation of hair matrix keratinocytes in situ compared with vehicle-treated controls, shortened the hair growth phase (anagen) in vitro and reduced hair shaft elongation. This was accompanied by the premature development of a catagen-like morphology of galanin-treated HFs. Conclusions We present the first evidence that human HFs are both a source and a functionally relevant target of galanin. Due to its hair growth-inhibitory properties in vitro, galanin application deserves further exploration as a potential new treatment strategy for unwanted hair growth (hirsutism, hypertrichosis).

https://doi.org/10.1111/j.1365-2133.2012.10890.x

28. Inui S., Itami S. Androgen actions on the human hair follicle: perspectives // Exp. Dermatol. 2013. Vol. 22, № 3. P. 168–171.

Androgens stimulate beard growth but suppress hair growth in androgenetic alopecia (AGA). This condition is known as 'androgen paradox'. Human pilosebaceous units possess enough enzymes to form the active androgens testosterone and dihydrotestosterone. In hair follicles, 5 alpha-reductase type 1 and 2, androgen receptors (AR) and AR coactivators can regulate androgen sensitivity of dermal papillae (DP). To regulate hair growth, androgens stimulate production of IGF-1 as positive mediators from beard DP cells and of TGF-beta 1, TGF-beta 2, dickkopf1 and IL-6 as negative mediators from balding DP cells. In addition, androgens enhance inducible nitric oxide synthase from occipital DP cells and stem cell factor for positive regulation of hair growth in beard and negative regulation of balding DP cells. Moreover, AGA involves crosstalk between androgen and Wnt/beta-catenin signalling. Finally, recent data on susceptibility genes have provided us with the impetus to investigate the molecular pathogenesis of AGA.

https://doi.org/10.1111/exd.12024

29. Iwabuchi T. et al. The topical penta-peptide Gly-Pro-Ile-Gly-Ser increases the proportion of thick hair in Japanese men with androgenetic alopecia // J. Cosmet. Dermatol. 2016. Vol. 15, № 2. P. 176–184.

BackgroundA penta-peptide, Gly-Pro-Ile-Gly-Ser (GPIGS), promotes proliferation of mouse hair keratinocytes and accelerates hair growth in mice. Aim of this studyThis study focused on the ability of the peptide to promote human hair growth. MethodsWe used a human hair keratinocyte proliferation assay and organ cultures of human hair follicle as in vitro systems. The lotions with and without the penta-peptide were administered to 22 Japanese men with androgenetic alopecia (AGA) for 4 months in a double-blind and randomized clinical study. ResultsThe penta-peptide significantly stimulated the proliferation of human hair keratinocytes at a concentration of 2.3 m (P < 0.01), and 5.0 m of this peptide had a marked effect on hair shaft elongation in the organ culture (P < 0.05). The change in the proportion of thick hair (60 m) compared to baseline in patients that received the peptide was significantly higher than in the placebo (P = 0.006). The change in the proportion of vellus hair (<40 m) was also significantly lower in the peptide group than in the placebo (P = 0.029). The penta-peptide also significantly improved the appearance of baldness (P = 0.020) when blinded reviewers graded photographs of the participants according to a standardized baldness scale. No adverse dermatological effects due to treatment were noted during this clinical study. ConclusionsThis penta-peptide promotes proliferation of human hair keratinocytes and hair shaft elongation of human hair follicles, in vitro. This peptide increases thick hair ratio in vivo, and this compound is useful for the improvement of AGA.

https://doi.org/10.1111/jocd.12216

30. Jahns A.C., Alexeyev O.A. Three dimensional distribution of Propionibacterium acnes biofilms in human skin // Exp. Dermatol. 2014. Vol. 23, № 9. P. 687–689.

Propionibacterium acnes is regarded as a common member of the human skin microbiota, often occurring in biofilms. Little is known about the size of bacterial biofilms in hair follicles as a few sections of biopsy tissue are routinely evaluated. Transversal sectioning provides a better opportunity for histological analyses of hair follicles which can be followed through the different morphological levels. Direct visualization of P.acnes biofilms in hundreds of consecutive sections allowed insight into the 3D distribution in human hair follicles as well as investigating the depth of biofilm distribution within hair follicles. Four distinct colonization patterns of P.acnes biofilms were revealed. Results have shown that an individual P.acnes biofilm can spread for 1900m in a terminal hair follicle. This information can be of help while designing potential antibiofilm treatment.

https://doi.org/10.1111/exd.12482

31. Jeong K.H. et al. Prostaglandin D2-Mediated DP2 and AKT Signal Regulate the Activation of Androgen Receptors in Human Dermal Papilla Cells // Int. J. Mol. Sci. 2018. Vol. 19, № 2. P. 556.

Prostaglandin D2 (PGD2) and prostaglandin D2 receptor 2 (DP2) is known to be an important factor in androgenetic alopecia (AGA). However, the effect of PGD2 in human dermal papilla cells (hDPCs) is not fully understood. The function of PGD2-induced expression of the androgen receptor (AR), DP2, and AKT (protein kinase B) signal were examined by using real time-PCR (qRT-PCR), western blot analysis, immunocytochemistry (ICC), and siRNA transfection system. PGD2 stimulated AR expression and AKT signaling through DP2. PGD2 stimulated AR related factors (transforming growth factor beta 1 (TGF1), Creb, lymphoid enhancer binding factor 1 (LEF1), and insulin-like growth factor 1, (IGF-1)) and AKT signaling (GSK3 and Creb) on the AR expression in hDPCs. However, these factors were down-regulated by DP2 antagonist (TM30089) and AKT inhibitor (LY294002) as well as DP2 knockdown in hDPCs decreased AR expression and AKT signaling. Finally, we confirmed that PGD2 stimulates the expression of AR related target genes, and that AKT and its downstream substrates are involved in AR expression on hDPCs. Taken together, our data suggest that PGD2 promotes AR and AKT signal via DP2 in hDPCs, thus, PGD2 and DP2 signal plays a critical role in AR expression. These findings support the additional explanation for the development of AGA involving PGD2-DP2 in hDPCs.

https://doi.org/10.3390/ijms19020556

32. Ji J.H. et al. The Ethnic Differences of the Damage of Hair and Integral Hair Lipid after Ultra Violet Radiation // Ann. Dermatol. 2013. Vol. 25, № 1. P. 54–60.

Background: Genetic factors account for the majority of differences in skin color and hair morphology across human populations. Although many studies have been conducted to examine differences in skin color across populations, few studies have examined differences in hair morphology. Objective: To investigate changing of integral hair lipids after ultraviolet (UV) irradiation in three human ethnic groups. Methods: We studied the UV irradiation induced hair damage in hairs of three human populations. UV irradiation had been performed with self-manufactured phototherapy system. Damaged hair samples were prepared at 12 and 48 hours after UVA (20 J/sec) and UVB (8 J/sec) irradiation. We evaluated the changes of hair lipid using scanning electron microscopy (SEM), transmission electron microscopy (TEM), lipid TEM and HP-TLC. After UV irradiation, hair surface damage was shown. Results: African hair showed more severe damage on hair surface than others. The lipid compositions across human populations were similar, but Asian hair had more integral hair lipids than other groups as a whole. Especially, free fatty acid contents were higher than other lipids. After UV irradiation, lipid contents were decreased. These patterns were shown in all human populations. Asian hair has more integral hair lipid than European or African hair. After UV irradiation, European and African hair samples exhibited more damage because they have less integral hair lipids. However, Asian hair samples have less damage. Conclusion: We conclude that integral hair lipid may protect the hair against the UV light. (Ann Dermatol 25(1) 54 similar to 60, 2013)

https://doi.org/10.5021/ad.2013.25.1.54

33. Jimenez F., Poblet E., Izeta A. Reflections on how wound healing-promoting effects of the hair follicle can be translated into clinical practice // Exp. Dermatol. 2015. Vol. 24, № 2. P. 91–94.

Clinicians have long reported that hair-bearing areas tend to heal more rapidly than those lacking hair follicles. In the past decade, numerous scientific studies have corroborated clinical evidence, showing a direct nexus between the human hair follicle and the wound healing process. The migration of epithelial follicular stem cells to the skin surface to help in the wound re-epithelialization and the effect of the hair cycle on the wound healing rate underline the influence of the hair follicle in the healing process. In clinical practice, non-healing wounds are pathologies of high prevalence with significant associated burden costs for the healthcare system. As the population ages, the prevalence of this pathology is expected to increase in future years. The recent advances in understanding the biology of hair follicle stem cells have created the challenges of using this newly acquired knowledge in practical therapeutic applications. Chronic leg ulcers are an example of the targeted pathologies that urgently need better therapies. In this essay, our aim is to raise interest in this question, reviewing what is known in relation to the connections between hair follicles and wound healing, and elaborating on future directions that the field might take, including implications for clinical practice.

https://doi.org/10.1111/exd.12521

34. Jing J. et al. Expression of decorin throughout the murine hair follicle cycle: hair cycle dependence and anagen phase prolongation // Exp. Dermatol. 2014. Vol. 23, № 7. P. 486–491.

Decorin is a prototypical member of the small leucine-rich proteoglycan (SLRP) family, which is involved in numerous biological processes. The role of decorin, as a representative SLRP, in hair follicle morphogenesis has not been elucidated. We present our initial findings on decorin expression patterns during induced murine hair follicle (HF) cycles. It was found that decorin expression is exclusively restricted to the epidermis, outer root sheath and sebaceous glands during the anagen phase, which correlates with the upregulation of decorin mRNA and protein expression in depilated murine dorsal skin. Furthermore, we used a functional approach to investigate the effects of recombinant human decorin (rhDecorin) via cutaneous injection into HFs at various murine hair cycle stages. The local injection of rhDecorin (100 mu g/ml) into the hypodermis of depilated C57BL/6 mice at anagen delayed catagen progression. In contrast, rhDecorin injection during the telogen phase caused the premature onset of anagen, as demonstrated by the assessment of the following parameters: (i) hair shaft length, (ii) follicular bulbar diameter, (iii) hair follicle cycling score and (iv) follicular phase percentage. Taken together, our results suggest that decorin may modulate follicular cycling and morphogenesis. In addition, this study also provides insight into the molecular control mechanisms governing hair follicular epithelial-mesenchymal interactions.

https://doi.org/10.1111/exd.12441

35. Kim D.H. et al. Effect of 27-MHz Radiofrequency on Hair Follicles: Histological Evaluation of Skin Treated Ex Vivo // Dermatol. Surg. 2015. Vol. 41, № 4. P. 466–472.

BACKGROUND A multitude of methods and treatments exist for cosmetic hair removal. Electroepilation is a commonly performed method of hair removal that is so-called "permanent"; however, there is a paucity of histological studies of the effects of radiofrequency (RF) on hair follicles. OBJECTIVE This study aimed to observe the destruction of human hair follicles and surrounding tissue after the treatment with 27.12-MHz RF, with more attention paid to the thermal destruction of bulge and bulb/dermal papilla. METHODS Human scalp specimens obtained during face-lift surgery were treated with 27.12-MHz RF. The probe tip was inserted into hair follicle, RF current was applied, and treated specimens were processed for histological analysis. RESULTS Significant damages were observed on treated hair follicles. Thermal damage was lance-shaped and extended over several hundred micrometers (100-400 mm). The location of destruction areas varied, likely depending on the point of insertion of the probe. The epidermis remained intact. CONCLUSION This study shows that the general mechanism of thermolysis is to generate damage to cells and tissues surrounding the insertion point of the filament. The results suggest that if the insertion point is close to the bulge region, there is a risk to destroy hair follicle epithelial stem cells.

https://doi.org/10.1097/DSS.0000000000000324

36. Kinori M. et al. Calcitonin gene-related peptide (CGRP) may award relative protection from interferon-?-induced collapse of human hair follicle immune privilege // Exp. Dermatol. 2012. Vol. 21, № 3. P. 223–226.

Interferon-? (IFN?)-induced collapse of hair follicle (HF) immune privilege (IP) is a key element in the pathogenesis of alopecia areata. In this pilot study, we investigated whether the immunosuppressive neuropeptide, calcitonin gene-related peptide (CGRP), can protect from and/or restore IFN?-induced HF-IP collapse. After showing that human scalp HFs express CGRP receptor-like receptor (CRLR) immunoreactivity, anagen HFs were cultured in the presence of IFN?, with CGRP added before or after. Adding CGRP after IFN? administration (restoration assay) failed to downregulate IFN?-induced ectopic MHC class I expression, while MHC class II expression was reduced. However, administering CGRP before IFN? application (protection assay) significantly reduced the IFN?-induced overexpression and ectopic expression of MHC class I and II and reduced the increased degranulation of perifollicular mast cells induced by IFN?. This suggests that CGRP may not restore HF-IP once it has collapsed, but may protect it from collapsing. Therefore, CRLR stimulation might help to retard AA progression.

https://doi.org/10.1111/j.1600-0625.2011.01432.x

37. Kwack M.H. et al. Dickkopf 1 Promotes Regression of Hair Follicles // J. Invest. Dermatol. 2012. Vol. 132, № 6. P. 1554–1560.

Recently, we suggested that Dickkopf 1 (DKK-1) is a pathogenic mediator involved in male pattern baldness. As premature catagen onset is a key characteristic of male pattern baldness, in this study, we evaluated whether DKK-1 has a role as a catagen inducer in hair cycling. Herein, we report that recombinant human DKK-1 (rhDKK-1) injection into the hypodermis of mice during anagen caused premature onset of catagen, whereas neutralizing DKK-1 antibody delayed anagen-to-catagen transition in mice. Moreover, treatment with rhDKK-1 led to a decrease in final hair follicle length, whereas DKK-1 antibody led to an increase compared with control animals. In addition, DKK-1 and DKK-1 messenger RNA expression is most upregulated in follicular keratinocytes of late anagen in depilation-induced hair cycle progression. Moreover, we observed that rhDKK-1 blocks canonical Wnt-mediated activation of beta-catenin signaling and induces the proapoptotic protein Bax, resulting in apoptosis in outer root sheath keratinocytes. Taken together, our data strongly suggest that DKK-1 is involved in anagen-to-catagen transition in the hair cycle by regulating the activity of follicular keratinocytes.

https://doi.org/10.1038/jid.2012.24

38. Langan E.A. et al. Dopamine is a novel, direct inducer of catagen in human scalp hair follicles in vitro // Br. J. Dermatol. 2013. Vol. 168, № 3. P. 520–525.

Background Although there are clinical reports of hair loss associated with levodopa and dopamine agonists, it is unclear whether dopamine exerts any direct effects on the human hair follicle (HF). Objectives Given the widespread use of dopamine agonists and antagonists in clinical medicine, we sought to determine whether dopamine exerts direct effects on human HF growth and/or pigmentation in vitro, and whether human HFs express dopamine receptors (DRs). Methods Microdissected human scalp HFs from women were treated in serum-free organ culture for 7 days with dopamine (10-1000 nmol L-1), and the effects on hair shaft production, HF cycling (i.e. anagen-catagen transition), hair matrix keratinocyte proliferation and apoptosis, and HF pigmentation were measured by quantitative (immuno-) histomorphometry. Results Dopamine had no consistent effect on hair shaft production, but did promote HF regression (catagen). It was also associated with significantly reduced proliferation of HF matrix keratinocytes (P < 0.01) and reduced intrafollicular melanin production. Dopamine receptor transcripts were identified in HFs and skin. Conclusions These data provide evidence that dopamine is an inhibitor of human hair growth, via the promotion of catagen induction, at least in vitro. This may offer a rational explanation for the induction of telogen effluvium in some women treated with dopamine agonists such as bromocriptine. Moreover, dopa-minergic agonists deserve further exploration as novel inhibitors of unwanted human hair growth (hirsutism, hypertrichosis).

https://doi.org/10.1111/bjd.12113

39. Larouche D. et al. Effect of intense pulsed light treatment on human skin in vitro: analysis of immediate effects on dermal papillae and hair follicle stem cells // Br. J. Dermatol. 2013. Vol. 169, № 4. P. 859–868.

BackgroundHair follicles house a permanent pool of epithelial stem cells. Intense pulsed light (IPL) sources have been successfully used for hair removal, but long-term hair reduction may require several treatments. Many questions remain regarding the impact of IPL treatment on the structure of the hair follicle, more specifically on hair follicular stem cells and dermal papilla cells, a group of specialized cells that orchestrate hair growth. ObjectivesTo characterize the destruction of human hair follicles and surrounding tissues following IPL treatment, with more attention paid to the bulge and the bulb regions. MethodsHuman scalp specimens of Fitzpatrick skin phototype II were exposed ex vivo to IPL pulses and were then processed for histological analysis, immunodetection of stem cell-associated keratin 19, and revelation of the endogenous alkaline phosphatase activity expressed in dermal papilla cells. ResultsHistological analysis confirmed that pigmented structures, such as the melanin-rich matrix cells of the bulb in anagen follicles and the hair shaft, are principally targeted by IPL treatment, while white hairs and epidermis remained unaffected. Damage caused by heat sometimes extended over the dermal papilla cells, while stem cells were mostly spared. ConclusionsIPL epilation principally targets pigmented structures. Our results suggest that, under the tested conditions, collateral damage does not deplete stem cells. Damage at the dermal papilla was observed only with high-energy treatment modalities. Extrapolated to frequently treated hairs, these observations explain why some hairs grow back after a single IPL treatment.

https://doi.org/10.1111/bjd.12477

40. Lecardonnel J. et al. Ageing and colony-forming efficiency of human hair follicle keratinocytes // Exp. Dermatol. 2013. Vol. 22, № 9. P. 604–606.

The decline of tissue regenerative potential of skin and hair is a hallmark of physiological ageing and may be associated with age-related changes in tissue-specific stem cells and/or their environment. Human hair follicles (hHF) contain keratinocytes having the property of stem cells such as clonogenic potential. Growth capacity of hHF keratinocytes shows that most of the colony-forming cells are classified as holoclones, meroclones or paraclones when analysed in a clonal assay (Cell, Volume 76, page 1063). Despite the well-known impact of ageing on human hair growth, little is known about changes in hHF keratinocyte clonogenic potential with age. This study aimed at assessing the clone-forming efficiency (CFE) of hHF keratinocytes from three age groups of human donors. It demonstrates that ageing affects hHF keratinocyte CFE.

https://doi.org/10.1111/exd.12204

41. Leiros G.J., Attorresi A.I., Balana M.E. Hair follicle stem cell differentiation is inhibited through cross-talk between Wnt/beta-catenin and androgen signalling in dermal papilla cells from patients with androgenetic alopecia // Br. J. Dermatol. 2012. Vol. 166, № 5. P. 1035–1042.

Background Hair follicle (HF) regeneration begins when signals from the mesenchyme-derived dermal papilla cells (DPC) reach multipotent epidermal stem cells in the bulge region. Wnt/beta-catenin signalling is known to affect mammalian hair growth positively. In androgenetic alopecia (AGA), androgens cause HF miniaturization through a mechanism that remains unclear. Circulating androgens act on DPC and alter paracrine factors that influence hair epithelial cells. Objectives To elucidate the role of androgens in dermal papilla-induced differentiation of HF stem cells. Methods HF stem cell differentiation was evaluated in a coculture model with DPC or culturing with media conditioned by DPC after activation of androgen and Wnt/b-catenin signalling pathways. To study the molecular cross-talk between the androgen and Wnt signalling pathway in DPC, we analysed the expression and activation of downstream Wnt signalling molecules in the presence of androgens. Results In a coculture model with human DPC from patients with AGA and HF stem cells, we observed that androgens abrogate hair differentiation evaluated by hair-specific keratin 6 expression. Wnt signalling activation restored the ability of androgen-treated DPC to induce differentiation. Androgen treatment revealed a significant decrease in the cytoplasmic/total beta-catenin protein ratio and upregulation of the activity of glycogen synthase kinase-3 beta in DPC, indicative of canonical Wnt pathway inhibition. Conclusions These results suggest that androgens deregulate DPC-secreted factors involved in normal HF stem cell differentiation via the inhibition of the canonical Wnt signalling pathway.

https://doi.org/10.1111/j.1365-2133.2012.10856.x

42. Lemasters J.J. et al. Compartmentation of Mitochondria! and Oxidative Metabolism in Growing Hair Follicles: A Ring of Fire // J. Invest. Dermatol. 2017. Vol. 137, № 7. P. 1434–1444.

Little is known about the energetics of growing hair follicles, particularly in the mitochondrially abundant bulb. Here, mitochondrial and oxidative metabolism was visualized by multiphoton and light sheet microscopy in cultured bovine hair follicles and plucked human hairs. Mitochondrial membrane potential (Delta psi), cell viability, reactive oxygen species (ROS), and secretory granules were assessed with parameter-indicating fluorophores. In growing follicles, lower bulb epithelial cells had high viability, and mitochondria were polarized. Most epithelially generated ROS co-localized with polarized mitochondria. As the imaging plane captured more central and distal cells, Delta psi disappeared abruptly at a transition to a nonfluorescent core continuous with the hair shaft. Approaching the transition, Delta Psi and ROS increased, and secretory granules disappeared. ROS and Delta psi were strongest in a circumferential paraxial ring at putative sites for formation of the outer cortex/cuticle of the hair shaft. By contrast, polarized mitochondria in dermal papillar fibroblasts produced minimal ROS. Plucked hairs showed a similar abrupt transition of degranulation/depolarization near sites of keratin deposition, as well as an ROS-generating paraxial ring of fire. Hair movement out of the follicle appeared to occur independently of follicular bulb bioenergetics by a tractor mechanism involving the inner and outer root sheaths.

https://doi.org/10.1016/j.jid.2017.02.983

43. Liu F. et al. Meta-analysis of genome-wide association studies identifies 8 novel loci involved in shape variation of human head hair // Hum. Mol. Genet. 2018. Vol. 27, № 3. P. 559–575.

Shape variation of human head hair shows striking variation within and between human populations, while its genetic basis is far from being understood. We performed a series of genome-wide association studies (GWASs) and replication studies in a total of 28 964 subjects from 9 cohorts from multiple geographic origins. A meta-analysis of three European GWASs identified 8 novel loci (1p36.23 ERRFI1/SLC45A1, 1p36.22 PEX14, 1p36.13 PADI3, 2p13.3 TGFA, 11p14.1 LGR4, 12q13.13 HOXC13, 17q21.2 KRTAP, and 20q13.33 PTK6), and confirmed 4 previously known ones (1q21.3 TCHH/TCHHL1/LCE3E, 2q35 WNT10A, 4q21.21 FRAS1, and 10p14 LINC00708/GATA3), all showing genome-wide significant association with hair shape (P < 5e-8). All except one (1p36.22 PEX14) were replicated with nominal significance in at least one of the 6 additional cohorts of European, Native American and East Asian origins. Three additional previously known genes (EDAR, OFCC1, and PRSS53) were confirmed at the nominal significance level. A multivariable regression model revealed that 14 SNPs from different genes significantly and independently contribute to hair shape variation, reaching a cross-validated AUC value of 0.66 (95% CI: 0.62-0.70) and an AUC value of 0.64 in an independent validation cohort, providing an improved accuracy compared with a previous model. Prediction outcomes of 2504 individuals from a multiethnic sample were largely consistent with general knowledge on the global distribution of hair shape variation. Our study thus delivers target genes and DNA variants for future functional studies to further evaluate the molecular basis of hair shape in humans.

https://doi.org/10.1093/hmg/ddx416

44. Locher H. et al. Hair follicle bulge cultures yield class III beta-tubulin-positive melanoglial cells // Histochem. Cell Biol. 2015. Vol. 144, № 1. P. 87–91.

Class III beta-tubulin (TUBB3)-positive cells from the hair follicle bulge are thought to be neuronal cells derived from a local neural crest stem cell. However, TUBB3 has recently been shown to be expressed in the melanocytic lineage. To evaluate the neural-crest-associated immunophenotype of TUBB3-positive cells from hair follicle bulge explants, we dissected hair follicle bulges out from mouse whisker pads and cultured for 1 month and assessed outgrowing cells by means of immunocytochemistry using the biomarkers TUBB3, nestin, NGFR, SOX9, TYRP1 and laminin. Large amounts of TUBB3-positive cells could be cultured that co-expressed nestin, NGFR, SOX9 and, to a lesser degree, TYRP1, matching a melanoglial phenotype. In addition, a small population of TUBB3-negative but laminin-positive cells was found, which presumably are of glial origin. It can be concluded that cells of melanoglial origin can easily be obtained from hair follicle bulge explants. These cells may be of use in experimental animal or human disease and wound healing models. Notably, the TUBB3-positive cells are of melanoglial rather than neuronal origin.

https://doi.org/10.1007/s00418-015-1312-8

45. Maatough A. et al. Human Hairless Protein Roles in Skin/Hair and Emerging Connections to Brain and Other Cancers // J. Cell. Biochem. 2018. Vol. 119, № 1. P. 69–80.

The mammalian hairless protein (HR) is a 130kDa nuclear transcription factor that is essential for proper skin and hair follicle function. Previous studies have focused on the role of HR in skin maintenance and hair cycling. However, the hairless gene (HR) is also expressed in brain and other tissues, where its role remains poorly understood. HR has been reported to contain functional domains that potentially serve in DNA binding, histone demethylation, nuclear translocation and protein-protein interactions. Indeed, HR has been shown to interact with and repress the action of the nuclear receptors for vitamin D and thyroid hormone as well as RAR-related orphan receptor alpha, possibly via recruitment of histone deacetylases. HR may also have important functions in non-skin tissues given that nearly 200 HR mutations have been identified in patients with various cancers, including prostate, breast, lung, melanoma, uterine, and glioma. This suggests that HR and/or mutants thereof have relevance to the growth and survival of cancer cells. For example, the reported intrinsic histone H3K9 demethylase activity of HR may activate dormant genes to contribute to carcinogenesis. Alternatively, the demonstrated ability of HR to interact with p53 and/or the p53 DNA response element to influence p53-regulated pathways may explain, at least in part, why many cancers express mutated HR proteins. In this review, we summarize the current knowledge of HR bioactions, how HR mutations may be contributing to alopecia as well as to cancer, and, finally, outline future directions in the study of this largely enigmatic nuclear protein. J. Cell. Biochem. 119: 69-80, 2018. (c) 2017 Wiley Periodicals, Inc.

https://doi.org/10.1002/jcb.26164

46. Maniatopoulou E., Bonovas S., Sitaras N. Isolation and Quantification of Glycosaminoglycans from Human Hair Shaft // Ann. Dermatol. 2016. Vol. 28, № 5. P. 533–539.

Background: There is evidence that glycosaminoglycans (GAGs) are present in the hair shaft within the follicle but there are no studies regarding GAGs isolation and measurement in the human hair shaft over the scalp surface, it means, in the free hair shaft. Objective: The purpose of our research was to isolate and measure the total GAGs from human free hair shaft. Methods: Seventy-five healthy individuals participated in the study, 58 adults, men and women over the age of 50 and 17 children (aged 4 similar to 9). GAGs in hair samples, received from the parietal and the occipital areas, were isolated with 4 M guanidine HCI and measured by the uronic acid-carbazole reaction assay. Results: GAGs concentration was significantly higher in the occipital area than in the parietal area, in all study groups. GAG levels from both areas were significantly higher in children than in adults. GAG levels were not associated with gender, hair color or type. Conclusion: We report the presence of GAGs in the human free hair shaft and the correlation of hair GAG levels with the scalp area and participants' age.

https://doi.org/10.5021/ad.2016.28.5.533

47. Matard B. et al. First evidence of bacterial biofilms in the anaerobe part of scalp hair follicles: a pilot comparative study in folliculitis decalvans // J. Eur. Acad. Dermatol. Venereol. 2013. Vol. 27, № 7. P. 853–860.

Background The cause of folliculitis decalvans (FD) remains unknown. We hypothesized that a bacterial biofilm could be involved in its pathogenesis. Objective To assess the presence or not of a bacterial biofilm in the hair roots of the scalp in FD. Patients and methods Hairs plucked from four patients and three controls were examined by field emission scanning electron microscopy (FESEM) and confocal laser scanning microscopy (CLSM). Results Bacterial communities organized as biofilms were observed both by FESEM and CLSM in the under infundibular part of hair follicles in all patients and in two of the three controls. In patients and controls, these biofilms were formed exclusively of bacilli of comparable shapes. Conclusion This pilot study provides the first evidence of the presence of bacterial biofilms in the infra infundibular part of human scalp hair follicles. These biofilms were detected both in FD patients and controls, suggesting their ubiquity as a commensal biofilm with a possible pathogenic shift in FD.

https://doi.org/10.1111/j.1468-3083.2012.04591.x

48. McElwee K.J. et al. What causes alopecia areata?: Section Editors: Ralf Paus, Manchester/Lubeck and Raymond Cho, San Francisco // Exp. Dermatol. 2013. Vol. 22, № 9. P. 609–626.

The pathobiology of alopecia areata (AA), one of the most frequent autoimmune diseases and a major unsolved clinical problem, has intrigued dermatologists, hair biologists and immunologists for decades. Simultaneously, both affected patients and the physicians who take care of them are increasingly frustrated that there is still no fully satisfactory treatment. Much of this frustration results from the fact that the pathobiology of AA remains unclear, and no single AA pathogenesis concept can claim to be universally accepted. In fact, some investigators still harbour doubts whether this even is an autoimmune disease, and the relative importance of CD8(+) T cells, CD4(+) T cells and NKGD2(+) NK or NKT cells and the exact role of genetic factors in AA pathogenesis remain bones of contention. Also, is AA one disease, a spectrum of distinct disease entities or only a response pattern of normal hair follicles to immunologically mediated damage? During the past decade, substantial progress has been made in basic AA-related research, in the development of new models for translationally relevant AA research and in the identification of new therapeutic agents and targets for future AA management. This calls for a re-evaluation and public debate of currently prevalent AA pathobiology concepts. The present Controversies feature takes on this challenge, hoping to attract more skin biologists, immunologists and professional autoimmunity experts to this biologically fascinating and clinically important model disease.

https://doi.org/10.1111/exd.12209

49. Michler J.K. et al. Horse hair follicles: A novel dermal stem cell source for equine regenerative medicine // Cytom. Part A. 2018. Vol. 93A, № 1. P. 104–114.

The easily accessible niche represented by skin and its appendages may serve as a promising source to complement modern regenerative medicine for horses. In humans and in animal models for human medicine, the hair follicle and its stem cell niches are well characterized. Since literature in this field of equine research is scarce, we sought to analyze cells of the dermal stem cell niche of the equine hair follicle morphologically and for a subset of markers useful for cell characterization via immunolabeling. We cultured equine forelock skin explants to obtain cultures with cells migrating from the hair follicles. Isolation of cells revealed typical fibroblast morphology with a strong tendency to aggregate and form spheroids. For immunofluorescent characterization of primary isolations, we tested an antibody panel consisting of lineage makers for the dermal compartment of the hair follicle, markers associated with an undifferentiated cell status and markers for epithelial cell types as negative controls. All antibodies used were also tested on equine skin sections. The isolated cells displayed clear profiles of dermal and undifferentiated cells. To substantiate our findings, we tested our primary isolations for established equine multipotent mesenchymal stromal cell antigen expression markers in flow cytometry experiments yielding strong convergence. The data presented here provide insights to a stem cell source in horses almost unnoticed to date. The basic investigations of the equine dermal hair follicle stem cell niche confirm the expression of standard markers used in other species and lay the foundation for future studies on this easily available adult stem cell source. (c) 2017 International Society for Advancement of Cytometry

https://doi.org/10.1002/cyto.a.23198

50. Mirmirani P. et al. Investigating the effects of metabolic dysregulation on hair follicles: a comparison of HIV-infected women with and without central lipohypertrophy // Int. J. Dermatol. 2014. Vol. 53, № 10. P. E443–E448.

Background Normal lipid metabolism and functioning of the peroxisome proliferator-activated receptor gamma (PPAR-gamma) in the sebaceous gland is critical to maintaining a normal hair follicle. Human immunodeficiency virus (HIV) infection affects lipid metabolism; some have hypothesized a link between PPAR-gamma function and lipodystrophy in HIV infection. Our objective was to determine whether lipodystrophy is associated with altered hair characteristics in HIV-infected women from the Women's Interagency HIV Study. Methods Hair characteristics and scalp inflammation were assessed by an interviewer-administered questionnaire. Central lipohypertrophy and peripheral lipoatrophy were defined by self-report of moderate to severe fat gain in central body sites and fat loss in peripheral body sites, respectively confirmed by clinical examination. Additional covariates considered in the analyses included demographics, behavioral characteristics, medical history, and HIV-related factors. Results There were 1037 women with data on all study variables; 76 women reported central lipohypertrophy, while only four women reported lipoatrophy. Women with central lipohypertrophy were more likely to be older, had a self-reported history of injection drug use, statin medication use, diabetes, elevated cholesterol, and have self-reported less hair and shorter eyelashes. After adjustment for age, central lipohypertrophy was associated with shorter eyelashes (OR 2.3; 95% CI 1.4-3.8). Conclusions Central lipohypertrophy was not associated with change in scalp hair texture or scalp inflammation in this cohort. Rather, we found an association between central lipohypertrophy and shorter eyelash length. This finding may be explained by an influence of prostaglandin E-2 mediators on eyelash follicles.

https://doi.org/10.1111/ijd.12044

51. Munkhbayar S. et al. Role of Arachidonic Acid in Promoting Hair Growth // Ann. Dermatol. 2016. Vol. 28, № 1. P. 55–64.

Background: Arachidonic acid (AA) is an omega-6 polyunsaturated fatty acid present in all mammalian cell membranes, and involved in the regulation of many cellular processes, including cell survival, angiogenesis, and mitogenesis. The dermal papilla, composed of specialized fibroblasts located in the bulb of the hair follicle, contributes to the control of hair growth and the hair cycle. Objective: This study investigated the effect of AA on hair growth by using in vivo and in vitro models. Methods: The effect of AA on. human dermal papilla cells (hDPCs) and hair shaft elongation was evaluated by MTT assay and hair follicle organ culture, respectively. The expression of various growth and survival factors in hDPCs were investigated by western blot or immunohistochemistry. The ability of AA to induce and prolong anagen phase in C57BL/6 mice was analyzed. Results: AA was found to enhance the viability of hDPCs and promote the expression of several factors responsible for hair growth, including fibroblast growth factor-7 (FGF-7) and FGF-10. Western blotting identified the role of AA in the phosphorylation of various transcription factors (ERK, CREB, and AKT) and increased expression of Bcl-2 in hDPCs. In addition, AA significantly promoted hair shaft elongation, with increased proliferation of matrix keratinocytes, during ex vivo hair follicle culture. It was also found to promote hair growth by induction and prolongation of anagen phase in telogen-stage C57BL/6 mice. Conclusion: This study concludes that AA plays a role in promoting hair growth by increasing the expression of growth factors in hDPCs and enhancing follicle proliferation and survival.

https://doi.org/10.5021/ad.2016.28.1.55

52. Nagasawa A. et al. t-Flavanone Improves the Male Pattern of Hair Loss by Enhancing Hair-Anchoring Strength: A Randomized, Double-Blind, Placebo-Controlled Study // Dermatol. Ther. 2016. Vol. 6, № 1. P. 59–68.

Introduction: trans-3,4'-Dimethyl-3-hydroxyflavanone (t-flavanone) is a derivative of astilbin that actively stimulates hair growth. The aim of the present study was to identify the mechanisms of action of t-flavanone on hair growth. Methods: A double-blind usage test was performed with healthy volunteers who had androgenic alopecia (AGA). The subjects were divided into three groups with equal average baldness. The members in each group applied a vasodilator-containing hair lotion supplemented with either 0, 0.1, or 0.3% (wt) t-flavanone twice a day for 30 weeks. The efficacy of t-flavanone was evaluated based on the parietal global and microscopic images. At week 30, the anchoring strength of hair was measured by the average peak force required for plucking out a single hair in a non-bald area using a digital force gauge. Desmoglein expression in the cultured human hair follicle was analyzed by Western blotting. Results: After 30 weeks, t-flavanone significantly improved AGA and enhanced the hair-anchoring strength in a hair diameter-independent manner. Culture of human hair follicles in vitro with t-flavanone resulted in the upregulation of desmoglein protein expression. Conclusions: The results of our in vivo and in vitro studies demonstrated that t-flavanone enhanced the cell-cell adhesions in hair follicles; thus, reinforcement of hair rooting may be a mechanism by which t-flavanone promotes hair growth.

https://doi.org/10.1007/s13555-016-0101-1

53. Nilforoushzadeh M. et al. Hair Follicle Generation by Injections of Adult Human Follicular Epithelial and Dermal Papilla Cells into Nude Mice // Cell J. 2017. Vol. 19, № 2. P. 259–268.

Nilforoushzadeh M. et al. Hair Follicle Generation by Injections of Adult Human Follicular Epithelial and Dermal Papilla Cells into Nude Mice // Cell J. 2017. Vol. 19, № 2. P. 259–268.

https://doi.org/10.22074/cellj.2016.3916

54. Oh J.W. et al. A Guide to Studying Human Hair Follicle Cycling In Vivo // J. Invest. Dermatol. 2016. Vol. 136, № 1. P. 34–44.

Hair follicles (HFs) undergo lifelong cyclical transformations, progressing through stages of rapid growth (anagen), regression (catagen), and relative "quiescence" (telogen). Given that HF cycling abnormalities underlie many human hair growth disorders, the accurate classification of individual cycle stages within skin biopsies is clinically important and essential for hair research. For preclinical human hair research purposes, human scalp skin can be xenografted onto immunocompromised mice to study human HF cycling and manipulate long-lasting anagen in vivo. Although available for mice, a comprehensive guide on how to recognize different human hair cycle stages in vivo is lacking. In this article, we present such a guide, which uses objective, well-defined, and reproducible criteria, and integrates simple morphological indicators with advanced, (immuno)-histochemical markers. This guide also characterizes human HF cycling in xenografts and highlights the utility of this model for in vivo hair research. Detailed schematic drawings and representative micrographs provide examples of how best to identify human HF stages, even in suboptimally sectioned tissue, and practical recommendations are given for designing human-on-mouse hair cycle experiments. Thus, this guide seeks to offer a benchmark for human hair cycle stage classification, for both hair research experts and newcomers to the field.

https://doi.org/10.1038/JID.2015.354

55. Ohyama M. et al. Restoration of the intrinsic properties of human dermal papilla in vitro // J. Cell Sci. 2012. Vol. 125, № 17. P. 4114–4125.

The dermal papilla (DP) plays pivotal roles in hair follicle morphogenesis and cycling. However, characterization and/or propagation of human DPs have been unsatisfactory because of the lack of efficient isolation methods and the loss of innate characteristics in vitro. We hypothesized that culture conditions sustaining the intrinsic molecular signature of the human DP could facilitate expansion of functional DP cells. To test this, we first characterized the global gene expression profile of microdissected, non-cultured human DPs. We performed a 'two-step' microarray analysis to exclude the influence of unwanted contaminants in isolated DPs and successfully identified 118 human DP signature genes, including 38 genes listed in the mouse DP signature. The bioinformatics analysis of the DP gene list revealed that WNT, BMP and FGF signaling pathways were upregulated in intact DPs and addition of 6-bromoindirubin-3'-oxime, recombinant BMP2 and basic FGF to stimulate these respective signaling pathways resulted in maintained expression of in situ DP signature genes in primarily cultured human DP cells. More importantly, the exposure to these stimulants restored normally reduced DP biomarker expression in conventionally cultured DP cells. Cell growth was moderate in the newly developed culture medium. However, rapid DP cell expansion by conventional culture followed by the restoration by defined activators provided a sufficient number of DP cells that demonstrated characteristic DP activities in functional assays. The study reported here revealed previously unreported molecular mechanisms contributing to human DP properties and describes a useful technique for the investigation of human DP biology and hair follicle bioengineering.

https://doi.org/10.1242/jcs.105700

56. Panhard S., Lozano I., Loussouarn G. Greying of the human hair: a worldwide survey, revisiting the “50” rule of thumb // Br. J. Dermatol. 2012. Vol. 167, № 4. P. 865–873.

Background While numerous papers have reported on the biological mechanisms of human hair pigmentation and greying, epidemiological descriptions of both natural hair colour and the greying process, worldwide, remain scarce. Objectives To assess hair colour and greying in a large world sample of human subjects, and to revisit the validity of the 50/50/50 rule of thumb, which states that 'at age 50 years, 50% of the population has at least 50% grey hair'. Methods The natural hair colour of 4192 healthy male and female volunteers was assessed using a sensorial expert evaluation through the comparison of each volunteer's hair with standard swatches. Hair colour was studied according to age, gender and ethnic or geographical origin. Results Overall we observed that between 45 and 65 years of age, 74% of people were affected by grey hair with a mean intensity of 27%. Men harboured significantly more grey hair than women. Both age at onset and rate of greying with age appeared to be clearly linked to ethnic/geographical origin. Subjects of Asian and African descent showed less grey hair than those of caucasian origin, at comparable ages, confirming previously reported data. Conclusions Calculating the percentage of people showing at least 50% grey hair coverage at age 50 years leads to a global range of 6-23%, according to ethnic/geographical origin and natural hair colour: well below that expressed by the '50' rule of thumb.

https://doi.org/10.1111/j.1365-2133.2012.11095.x

57. Peterson S.C. et al. Basal Cell Carcinoma Preferentially Arises from Stem Cells within Hair Follicle and Mechanosensory Niches // Cell Stem Cell. 2015. Vol. 16, № 4. P. 400–412.

Basal cell carcinoma (BCC) is characterized by frequent loss of PTCH1, leading to constitutive activation of the Hedgehog pathway. Although the requirement for Hedgehog in BCC is well established, the identity of disease-initiating cells and the compartments in which they reside remain controversial. By using several inducible Cre drivers to delete Ptch1 in different cell compartments in mice, we show here that multiple hair follicle stem cell populations readily develop BCC-like tumors. In contrast, stem cells within the interfollicular epidermis do not efficiently form tumors. Notably, we observed that innervated Gli1-expressing progenitors within mechanosensory touch dome epithelia are highly tumorigenic. Sensory nerves activate Hedgehog signaling in normal touch domes, while denervation attenuates touch dome-derived tumors. Together, our studies identify varying tumor susceptibilities among different stem cell populations in the skin, highlight touch dome epithelia as "hot spots'' for tumor formation, and implicate cutaneous nerves as mediators of tumorigenesis.

https://doi.org/10.1016/j.stem.2015.02.006

58. Plikus M.V. At the dawn of hair research - testing the limits of hair follicle regeneration // Exp. Dermatol. 2014. Vol. 23, № 5. P. 314–315.

In the late 1960s, tissue recombination studies by Roy Oliver on the model of rat vibrissae provided invaluable information about the morphogenetic properties of hair follicles. Now more than ever, the field is hopeful that a clinically reproducible procedure for cell-based hair regeneration is achievable. Highly inductive mesenchymal cells are thought to be the key ingredient necessary to achieve robust hair regeneration, and efforts are underway to develop protocols to improve the naturally low inductive properties of human dermal papilla cells. In this respect, the original studies by Oliver provide essential rodent research benchmarks, which current-day human studies should aim to reach.

https://doi.org/10.1111/exd.12334

59. Purba T.S. et al. Human epithelial hair follicle stem cells and their progeny: Current state of knowledge, the widening gap in translational research and future challenges // Bioessays. 2014. Vol. 36, № 5. P. 513–525

Epithelial hair follicle stem cells (eHFSCs) are required to generate, maintain and renew the continuously cycling hair follicle (HF), supply cells that produce the keratinized hair shaft and aid in the reepithelialization of injured skin. Therefore, their study is biologically and clinically important, from alopecia to carcinogenesis and regenerative medicine. However, human eHFSCs remain ill defined compared to their murine counterparts, and it is unclear which murine eHFSC markers really apply to the human HF. We address this by reviewing current concepts on human eHFSC biology, their immediate progeny and their molecular markers, focusing on Keratin 15 and 19, CD200, CD34, PHLDA1, and EpCAM/Ber-EP4. After delineating how human eHFSCs may be selectively targeted experimentally, we close by defining as yet unmet key challenges in human eHFSC research. The ultimate goal is to transfer emerging concepts from murine epithelial stem cell biology to human HF physiology and pathology.

https://doi.org/10.1002/bies.201300166

60. Purba T.S. et al. Mapping the expression of epithelial hair follicle stem cell-related transcription factors LHX2 and SOX9 in the human hair follicle // Exp. Dermatol. 2015. Vol. 24, № 6. P. 462–467.

In the murine hair follicle (HF), the transcription factors LHX2 and SOX9 are implicated in epithelial hair follicle stem cell (eHFSC) self-renewal and the maintenance of eHFSC niche characteristics. However, the exact expression patterns of LHX2 and SOX9 in the human HF are unclear. Therefore, we have quantitatively mapped the localisation of known human eHFSC markers keratin 15 (K15) and keratin 19 (K19) in the outer root sheath (ORS) of male occipital scalp anagen HFs and related this to the localisation of LHX2 and SOX9 protein expression. As expected, K15(+) and K19(+) cells represented two distinct progenitor cell populations in the bulge and in the proximal bulb ORS (pbORS). Interestingly, cell fluorescence for K19 was significantly stronger within the pbORS versus the bulge, and vice versa for K15, describing a hitherto unrecognised differential expression pattern. LHX2 and SOX9 expressing cells were distributed throughout the ORS, including the bulge, but were not restricted to it. SOX9 expression was most prominent in the ORS immediately below the human bulge, whereas LHX2(+) cells were similarly distributed between the sub-bulge and pbORS, that is compartments not enriched with quiescent eHFSCs. During catagen development, the intensity of LHX2 and SOX9 protein expression increased in the proximal HF epithelium. Double immunostaining showed that the majority ofSOX9(+) cells in the human anagen HF epithelium did not co-express K15, K19 or LHX2. This expression profile suggests that LHX2 and SOX9 highlight distinct epithelial progenitor cell populations, in addition to K15(+) or K19(+) cells, that could play an important role in the maintenance of the human HF epithelium.

https://doi.org/10.1111/exd.12700

61. Rastegar H. et al. Herbal Extracts Induce Dermal Papilla Cell Proliferation of Human Hair Follicles // Ann. Dermatol. 2015. Vol. 27, № 6. P. 667–675.

Background: The number of people suffering from balding or hair thinning is increasing, despite the advances in various medical therapies. Therefore, it is highly important to develop new therapies to inhibit balding and increase hair proliferation. Objective: We investigated the effects of herbal extracts commonly used for improving balding in traditional medicine to identify potential agents for hair proliferation. Methods: The expression levels of 5 alpha -reductase isoforms (type I and II) were analyzed using quantitative real-time reverse transcription polymerase chain reaction in the human follicular dermal papilla cells (DPCs). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylteterazolium bromide and bromodeoxyuridine tests were used to evaluate the cell proliferation effect of herbal extracts in DPCs. The expression levels of extracellular signal-regulated kinase (ERK), Akt, cycl in D1, cyclin-dependent kinase 4 (Cdk4), B-cell lymphoma (Bcl-2) and Bcl-2-associated X protein (Bax) were measured using western blot analysis. Results: The 5 alpha -reductase isoform mRNAs and proteins were detected in the cultured DPCs, and the expression level of 5 alpha -R2 in DPCs in the presence of the herbal extracts was gradually decreased. Herbal extracts were found to significantly increase the proliferation of human DPCs at concentrations ranging from 1.5% to 4.5%. These results show that the herbal extracts tested affected the protein expressions of ERK, Akt, cyclin D1, Cdk4, Bcl-2, and Bax in DPCs. Conclusion: These results suggest that herbal extracts exert positive effects on hair proliferation via ERK, Akt, cycl in D1, and Cdk4 signaling in DPCs; they also suggest that herbal extracts could be a great alternative therapy for increasing hair proliferation.

https://doi.org/10.5021/ad.2015.27.6.667

62. Ryu S. et al. Mycophenolate Antagonizes IFN-gamma-Induced Catagen-Like Changes via beta-Catenin Activation in Human Dermal Papilla Cells and Hair Follicles // Int. J. Mol. Sci. 2014. Vol. 15, № 9. P. 16800–16815.

Recently, various immunosuppressant drugs have been shown to induce hair growth in normal hair as well as in alopecia areata and androgenic alopecia; however, the responsible mechanism has not yet been fully elucidated. In this study, we investigate the influence of mycophenolate (MPA), an immunosuppressant, on the proliferation of human dermal papilla cells (hDPCs) and on the growth of human hair follicles following catagen induction with interferon (IFN)-gamma. IFN-gamma was found to reduce beta-catenin, an activator of hair follicle growth, and activate glycogen synthase kinase (GSK)-3 beta, and enhance expression of the Wnt inhibitor DKK-1 and catagen inducer transforming growth factor (TGF)-beta 2. IFN-gamma inhibited expression of ALP and other dermal papillar cells (DPCs) markers such as Axin2, IGF-1, and FGF 7 and 10. MPA increased beta-catenin in IFN-gamma-treated hDPCs leading to its nuclear accumulation via inhibition of GSK3 beta and reduction of DKK-1. Furthermore, MPA significantly increased expression of ALP and other DPC marker genes but inhibited expression of TGF-beta 2. Therefore, we demonstrate for the first time that IFN-gamma induces catagen-like changes in hDPCs and in hair follicles via inhibition of Wnt/beta-catenin signaling, and that MPA stabilizes beta-catenin by inhibiting GSK3 beta leading to increased beta-catenin target gene and DP signature gene expression, which may, in part, counteract IFN-gamma-induced catagen in hDPCs.

https://doi.org/10.3390/ijms150916800

63. Savkovic V. et al. Improved method of differentiation, selection and amplification of human melanocytes from the hair follicle cell pool // Exp. Dermatol. 2012. Vol. 21, № 12. P. 948–950.

Hair root harbours a complex cell pool with an immense developmental potential. Several lineages, including skin, can be differentiated from the multipotent to pluripotent cells of outer root sheath (ORS) of hair follicle. Outer root sheath presents the most opulent non-invasively gained adult stem cell source known. For the purposes of cultivating melanocytes designated for graft-based treatments of depigmentation disorders, we have established an ex vivo/in vitro cultivation method by introducing several methodological improvements to the ORS explant method of Dieckmann. As a result, we gained a higher, purer yield of differentiated melanocytes in half the time (at least 106 of 95% pure cells in 4 weeks). This reliable cultivation procedure begins with the epilation of 60 hairs and yields high numbers of ORS melanocytes that could be used for grafting applications. The procedure not only utilises the developmental potential of hair root cell pool and favors differentiation into melanocytes, but also contributes to the general trend of minimal-to-non-invasive strategies for regenerative medicine.

https://doi.org/10.1111/exd.12038

64. Serrano G. et al. Microneedling dilates the follicular infundibulum and increases transfollicular absorption of liposomal sepia melanin // CLIN. COSMET. INVESTIG. DERMATOL. 2015. Vol. 8. P. 313–318.

Encapsulation of chemicals in liposomes and microneedling are currently used techniques to enhance the penetration of several substances through skin and hair. In this study, we apply a liposomal melanin-fluorescein compound to an ex vivo model of human skin, using a new electrical microneedling device (Nanopore turbo roller). The product was applied by hand massage (A) or with the assistance of the electrical roller for 2 minutes (B). An additional test was performed free of product and with only the E-roller (C). Histological changes and product absorption were evaluated by optical and fluorescent microscopy 60 and 90 minutes after the treatment. Site B showed larger deposits of melanin-fluorescein at superficial and deep levels of hair structures in comparison to site A. Light, epidermal deposits of the melanin-fluorescein complex were also observed. Sites B and C showed a significant widening (47%) of the follicular infundibulum which could explain the increased penetration of the formulation. Microneedling also removed the scales and sebum residues in the neighborhood of the infundibulum. Targeting hair follicles with melanin may be useful to dye poorly pigmented hairs, improving laser hair removal. The procedure accelerates the delivery of melanin into hair structures allowing an even absorption, larger pigment deposits, and deeper penetration of the formulation into the hair.

https://doi.org/10.2147/CCID.S77228

65. Sextius P. et al. Polygonum multiflorum Radix extract protects human foreskin melanocytes from oxidative stress in vitro and potentiates hair follicle pigmentation ex vivo // Int. J. Cosmetic Sci. 2017. Vol. 39, № 4. P. 419–425.

ObjectiveTo examine the ability of an extract from traditional Chinese medicine, Polygonum multiflorum Radix, to protect melanocyte viability from oxidative stress, a key mechanism in the initiation and progression of hair greying. MethodsTo assess the antioxidant capacity of Polygonum multiflorum Radix extract, primary human foreskin melanocytes were treated with a commercially available Polygonum multiflorum Radix extract added to culture medium and exposed to hydrogen peroxide (H2O2), using intracellular reactive oxygen species concentrations and glutathione/protein ratios as endpoints. To improve solubility for cosmetic uses, a new Polygonum multiflorum Radix extract was derived. As hair greying is the consequence of melanocyte disappearance in an oxidative stress environment, we checked whether the antioxidant capacity of the new Polygonum multiflorum Radix extract could preserve melanocyte viability in response to H2O2-induced oxidative stress, and preserve pigmentation within exvivo human hair follicles. ResultsIn vitro treatment of primary human foreskin melanocytes with traditional available Polygonum multiflorum Radix extract resulted in decreased intracellular ROS accumulation in response to H2O2 exposure with a concomitant preservation of glutathione-to-protein ratio, consistent with a protective response against H2O2 exposure and demonstrating the promise of this extract for protecting melanocytes against oxidative stress. Melanocytes treated with the improved Polygonum multiflorum Radix extract exhibited attenuated H2O2-induced cell death, demonstrating a clear cytoprotective effect. Treatment of exvivo human hair follicles with the improved Polygonum multiflorum Radix extractresulted in a higher level of melanin compared to vehicle-treated controls, demonstrating an exvivo protective effect on hair pigmentation. ConclusionPolygonum multiflorum Radix extract protects invitro primary human foreskin melanocytes from the deleterious effects of H2O2 exposure and improves pigmentation within exvivo human hair follicles, demonstrating the utility of Polygonum multiflorum Radix extract as a potential active ingredient for the protection of melanocytes against premature death. This data provides invitro mechanistic evidence consistent with existing invivo studies for the use of Polygonum multiflorum Radix extract as a strategy for the prevention of oxidative stress-induced hair greying, in line with traditional Polygonum multiflorum Radix uses.

https://doi.org/10.1111/ics.12391

66. Shin H. et al. Enhancement of Human Hair Growth Using Ecklonia cava Polyphenols // Ann. Dermatol. 2016. Vol. 28, № 1. P. 15–21.

Background: Ecklonia cava is a brown alga that contains various compounds, including carotenoids, fucoidans, and phlorotannins. E. cava polyphenols (ECPs) are known to increase fibroblast survival. The human dermal papilla cell (hDPC) has the properties of mesenchymal-origin fibroblasts. Objective: This study aims, to investigate the effect of ECPs on human hair growth promotion in vitro and ex vivo. Methods: MTT assays were conducted to examine the effect of ECPs on hDPC proliferation. Hair growth was measured using ex-vivo hair follicle cultures. Real-time polymerase chain reaction was performed to evaluate the mRNA expression of various growth factors in ECP-treated hDPCs. Results: Treatment with 10 mu g/ml purified polyphenols from E. cava (PPE) enhanced the proliferation of hDPCs 30.3% more than in the negative control (p<0.001). Furthermore, 0.1 mu g/ml PPE extended the human hair shaft 30.8% longer than the negative control over 9 days (p<0.05). Insulin-like growth factor-1 (IGF-1) mRNA expression increased 3.2-fold in hDPCs following treatment with 6 mu g/ml PPE (p<0.05). Vascular endothelial growth factor (VEGF) mRNA expression was also increased 2.0-fold by 3 mu g/ml PPE (p<0.05). Treatment with 10 mu g/ml PPE reduced oxidative stress in hDPCs (p<0.05). Conclusion: These results suggest that PPE could enhance human hair growth. This can be explained by hDPC proliferation coupled with increases in growth factors such as IGF-1 and VEGF. Reducing oxidative stress is also thought to help increase hDPCs. These favorable results suggest that PPE is a promising therapeutic candidate for hair loss.

https://doi.org/10.5021/ad.2016.28.1.15

67. Shin S. et al. Epigallocatechin Gallate-Mediated Alteration of the MicroRNA Expression Profile in 5 alpha-Dihydrotestosterone-Treated Human Dermal Papilla Cells // Ann. Dermatol. 2016. Vol. 28, № 3. P. 327–334.

Background: Dihydrotestosterone (DHT) induces androgenic alopecia by shortening the hair follicle growth phase, resulting in hair loss. We previously demonstrated how changes in the microRNA (miRNA) expression profile influenced DHT-mediated cell death, cell cycle arrest, cell viability, the generation of reactive oxygen species (ROS), and senescence. Protective effects against DHT have not, however, been elucidated at the genome level. Objective: We showed that epigallocatechin gal late (EGCG), a major component of green tea, protects DHT-induced cell death by regulating the cellular miRNA expression profile. Methods: We used a miRNA microarray to identify miRNA expression levels in human dermal papilla cells (DPCs). We investigated whether the miRNA expression influenced the protective effects of EGCG against DHT-induced cell death, growth arrest, intracellular ROS levels, and senescence. Results: EGCG protected against the effects of DHT by altering the miRNA expression profile in human DPCs. In addition, EGCG attenuated DHT-mediated cell death and growth arrest and decreased intracellular ROS levels and senescence. A bioinformatics analysis elucidated the relationship between the altered miRNA expression and EGCG-mediated protective effects against DHT. Conclusion: Overall, our results suggest that EGCG ameliorates the negative effects of DHT by altering the miRNA expression profile in human DPCs.

https://doi.org/10.5021/ad.2016.28.3.327

68. Son S. et al. Magnetofection Mediated Transient NANOG Overexpression Enhances Proliferation and Myogenic Differentiation of Human Hair Follicle Derived Mesenchymal Stem Cells // Bioconjugate Chem. 2015. Vol. 26, № 7. P. 1314–1327.

We used magnetofection (MF) to achieve high transfection efficiency into human mesenchymal stem cells (MSCs). A custom-made magnet array, matching well-to-well to a 24-well plate, was generated and characterized. Theoretical predictions of magnetic force distribution within each well demonstrated that there was no magnetic field interference among magnets in adjacent wells. An optimized protocol for efficient gene delivery to human hair follicle derived MSCs (hHF-MSCs) was established using an egfp-encoding plasmid, reaching approximately 60% transfection efficiency without significant cytotoxicity. Then we applied the optimized MF protocol to express the pluripotency-associated transcription factor NANOG, which was previously shown to reverse the effects of organismal aging on MSC proliferation and myogenic differentiation capacity. Indeed, MF-mediated NANOG delivery increased proliferation and enhanced the differentiation of hHF-MSCs into smooth muscle cells (SMCs). Collectively, our results show that MF can achieve high levels of gene delivery to MSCs and, therefore, may be employed to moderate or reverse the effects of cellular senescence or reprogram cells to the pluripotent state without permanent genetic modification.

https://doi.org/10.1021/bc5005203

69. Sriwiriyanont P. et al. Morphogenesis of chimeric hair follicles in engineered skin substitutes with human keratinocytes and murine dermal papilla cells // Exp. Dermatol. 2012. Vol. 21, № 10. P. 783–785.

Engineered skin substitutes (ESS) have been used successfully to treat life-threatening burns, but lack cutaneous appendages. To address this deficiency, dermal constructs were prepared using collagen-glycosaminoglycan scaffolds populated with murine dermal papilla cells expressing green fluorescent protein (mDPC-GFP), human dermal papilla cells (hDPC) and/or human fibroblasts (hF). Subsequently, human epidermal keratinocytes (hK) or hK genetically modified to overexpress stabilized beta-catenin (hK') were used to prepare ESS epithelium. After 10 days incubation at air-liquid interface, ESS were grafted to athymic mice and were evaluated for 6 weeks. Neofollicles were observed in ESS containing mDPC-GFP, but not hDPC or hF, independent of whether or not the hK were genetically modified. Based on detection of GFP fluorescence, mDPC were localized to the dermal papillae of the well-defined follicular structures of grafted ESS. In addition, statistically significant increases in LEF1, WNT10A and WNT10B were found in ESS with neofollicles. These results demonstrate a model for generation of chimeric hair in ESS.

https://doi.org/10.1111/exd.12003

70. Takahashi T. et al. Improvement of androgenetic alopecia with topical Sophora flavescens Aiton extract, and identification of the two active compounds in the extract that stimulate proliferation of human hair keratinocytes // Clin. Exp. Dermatol. 2016. Vol. 41, № 3. P. 302–307.

BackgroundAndrogenetic alopecia (AGA) is a hair loss disorder that commonly affects middle-aged men. To date, the properties of a number of natural or synthetic substances have been investigated for their ability to improve the condition. AimTo evaluate the hair growth-promoting activities of an extract from the root of Sophora flavescens Aiton. MethodsWe used a human hair keratinocyte proliferation assay and ex vivo organ cultures of human hair follicle to examine the potential of the extract to stimulate hair growth via anagen elongation. We isolated the compounds promoting the growth of epithelial cells, and determined their chemical structures. A randomized, double-blinded, placebo-controlled clinical study for S. flavescens extract was carried out for 6 months with patients with AGA. ResultsThe extract stimulated the proliferation of hair keratinocytes at a concentration of 0.1 ng/mL, while 100 ng/mL of the extract had a marked effect on hair shaft elongation in an organ culture of human hair follicle. Cell proliferation assay-directed fractionation led to the identification of two pterocarpan derivatives, L-maackiain and medicarpin, as active compounds that promote the proliferation of human hair keratinocytes. Studies in human subjects showed that improvement in the inspected alopecia scores in the lotion plus extract group were significant over a period of 6 months (P < 0.01). ConclusionsS. flavescens root extract is effective for the treatment of AGA. The isolated two pterocarpans might have important role in this effect.

71. Tohgi N. et al. Human hair-follicle associated pluripotent (hHAP) stem cells differentiate to cardiac-muscle cells // Cell Cycle. 2017. Vol. 16, № 1. P. 95–99.

We have previously demonstrated that nestin-expressing hair follicle-associated-pluripotent (HAP) stem cells are located in the bulge area. HAP stem cells have been previously shown to differentiate to neurons, glial cells, keratinocytes, smooth-muscle cells, melanocytes and cardiac-muscle cells in vitro. Subsequently, we demonstrated that HAP stem cells could effect nerve and spinal cord regeneration in mouse models, differentiating to Schwann cells and neurons. In previous studies, we established an efficient protocol for the differentiation of cardiac-muscle cells from mouse HAP stem cells. In the present study, we isolated the upper part of human hair follicles containing human HAP (hHAP) stem cells. The upper parts of human hair follicles were suspended in DMEM containing 10% FBS where they differentiated to cardiac-muscle cells as well as neurons, glial cells, keratinocytes and smooth-muscle cells. This method is appropriate for future use with human hair follicles to produce hHAP stem cells in sufficient quantities for future heart, nerve and spinal cord regeneration in the clinic.

https://doi.org/10.1080/15384101.2016.1253642

72. Veraitch O. et al. Human Induced Pluripotent Stem Cell-Derived Ectodermal Precursor Cells Contribute to Hair Follicle Morphogenesis In Vivo // J. Invest. Dermatol. 2013. Vol. 133, № 6. P. 1479–1488.

Well-orchestrated epithelial-mesenchymal interactions are crucial for hair follicle (HF) morphogenesis. In this study, ectodermal precursor cells (EPCs) with the capacity to cross talk with hair-inductive dermal cells were generated from human induced pluripotent stem cells (hiPSCs) and assessed for HF-forming ability in vivo. EPCs derived from three hiPSC lines generated with 4 or 3 factors (POU5F1, SOX2, KLF4 +/- MYC) mostly expressed keratin 18, a marker of epithelial progenitors. When cocultured with human dermal papilla (DP) cells, a 4 factor 201B7 hiPSC-EPC line upregulated follicular keratinocyte (KC) markers more significantly than normal human adult KCs (NHKCs) and other hiPSC-EPC lines. DP cells preferentially increased DP biomarker expression in response to this line. Interestingly, 201B7 hiPSCs were shown to be ectodermal/epithelial prone, and the derived EPCs were putatively in a wingless-type MMTV integration site family (WNT)-activated state. Importantly, co-transplantation of 201B7 hiPSC-EPCs, but not NHKCs, with trichogenic mice dermal cells into immunodeficient mice resulted in HF formation. Human HF stem cell markers were detected in reconstituted HFs; however, a low frequency of human-derived cells implied that hiPSC-EPCs contributed to HF morphogenesis via direct repopulation and non-cell autonomous activities. The current study suggests a, to our knowledge, previously unrecognized advantage of using hiPSCs to enhance epithelial-mesenchymal interactions in HF bioengineering.

https://doi.org/10.1038/jid.2013.7

73. Vidali S. et al. Hypothalamic-Pituitary-Thyroid Axis Hormones Stimulate Mitochondrial Function and Biogenesis in Human Hair Follicles // J. Invest. Dermatol. 2014. Vol. 134, № 1. P. 33–42.

Thyroid hormones regulate mitochondria! function. As other hypothalamic pituitary thyroid (HPT) axis hormones, i.e., thyrotropin-releasing hormone (TRH) and thyrotropin (TSH), are expressed in human hair follicles (HFs) and regulate mitochondrial function in human epidermis, we investigated in organ-cultured human scalp HFs whether TRH (30 nm), TSH (10 mU ml(-1)), thyroxine (T-4) (100 nm), and triiodothyronine (T-3) (100 pm) alter intrafollicular mitochondrial energy metabolism. All HPT-axis members increased gene and protein expression of mitochondrial-encoded subunit 1 of cytochrome c oxidase (MTCO1), a subunit of respiratory chain complex IV, mitochondrial transcription factor A (TFAM), and Porin. All hormones also stimulated intrafollicular complex I/IV activity and mitochondrial biogenesis. The TSH effects on MTCO1, TFAM, and porin could be abolished by K1-70, a TSH-receptor antagonist, suggesting a TSH receptor mediated action. Notably, as measured by calorimetry, T-3 and TSH increased follicular heat production, whereas T-3/T-4 and TRH stimulated ATP production in cultured HF keratinocytes. HPT-axis hormones did not increase reactive oxygen species (ROS) production. Rather, T-3 and T-4 reduced ROS formation, and all tested HPT-axis hormones increased the transcription of ROS scavengers (catalase, superoxide dismutase 2) in HF keratinocytes. Thus, mitochondrial biology, energy metabolism, and redox state of human HFs are subject to profound (neuro-)endocrine regulation by HPT-axis hormones. The neuroendocrine control of mitochondrial biology in a complex human mini-organ revealed here may be therapeutically exploitable.

https://doi.org/10.1038/jid.2013.286

74. Vogt A. et al. Hair follicle targeting, penetration enhancement and Langerhans cell activation make cyanoacrylate skin surface stripping a promising delivery technique for transcutaneous immunization with large molecules and particle-based vaccines // Exp. Dermatol. 2015. Vol. 24, № 1. P. 73–75.

Transcutaneous immunization (TCI) requires targeting of a maximum number of skin antigen-presenting cells as non-invasive as possible on small skin areas. In two clinical trials, we introduced cyanoacrylate skin surface stripping (CSSS) as a safe method for TCI. Here, using ex vivo human skin, we demonstrate that one CSSS procedure removed only 30% of stratum corneum, but significantly increased the penetration of 200nm polystyrene particles deep into vellus and intermediate hair follicles from where they could not been retrieved by conventional tape stripping. Two subsequent CSSS had no striking additional effect. CSSS increased particle penetration in superficial stratum corneum and induced Langerhans cell activation. Formulation in amphiphilic ointment or massage did not substantially influences the interfollicular penetration profiles. Hair follicle (HF) targeting by CSSS could become a highly effective tool for TCI when combined with carrier-based delivery and is gaining new attention as our understanding on the HF immune system increases.

https://doi.org/10.1111/exd.12589

75. Vogt A., Blume-Peytavi U. Selective hair therapy: bringing science the fiction // Exp. Dermatol. 2014. Vol. 23, № 2. P. 83–86.

Investigations on carrier-based drug delivery systems for higher selectivity in hair therapy have clearly evolved from dye release and model studies to highly sophisticated approaches, many of which specifically tackle hair indications and the delivery of hair-relevant molecules. Here, we group recent hair disease-oriented work into efforts towards (i) improved delivery of conventional drugs, (ii) delivery of novel drug classes, for example biomolecules and (iii) targeted delivery on the cellular/molecular level. Considering the solid foundation of experimental work, it does not take a large step outside the current box of thinking to follow the idea of using large carriers (>500nm, unlikely to penetrate as a whole) for follicular penetration, retention and protection of sensitive compounds. Yet, reports on particles <200nm being internalized by keratinocytes and dendritic cells at sites of barrier disruption (e.g., hair follicles) combined with recent advances in nanodermatology add interesting new facets to the possibilities carrier technologies could offer, for example, unprecedented levels of selectivity. The authors provide thought-provoking ideas on how smart delivery technologies and advances in our molecular understanding of hair pathophysiology could result in a whole new era of hair therapeutics. As the field still largely remains in preclinical investigation, determined efforts towards production of medical grade material and truly translational work are needed to demonstrate surplus value of carrier systems for clinical applications.

https://doi.org/10.1111/exd.12318

76. Wu Z. et al. Identification of Differentially Expressed miRNAs between White and Black Hair Follicles by RNA-Sequencing in the Goat (Capra hircus) // Int. J. Mol. Sci. 2014. Vol. 15, № 6. P. 9531–9545.

MicroRNAs (miRNAs) play a key role in many biological processes by regulating gene expression at the post-transcriptional level. A number of miRNAs have been identified from livestock species. However, compared with other animals, such as pigs and cows, the number of miRNAs identified in goats is quite low, particularly in hair follicles. In this study, to investigate the functional roles of miRNAs in goat hair follicles of goats with different coat colors, we sequenced miRNAs from two hair follicles samples (white and black) using Solexa sequencing. A total of 35,604,016 reads were obtained, which included 30,878,637 clean reads (86.73%). MiRDeep2 software identified 214 miRNAs. Among them, 205 were conserved among species and nine were novel miRNAs. Furthermore, DESeq software identified six differentially expressed miRNAs. Quantitative PCR confirmed differential expression of two miRNAs, miR-10b and miR-211. KEGG pathways were analyzed using the DAVID website for the predicted target genes of the differentially expressed miRNAs. Several signaling pathways including Notch and MAPK pathways may affect the process of coat color formation. Our study showed that the identified miRNAs might play an essential role in black and white follicle formation in goats.

https://doi.org/10.3390/ijms15069531

77. Xiong Y. et al. Identification of Wnt/beta-catenin signaling pathway in dermal papilla cells of human scalp hair follicles: TCF4 regulates the proliferation and secretory activity of dermal papilla cell // J. Dermatol. 2014. Vol. 41, № 1. P. 84–91.

It is clear that the dermal papilla cell (DPC), which is located at the bottom of the hair follicle, is a special mesenchymal component, and it plays a leading role in regulating hair follicle development and periodic regeneration. Recent studies showed that the Wnt signaling pathway through -catenin (canonical Wnt signaling pathway) is an essential component in maintaining the hair-inducing activity of the dermal papilla and growth of hair papilla cells. However, the intrinsic pathways and regulating mechanism are largely unknown. In the previous work, we constructed a cDNA subtractive library of DPC and first found that the TCF4 gene, as a key factor of Wnt signaling pathway, was expressed as the upregulated gene of the hair follicle in low-passage DPC. This study was to explore the role of TCF4 in regulating the proliferation and secretory activity of DPC. We constructed a pcDNA3.0-TCF4 expression vector and transfected it into DPC to achieve stable expression by bangosome 2000. Furthermore, we used the method of chemosynthesis to synthesize three pairs of TCF4 siRNA and transfected them into DPC. Meanwhile, we compared the transfection group and non-transfection group. We first proposed that there was expression difference in TCF4 in DPC under different biological condition. This study may have a high impact on the molecular mechanism of follicular lesions and provide a new vision for the treatment of clinic diseases.

https://doi.org/10.1111/1346-8138.12313

78. Yang R., Xu X. Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles // J. Vis. Exp. 2013. № 74. P. UNSP e3194.

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells(1). This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells(2), melanocyte stem cells(3) and neural crest like stem cells(4-7). Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles(4,5). These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury(8). Furthermore, peripheral nerves have been repaired with stem cell grafts(9), and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination(10). Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models. Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

https://doi.org/10.3791/3194

79. Yoon J.-S. et al. Development of a Model for Chemotherapy-Induced Alopecia: Profiling of Histological Changes in Human Hair Follicles after Chemotherapy // J. Invest. Dermatol. 2016. Vol. 136, № 3. P. 584–592.

Optimized research models are required to further understand the pathogenesis and prophylaxis of chemotherapy-induced alopecia. Our aim was to develop a mouse model for chemotherapy-induced alopecia by follicular unit transplantation of human hair follicles onto immunodeficient mice. Twenty-two weeks after transplantation, a single dose of cyclophosphamide (Cph) was administered to mice in the Cph100 (100 mg/kg) and Cph150 (150 mg/kg) groups. On day 6, hair follicles showed dystrophic changes, with swollen dermal papilla and ectopic melanin clumping in the hair bulb. In addition, upregulated expression of apoptotic regulators [P53, Fas/Fas-ligand, tumor necrosis factor-related apoptosis-inducing ligand/tumor necrosis factor-related apoptosis-inducing ligand receptor (TRAIL/TRAIL receptor), and Bax], increased apoptotic matrix keratinocytes, downregulated Ki67 expression, and decreased melanogenic protein in the hair bulb were noted in both groups. After 12 treatment days, hair follicles in Cph100 mice appeared to diminish dystrophic changes. In contrast, hair follicles of Cph150 mice prematurely entered a dystrophic catagen phase after 9 treatment days, and immunofluorescence staining for Ki67 and melanogenic protein expressions was barely visible. Two hair follicle damage response pathways were observed in this model, namely dystrophic anagen (Cph100) and catagen (Cph150) pathways. Our model might be useful for further understanding the impact of chemotherapy on human hair follicles.

https://doi.org/10.1038/JID.2015.358

80. Yoshikawa K. et al. Multipotent stem cells are effectively collected from adult human cheek skin // Biochem. Biophys. Res. Commun. 2013. Vol. 431, № 1. P. 104–110.

Skin-derived precursor (SKP) cells are a valuable resource for tissue engineering and regenerative medicine, because they represent multipotent stem cells that differentiate into neural and mesodermal progenies. Previous studies suggest that the stem cell pool decreases with age. Here, we show that human multipotent SKP cells can be efficiently collected from adult cheek/chin skin, even in aged individuals of 70-78 years. SKP cells were isolated from 38 skin samples by serum-free sphere culture and examined for the ability to differentiate into neural and mesodermal lineages. The number of spheres obtained from adult facial skin was significantly higher than that of trunk or extremity skin. SKP cells derived from cheek/chin skin exhibited a high ability to differentiate into neural and mesodermal cells relative to those derived from eyelid, trunk, or extremity skin. Furthermore, cheek/chin skin SKP cells were shown to express markers for undifferentiated stem cells, including a high expression level of the Sox9 gene. These results indicate that cheek/chin skin is useful for the recovery of multipotent stem cells for tissue engineering and regenerative therapy. (C) 2012 Elsevier Inc. All rights reserved.

https://doi.org/10.1016/j.bbrc.2012.12.069

81. Yoshikawa Y. et al. Transcriptional Analysis of Hair Follicle-Derived Keratinocytes from Donors with Atopic Dermatitis Reveals Enhanced Induction of IL32 Gene by IFN-gamma // Int. J. Mol. Sci. 2013. Vol. 14, № 2. P. 3215–3227.

We cultured human hair follicle-derived keratinocytes (FDKs) from plucked hairs. To gain insight into gene expression signatures that can distinguish atopic dermatitis from non-atopic controls without skin biopsies, we undertook a comparative study of gene expression in FDKs from adult donors with atopic dermatitis and non-atopic donors. FDK primary cultures (atopic dermatitis, n = 11; non-atopic controls, n = 7) before and after interferon gamma (IFN-gamma) treatment were used for microarray analysis and quantitative RT-PCR. Comparison of FDKs from atopic and non-atopic donors indicated that the former showed activated pathways with innate immunity and decreased pathways of cell growth, as indicated by increased NLRP2 expression and decreased DKK1 expression, respectively. Treatment with IFN-gamma induced the enhanced expression of IL32, IL1B, IL8, and CXCL1 in the cells from atopic donors compared to that in cells from non-atopic donors at 24 h after treatment. IL1B expression in FDKs after IFN-gamma treatment correlated with IL32 expression. We hypothesized that overexpression of IL32 in hair follicle keratinocytes of patients with atopic dermatitis would lead to the excessive production of IL1 beta and that the activation of IL1 beta from pro-IL1 beta by inflammasome complex, in which NLRP2 protein might be involved, would be augmented. This is the first report to show enhanced induction of cytokine/chemokine genes by IFN-gamma in atopic dermatitis using cultured FDKs.

https://doi.org/10.3390/ijms14023215

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