RESUMEN
Telomerase is present in cells with numerous or even un-limited replicative cycles, and some studies suggest it is a stemness marker. In order to determine whether this is the case for the human hair bulbs, an immunohistochemical and ultrastructural study has been carried out using antibodies against telomerase and PCNA (a cell proliferation marker). The observed labeling is similar for the two antibodies here utilized and is mainly nuclear. More frequent telomerase-positive cells are seen in the matrix epithelium of anagen hair bulbs but sparse labeled cells are also seen in the outer root sheath. In late catagen and also in telogen hair follicles only sparse labeled cells are present in the outer root sheath and few cells also in the secondary germinal epithelium formed at the base of the hair bulb in telogen. Electron microscopic immunogold shows a prevalent nuclear distribution and a lower cytoplasmic distribution in sparse cells of anagen bulb matrix that contain few keratin bundles. The nuclear localization is generally seen over the euchromatin or in areas occupied by more compact chromatin that may indicate an activity of telomerase in chromatin assemblage or dis-assemblage. The study concludes that the localization of telomerase is present in cells undergoing proliferation, namely transit amplifying cells of the outer root sheath that are sparsely detected in the lowermost secondary germinal hair bulb also in telogen.
Asunto(s)
Proliferación Celular , Folículo Piloso , Telomerasa , Humanos , Telomerasa/metabolismo , Folículo Piloso/metabolismo , Folículo Piloso/ultraestructura , Folículo Piloso/citología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Inmunohistoquímica , Cabello/metabolismo , Cabello/ultraestructura , Cabello/citología , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructuraRESUMEN
Niche signals maintain stem cells in a prolonged quiescence or transiently activate them for proper regeneration1. Altering balanced niche signalling can lead to regenerative disorders. Melanocytic skin nevi in human often display excessive hair growth, suggesting hair stem cell hyperactivity. Here, using genetic mouse models of nevi2,3, we show that dermal clusters of senescent melanocytes drive epithelial hair stem cells to exit quiescence and change their transcriptome and composition, potently enhancing hair renewal. Nevus melanocytes activate a distinct secretome, enriched for signalling factors. Osteopontin, the leading nevus signalling factor, is both necessary and sufficient to induce hair growth. Injection of osteopontin or its genetic overexpression is sufficient to induce robust hair growth in mice, whereas germline and conditional deletions of either osteopontin or CD44, its cognate receptor on epithelial hair cells, rescue enhanced hair growth induced by dermal nevus melanocytes. Osteopontin is overexpressed in human hairy nevi, and it stimulates new growth of human hair follicles. Although broad accumulation of senescent cells, such as upon ageing or genotoxic stress, is detrimental for the regenerative capacity of tissue4, we show that signalling by senescent cell clusters can potently enhance the activity of adjacent intact stem cells and stimulate tissue renewal. This finding identifies senescent cells and their secretome as an attractive therapeutic target in regenerative disorders.
Asunto(s)
Cabello , Melanocitos , Transducción de Señal , Animales , Ratones , Cabello/citología , Cabello/crecimiento & desarrollo , Folículo Piloso/citología , Folículo Piloso/fisiología , Receptores de Hialuranos/metabolismo , Melanocitos/citología , Melanocitos/metabolismo , Nevo/metabolismo , Nevo/patología , Osteopontina/metabolismo , Células Madre/citologíaRESUMEN
Liposomal honokiol isolated from the genus Magnolia has been found to have antiangiogenic, anti-inflammatory and antitumor properties. However, there has no report on its role in hair growth. Hair follicles are life-long cycled organelles that go through from anagen, catagen and telogen stages and are regulated by diverse signaling pathways, including Wnt/ß-catenin, Notch, Epidermal growth factor (EGF) and Sonic hegehog (SHH). Wnt signals are essential for the initiation of hair follicle placode development and a new potential target of hair loss treatment. This study was designed to investigate the effect of liposomal honokiol (Lip-honokiol) on inducing hair anagen. We identified the hair grew out in advance in the shaving area of C57BL/6N mice after the treatment of liposomal honokiol (Lip-honokiol) by daily abdominal injection. We first demonstrated that Lip-Honokiol activated the Wnt3a/ß-catenin pathway and downregulated the transforming growth factor-ß1 (TGF-ß1) to promote hair growth in mice via immunohistochemistry and immunofluorescence staining. These findings suggest that Lip-honokiol activated the Wnt/ß-catenin pathway and accelerated the transfer from the telogen to anagen stage and finally promoted the hair growth.
Asunto(s)
Compuestos de Bifenilo/administración & dosificación , Folículo Piloso/efectos de los fármacos , Folículo Piloso/metabolismo , Lignanos/administración & dosificación , Factor de Crecimiento Transformador beta1/biosíntesis , Proteína Wnt3A/biosíntesis , beta Catenina/biosíntesis , Animales , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Femenino , Cabello/citología , Cabello/efectos de los fármacos , Cabello/metabolismo , Folículo Piloso/citología , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta1/antagonistas & inhibidoresRESUMEN
Methotrexate (MTX), a folate antagonist, is the anchor drug used to treat several diseases. Therapeutic effects are attributed to intracellular levels of various methotrexate conjugates that are present in the cell as polyglutamates (MTX-Glu). The present study was conducted to develop a new liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based assay to separately quantitate the MTX-Glu in hair cells, red blood cells, and serum using internal standards. Sample preparation consisted of extraction with an organic solution followed by solid-phase extraction. The presented methodology was applied for the analysis of methotrexate and its polyglutamates in hair cells, red blood cells, and serum obtained from clinical patients. The developed LC-ESI-MS/MS method for the quantitative measurement of MTX-Glu was both sensitive and precise within the clinically relevant range. This method is possibly be superior with respect to sensitivity, selectivity, and speed than all previously described approaches and can be easily applied in routine clinical tests owing to the combination of a simple pretreatment process with robust LC-MS/MS.
Asunto(s)
Metotrexato/análisis , Cromatografía Líquida de Alta Presión , Eritrocitos , Cabello/química , Cabello/citología , Cabello/metabolismo , Humanos , Metotrexato/metabolismo , Plasma/química , Plasma/citología , Plasma/metabolismo , Ácido Poliglutámico/análisis , Reproducibilidad de los Resultados , Extracción en Fase Sólida , Espectrometría de Masas en TándemRESUMEN
The dermal papilla comprises mesenchymal cells in hair follicles, which play the main role in regulating hair growth. Maintaining the potential hair inductivity of dermal papilla cells (DPCs) and dermal sheath cells during cell culture is the main factor in in vitro morphogenesis and regeneration of hair follicles. Using common methods for the cultivation of human dermal papilla reduces the maintenance requirements of the inductive capacity of the dermal papilla and the expression of specific dermal papilla biomarkers. Optimizing culture conditions is therefore crucial for DPCs. Moreover, exosomes appear to play a key role in regulating the hair follicle growth through a paracrine mechanism and provide a functional method for treating hair loss. The present review investigated the biology of DPCs, the molecular and cell signaling mechanisms contributing to hair follicle growth in humans, the properties of the dermal papilla, and the effective techniques in maintaining hair inductivity in DPC cultures in humans as well as hair follicle bioengineering.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Dermis/citología , Dermis/crecimiento & desarrollo , Folículo Piloso/citología , Folículo Piloso/crecimiento & desarrollo , Dermis/metabolismo , Cabello/citología , Cabello/crecimiento & desarrollo , Cabello/metabolismo , Folículo Piloso/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Regeneración/fisiologíaRESUMEN
Keratinocytes, as a primary somatic cell source, offer exceptional advantages compared to fibroblasts, which are commonly used for reprogramming. Keratinocytes can beat fibroblasts in reprogramming efficiency and reprogramming time and, in addition, can be easily and non-invasively harvested from human hair roots. However, there is still much to know about acquiring keratinocytes and maintaining them in cell culture. In this article, we want to offer readers the profound knowledge that we have gained since our initial use of keratinocytes for reprogramming more than 10 years ago. Here, all hints and tricks, from plucking the hair roots to growing and maintaining keratinocytes, are described in detail. Additionally, an overview of the currently used reprogramming methods, viral and non-viral, is included, with a special focus on their applicability to keratinocytes. This overview is intended to provide a brief but comprehensive insight into the field of keratinocytes and their use for reprogramming into induced pluripotent stem cells (iPSCs). © 2020 The Authors.
Asunto(s)
Técnicas de Reprogramación Celular , Cabello/citología , Células Madre Pluripotentes Inducidas/citología , Queratinocitos/citología , Diferenciación Celular , Células Cultivadas , HumanosRESUMEN
Rab27a/b are known to play an important role in the transport of melanosomes, with their knockout causing silvery gray hair. However, the relationship between Rab27a/b and hair growth is not well known. To evaluate the role of Rab27a/b in hair cycle, we investigated the expression of Rab27a/b during hair cycling and human outer root sheath (hORS) cells. The expression of Rab27a in ORS cells was mainly detected at the anagen, whereas expression of Rab27b in ORS, and epidermal cells was strongly expressed at the telogen. Additionally, Rab27a/b were expressed in the Golgi of hORS cells. To evaluate the role of Rab27a/b in hair growth, telogen-to-anagen transition animal and vibrissae hair follicles (HFs) organ culture models were assayed using Rab27a/b siRNAs. The knockdown of Rab27a or Rab27b suppressed or promoted hair growth, respectively. These results were also confirmed in human dermal papilla cells (hDPCs) and hORS cells, showing the opposite mitogenic effects. Moreover, Rab27b knockdown increased the expression levels of various growth factors in the hDPCs and hORS cells. Overall, the opposite temporal expression patterns during hair cycling and roles for hair growth of Rab27a/b suggested that Rab27a/b might regulate the hair cycle. Therefore, our study may provide a novel solution for the development of hair loss treatment by regulating Rab27a/b levels.
Asunto(s)
Cabello/crecimiento & desarrollo , Proteínas de Unión al GTP rab/antagonistas & inhibidores , Proteínas rab27 de Unión a GTP/antagonistas & inhibidores , Animales , Dermis/citología , Cabello/citología , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Modelos Biológicos , Regulación hacia Arriba , Vibrisas/crecimiento & desarrollo , Proteínas de Unión al GTP rab/metabolismo , Proteínas rab27 de Unión a GTP/metabolismoRESUMEN
Piloerection (goosebumps) requires concerted actions of the hair follicle, the arrector pili muscle (APM), and the sympathetic nerve, providing a model to study interactions across epithelium, mesenchyme, and nerves. Here, we show that APMs and sympathetic nerves form a dual-component niche to modulate hair follicle stem cell (HFSC) activity. Sympathetic nerves form synapse-like structures with HFSCs and regulate HFSCs through norepinephrine, whereas APMs maintain sympathetic innervation to HFSCs. Without norepinephrine signaling, HFSCs enter deep quiescence by down-regulating the cell cycle and metabolism while up-regulating quiescence regulators Foxp1 and Fgf18. During development, HFSC progeny secretes Sonic Hedgehog (SHH) to direct the formation of this APM-sympathetic nerve niche, which in turn controls hair follicle regeneration in adults. Our results reveal a reciprocal interdependence between a regenerative tissue and its niche at different stages and demonstrate sympathetic nerves can modulate stem cells through synapse-like connections and neurotransmitters to couple tissue production with demands.
Asunto(s)
Nervio Accesorio/fisiología , Folículo Piloso/citología , Cabello/crecimiento & desarrollo , Proteínas Hedgehog/metabolismo , Norepinefrina/metabolismo , Transducción de Señal/genética , Células Madre/metabolismo , Células Madre/fisiología , Nervio Accesorio/citología , Animales , Ciclo Celular/genética , Frío , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Cabello/citología , Cabello/fisiología , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Piloerección , RNA-Seq , Receptores Adrenérgicos beta 2/deficiencia , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Nicho de Células Madre , Células Madre/citología , Sistema Nervioso Simpático/citología , Sistema Nervioso Simpático/fisiología , Sinapsis/fisiologíaRESUMEN
The skin is a multilayered organ, equipped with appendages (that is, follicles and glands), that is critical for regulating body temperature and the retention of bodily fluids, guarding against external stresses and mediating the sensation of touch and pain1,2. Reconstructing appendage-bearing skin in cultures and in bioengineered grafts is a biomedical challenge that has yet to be met3-9. Here we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use stepwise modulation of the transforming growth factor ß (TGFß) and fibroblast growth factor (FGF) signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During an incubation period of 4-5 months, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis and pigmented hair follicles that are equipped with sebaceous glands. A network of sensory neurons and Schwann cells form nerve-like bundles that target Merkel cells in organoid hair follicles, mimicking the neural circuitry associated with human touch. Single-cell RNA sequencing and direct comparison to fetal specimens suggest that the skin organoids are equivalent to the facial skin of human fetuses in the second trimester of development. Moreover, we show that skin organoids form planar hair-bearing skin when grafted onto nude mice. Together, our results demonstrate that nearly complete skin can self-assemble in vitro and be used to reconstitute skin in vivo. We anticipate that our skin organoids will provide a foundation for future studies of human skin development, disease modelling and reconstructive surgery.
Asunto(s)
Cabello/citología , Cabello/crecimiento & desarrollo , Organoides/citología , Células Madre Pluripotentes/citología , Piel/citología , Animales , Ectodermo/citología , Femenino , Cabello/trasplante , Color del Cabello , Folículo Piloso/citología , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/inervación , Folículo Piloso/trasplante , Cabeza , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Organoides/crecimiento & desarrollo , Organoides/inervación , Organoides/trasplante , RNA-Seq , Análisis de la Célula Individual , Piel/crecimiento & desarrollo , Piel/inervación , Trasplante de PielRESUMEN
Conventional therapeutic applications of mesenchymal stromal cells (MSCs) focus on cell replacement and differentiation; however, increasing evidence suggests that most of their therapeutic effects are carried out by their various secretions. This study investigated the application of conditioned medium (CM) from human umbilical cord blood-derived MSCs (hUCB-MSCs) to improve hair growth and developed a method to reliably produce this optimized CM. Primed MSC-derived CM (P-CM) with combinations of TGF-ß1 and LiCl was optimized by comparing its effects on the cell viability of dermal papilla cells (DPCs). P-CM significantly increased the viability of DPCs compared to CM. The secretion of vascular endothelial growth factor (VEGF) in DPCs was regulated by the macrophage migration inhibitory factor (MIF) in the P-CM secreted by MSCs. These findings suggest that P-CM can improve the efficacy in hair growth via a paracrine mechanism and that MIF in P-CM exerts hair growth-promoting effects via a VEGF-related ß-catenin and p-GSK-3ß [SER9] signaling pathway. Furthermore, clinical trials have shown that 5% P-CM improved androgenetic alopecia through producing an increased hair density, thickness, and growth rate, suggesting that this topical agent may be a novel and effective treatment option for patients with androgenetic alopecia.
Asunto(s)
Medios de Cultivo Condicionados/química , Sangre Fetal/citología , Cabello/crecimiento & desarrollo , Factores Inhibidores de la Migración de Macrófagos/farmacología , Células Madre Mesenquimatosas/citología , Adulto , Alopecia/patología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Femenino , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Cabello/citología , Cabello/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Cloruro de Litio/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Persona de Mediana Edad , Modelos Biológicos , Factor de Crecimiento Transformador beta1/farmacología , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adulto Joven , beta Catenina/metabolismoRESUMEN
Context: Although Salvia plebeia (SP) R. Brown (Labiatae) is known to possess various biological activities, the effects of SP on hair growth have not been elucidated.Objective: To investigate the hair growth potential of SP extract by using human dermal papilla cells (hDPCs) and C57BL/6 mice.Materials and methods: The entire SP plant sample was ground into powder and extracted with 99.9% methyl alcohol. Various concentrations of SP extract were added to hDPCs to evaluate the proliferation, migration, and factors related to hair growth and cycling. Effect of topical SP administration on hair regrowth was tested in vivo in male C57BL/6 mice for 21 days.Results: SP extract significantly increased the proliferation of cultured hDPCs at doses of 15.6 and 31.3 µg/mL compared to control group by 123% and 132%, respectively. Expression of hepatocyte growth factor increased while the level of TGF-ß1 and SMAD2/3 decreased when treated with SP extract. At the molecular level, the extract activated Wnt/ß-catenin signalling by raising ß-catenin and phospho-GSK3ß expression. SP extract also exerted anti-apoptotic and proliferative effects in hDPCs by increasing the Bcl-2/Bax ratio and activating cell proliferation-related proteins, ERK and Akt. Finally, the extract caused an induction of the anagen phase leading to significantly enhanced hair growth in treated male mice.Discussion and conclusion: Our results indicate that SP extract has the capacity to activate hDPCs into a proliferative state to promote hair growth. Further research is necessary to determine the bioactive components and their mechanisms of action responsible for SP-related hair growth effect.
Asunto(s)
Folículo Piloso/efectos de los fármacos , Folículo Piloso/crecimiento & desarrollo , Extractos Vegetales/farmacología , Salvia , Animales , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Cabello/citología , Cabello/efectos de los fármacos , Cabello/crecimiento & desarrollo , Folículo Piloso/citología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Extractos Vegetales/aislamiento & purificaciónRESUMEN
Alopecia is a common and distressing condition, and developing new therapeutic agents to prevent hair loss is important. Human umbilical cord bloodderived mesenchymal stem cells (hUCBMSCs) have been studied intensively in regenerative medicine. However, the therapeutic potential of these cells against hair loss and hair organ damage remains unclear, and the effects of hUCBMSC transplantation on hair loss require evaluation. The current study aimed to investigate the effects of hUCBMSCs on hair regression in vivo and restoration of anagen conduction on hair growth in vitro. The effects of hUCBMSCs were explored in mouse catagen induction models using a topical treatment of 0.1% dexamethasone to induce hair regression. Dexamethasone was also used to simulate a stress environment in vitro. The results demonstrated that hUCBMSCs significantly prevented hair regression induced by dexamethasone topical stimulation in vivo. Additionally, hUCBMSCs significantly increased the proliferation of human dermal papilla cells (hDPCs) and HaCaT cells, which are key constituent cells of the hair follicle. Stimulation of vascular endothelial growth factor secretion and decreased expression of DKK1 by hUCBMSCs were also observed in hDPCs. Restoration of cell viability by hUCBMSCs suggested that these cells exerted a protective effect on glucocorticoid stressassociated hair loss. In addition, antiapoptotic effects and regulation of the autophagic flux recovery were observed in HaCaT cells. The results of the present study indicated that hUCBMSCs may have the capacity to protect hair follicular dermal papilla cells and keratinocytes, thus preventing hair loss. Additionally, the protective effects of hUCBMSCs may be resistant to dysregulation of autophagy under harmful stress.
Asunto(s)
Antiinflamatorios/efectos adversos , Dexametasona/efectos adversos , Folículo Piloso/citología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Células Cultivadas , Femenino , Sangre Fetal/citología , Cabello/citología , Cabello/efectos de los fármacos , Cabello/crecimiento & desarrollo , Cabello/ultraestructura , Folículo Piloso/efectos de los fármacos , Folículo Piloso/ultraestructura , Humanos , Ratones Endogámicos C57BLRESUMEN
Drug overdose (DO) is considered one of the current issues of intravenous (IV) infusion particularly resulting in serious injuries and deaths. Malfunction of infusion pumps is reported as the main cause of the drug overdose. Live monitoring and flow rate calculation by health professionals have been practicing to avoid DO. However, human errors and miscalculations are inevitable. A secondary measurement tool is required to avoid the risk of OD when infusion pump malfunctions cannot be detected immediately. Here, inspired by nature, we developed a real-time monitoring device through which an administrator can review, evaluate, and modify the IV infusion process. Our flow sensor possesses an erected polymer hair cell on a multi-layered silicon base forming from a patterned gold strained gauge layer on a piezoresistive liquid crystal polymer (LCP) membrane. Gold strain gauges on an LCP membrane have been used instead of a piezoresistive silicon membrane as the sensing element. The combination of gold strain gauges and LCP membrane provides better sensitivity than a piezoresistive silicon membrane of the same dimensions and thickness. We also miniaturized our biocompatible sensor such that it can be possible to install it inside the IV tube in contact with the liquid providing an in-suite online flow monitoring. The proposed LCP membrane sensor is compared with two commercially available IV sensors to validate its flow sensing ability. The experimental results demonstrate that the proposed sensor provides a low threshold detection limit of 5 mL/hr, which betters the performance of other commercial sensors at low flow rates.
Asunto(s)
Materiales Biocompatibles/química , Oro/química , Infusiones Intravenosas/instrumentación , Infusiones Intravenosas/métodos , Polímeros/química , Silicio/química , Animales , Técnicas Biosensibles , Línea Celular , Simulación por Computador , Diseño de Equipo , Peces , Gryllidae , Cabello/citología , Humanos , Cinética , Límite de Detección , Propiedades de SuperficieRESUMEN
The dermal papilla (DP) is a key mesenchymal compartment of hair follicles that orchestrates mesenchymal-epithelial interaction regulating hair growth cycles. In the present study, we demonstrate that a TALE-family transcription factor, Meis1, is selectively localized in the nucleus of the DP in the anagen phase of the hair cycle. By using an ex vivo organ culture of vibrissae follicles, conditional Meis1 loss causes retardation in hair growth, accompanied by defects in cell proliferation of hair matrix cells. This cell proliferation defect is partly rescued by the addition of culture supernatants derived from Meis1-sufficient but not -deficient DP cells. These findings indicate that nuclear Meis1 in DP activate genes involved in secretion of some unknown factors, which promote proliferation of hair matrix cells in the anagen phase of the hair cycle.
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Proliferación Celular , Dermis/metabolismo , Folículo Piloso/metabolismo , Cabello/metabolismo , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/metabolismo , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Dermis/citología , Regulación de la Expresión Génica , Cabello/citología , Cabello/crecimiento & desarrollo , Folículo Piloso/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , Técnicas de Cultivo de Órganos , Vibrisas/citología , Vibrisas/metabolismoRESUMEN
The objective of the present study was to optimize genotyping of nuclear DNA contained in human hair. The most efficient procedures for DNA isolation and typing are described taking into consideration the hair growth phase, the epithelial tissue conditions, and the number of nuclei in the near-root ends of the hair. The recommendations for the expert interpretation of the results of the molecular-genetic investigations are proposed.
Asunto(s)
Núcleo Celular/genética , Dermatoglifia del ADN , ADN/aislamiento & purificación , Genotipo , Cabello/citología , HumanosRESUMEN
The aim of the present study was to accomplish de novo generation of reconstituted human skin with enriched hair follicles. Dermal papillae (DP) are known to play a crucial organizing role in hair follicle induction. However, generation of enriched human hair follicles using cultured DP cells has not been accomplished because DP cells easily lose their hair-inducing ability with culturing. To enhance the hair-inducing ability of DP cells, Wnt signaling pathway activation or three-dimensional (3D) spheroid culture methods were employed in previous studies. Herein, we assessed effects of the canonical Wnt/ß-catenin signaling activator CHIR99021 and found that it enhanced the expression of DP signature genes associated with hair-inducing ability. Further comparison of three different 3D culture methods revealed the highest expression of DP signature genes in spheroids generated by a floating drop method compared with other methods. CHIR99021 synergistically increased expression of DP signature genes in combination with floating drop culture. "Reconstituted skin assay" prepared using the most promising CHIR99021-stimulated 3D spheroids showed enrichment for human hair follicles. Labeled DP spheroids and derived cells were primarily found to be DP and dermal sheath cup (DSC) cells, implying organization of hair formation by DP spheroids. Finally, to evaluate the functional features of generated human skin and hair follicles, we injected human DSC cells, which reportedly show DP precursor behavior, and exhibit hair-inducing ability through incorporation into hair follicles, into mice. Histological studies revealed injected DSC cells in dermal sheath of hair follicles, consistent with a previous report, thus verifying the functionality of generated skin and hair follicles. Collectively, our findings demonstrate that DP spheroids synergistically stimulated by CHIR99021 and 3D culture contributed to hair follicle formation, thus making it possible to generate reconstituted hair follicle-enriched human skin with functional features.
Asunto(s)
Dermis/efectos de los fármacos , Folículo Piloso/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , Piel/efectos de los fármacos , Esferoides Celulares/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Animales , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Dermis/citología , Dermis/metabolismo , Expresión Génica/efectos de los fármacos , Cabello/citología , Cabello/efectos de los fármacos , Cabello/metabolismo , Folículo Piloso/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Factor de Unión 1 al Potenciador Linfoide/genética , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Ratones , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Piel/citología , Piel/metabolismo , Esferoides Celulares/metabolismo , Vía de Señalización Wnt/genéticaRESUMEN
Adiponectin (APN), released mainly from adipose tissue, is a well-known homeostatic factor for regulating glucose levels, lipid metabolism, and insulin sensitivity. A recent study showed that human hair follicles express APN receptors and the presence of APN-mediated hair growth signaling, thereby suggesting that APN is a potent hair growth-promoting adipokine. Previously, kojyl cinnamate ester derivatives (KCEDs) were synthesized in our institute as new anti-aging or adiponectin-/adipogenesis-inducing compounds. Here, we tested the activity of these derivatives to induce endogenous APN secretion. Among the derivatives, KCED-1 and KCED-2 showed improved activity in inducing APN mRNA expression, secretion of APN protein, and adipogenesis in human subcutaneous fat cells (hSCFs) when compared with the effects of Seletinoid G, a verified APN inducer. When human follicular dermal papilla cells were treated with the culture supernatant of KCED-1- or KCED-2-treated hSCFs, the mRNA expression of APN-induced hair growth factors such as insulin-like growth factor, hepatocyte growth factor, and vascular endothelial growth factor was upregulated compared with that in the control. Taken together, our study shows that among kojyl cinnamate ester derivatives, KCED-1, KCED-2, as well as Seletinoid G are effective inducers of endogenous APN production in subcutaneous fat tissues, which may in turn contribute to the promotion of hair growth in the human scalp.
Asunto(s)
Adiponectina/genética , Cinamatos/farmacología , Folículo Piloso/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Adiponectina/metabolismo , Línea Celular , Cinamatos/química , Ésteres/química , Ésteres/farmacología , Cabello/citología , Cabello/efectos de los fármacos , Cabello/crecimiento & desarrollo , Cabello/metabolismo , Folículo Piloso/citología , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismoRESUMEN
We demonstrated that the topical combined use of sponge Haliclona sp. spicules (SHS) and flexible liposomes (FL), referred to as SFLS (SHS-Flexible Liposomes combined System), can result in synergy to improve the skin absorption and deposition of hyaluronic acid (HA), especially in deep skin layers, both in vitro and in vivo. SHS treatment can result in skin micro-channels which are continuous, deep enough (48.6 ± 13.5 µm) and available in large quantities (850 ± 125 micro-channels per mm2). These micro-channels gradually closed up in 120 h and also allowed the intact vesicles of flexible liposomes and vesicle-bound or vesicle-encapsulated HA to penetrate into the skin-deep layers under the driving force of transdermal osmotic gradients. Specifically, SFLS topical application enhanced the penetration of FITC-HA (MW: 250 kDa) into porcine skin in vitro up to 23.2 ± 3.7%, which is 19.4 ± 3.1-fold (p < 0.001) that of a Phosphate Buffered Saline (PBS) group, 3.4 ± 0.5-fold (p < 0.01) that of an SHS group and 3.6 ± 0.6-fold (p < 0.01) that from the combined use of a Dermaroller and flexible liposomes. Moreover, SFLS can lead to significantly enhanced skin deposition of HA in all skin layers, especially in deep skin layers: up to 86.8 ± 4.1% of HA absorbed by skin was accumulated in deep skin layers. The effectiveness of SFLS topical application was also confirmed in vivo by using BALB/c mice. In addition, a skin irritation and toxicity study showed that the SFLS treatment may cause very minimal redness and the skin can recover in a short time. In sum, the combined use of SHS and FL (SFLS) offers a promising strategy to safely and effectively improve the skin delivery of hydrophilic biomacromolecules such as HA.
Asunto(s)
Ácido Hialurónico/química , Poríferos/química , Piel/química , Animales , Femenino , Cabello/química , Cabello/citología , Ácido Hialurónico/administración & dosificación , Liposomas/química , Ratones , Ratones Endogámicos BALB C , Piel/citología , Absorción Cutánea , PorcinosRESUMEN
There have been many studies about the formation, storage, transport and degradation of melanosomes in epidermal melanocytes but studies of melanocytes and melanosomes in fetal hair follicles (HFs) have been limited and ambiguous. The goal of this study was to investigate the distribution of melanocytes and the degradation of melanosomes in fetal HFs. After obtaining approval and informed consent for the study, a scalp specimen from a 5 month gestational age fetus was obtained and was divided into two parts. One part was subjected to immunohistochemical staining with the melanocyte-specific marker HMB-45 and was then observed by light microscopy to detect the distribution of melanocytes in HFs. The other part underwent conventional processing for transmission electron microcopy (TEM). Subsequently, the morphology of melanosomes in HF melanocytes and their degradation in cortical keratinocytes were observed. Immunohistochemically, scattered round melanocytes lacking dendrites were mainly observed along the outer root sheath of the lower part of the HF. A few fusiform or tri-dendritic melanocytes were located at the bottom of the hair bulbs. Significantly melanized melanocytes with multiple dendrites were concentrated in the pigmented area in the center of the hair bulbs, only above the dermal papilla. Analysis by TEM revealed melanocytes containing melanosomes at all stages of development. Autophagosomes containing stage mature IV melanosomes were observed in some melanocytes. Many phagolysosomes containing numerous melanosomes were observed in the cortical keratinocytes. Some phagolysosomes were concentrically surrounded by 3-5 layers of endoplasmic reticulum. Melanosomes that had been degraded or were being degraded in phagolysosomes in keratinocytes had lost their integrity and had become an ill-defined melanosomal dust that were arranged irregularly. Partial melanin particles were released into the cytosol. Melanocytes in different regions of fetal HFs had different morphologies and were at various stages of differentiation. Fetal HF melanocytes contained not only melanosomes at different developmental stages, but autophagosomes were seen occasionally. Melanosomes were degraded into irregular pigment particles in the phagolysosomes of cortical keratinocytes. These results provide important clues to elucidate the mechanism of melanosome biodegradation.