RESUMEN
In a clinical study of autologous cell-based therapy using dermal sheath cup (DSC) cells, the treatment of hair loss showed improvements. However, the outcomes were variable. Here, correlations between marker gene expression in DSC cells and treatment outcomes were assessed to predict therapeutic efficacy. Overall, 32 DSC cell lines were used to evaluate correlations between marker gene expression and treatment outcomes. Correlations between vascular pericyte and preadipocyte marker expression and treatment outcomes were inconsistent. As smooth muscle cell markers, MYOCD correlated negatively with treatment outcomes and SRF consistently demonstrated an inverse correlation. Additionally, CALD1 correlated negatively and ACTA2 correlated inversely with treatment outcomes. DSC cell lines were divided into good and moderate/poor responders to further investigate the correlations. SRF and CALD1 were lower in a good responder compared with a moderate responder. Next, DSC cells were differentiated toward dermal papilla cells. Dermal papilla markers SOX2 and LEF1 before differentiation had moderate positive and inverse correlations with the treatment outcome, respectively. SOX2 after differentiation more consistently demonstrated a positive correlation. Significant downregulation of smooth muscle-related genes was also observed after differentiation. These findings revealed putative markers for preclinical evaluation of DSC cells to improve hair loss.
Asunto(s)
Alopecia , Folículo Piloso , Alopecia/genética , Alopecia/metabolismo , Alopecia/terapia , Biomarcadores/metabolismo , Células Cultivadas , Femenino , Folículo Piloso/metabolismo , Humanos , Masculino , Miocitos del Músculo Liso/metabolismo , Piel/metabolismo , Resultado del TratamientoRESUMEN
BACKGROUND: Few effective treatments are available for male pattern hair loss (MPHL) or, especially, for female pattern hair loss (FPHL). Recently, cell-based therapies using autologous or allogeneic cells have been used clinically. OBJECTIVE: We examined the safety and efficacy of autologous cell-based therapy using dermal sheath cup (DSC) cells to treat MPHL and FPHL. METHODS: DSCs dissected from occipital hair follicles were cultured to manufacture DSC cells. Participants with MPHL or FPHL received single injections of 7.5 × 106, 1.5 × 106, or 3.0 × 105 DSC cells or a placebo in 4 randomized separate regions on the scalp, and hair densities and diameters were measured for 3, 6, 9, and 12 months. RESULTS: Fifty men and 15 women aged 33 to 64 years were injected with DSC cells. Total hair density and cumulative hair diameter at the 3.0 × 105 DSC cells injection site was significantly increased compared with the placebo after 6 and 9 months. Men and women showed similar improvements, and there were no serious adverse events. LIMITATIONS: No lower cell numbers were tested, and the positive effect was temporary until 9 months. CONCLUSION: The results suggest that cell therapy with autologous DSC cells may be useful as a new therapeutic method for treating MPHL and FPHL.
Asunto(s)
Alopecia/terapia , Trasplante de Células , Folículo Piloso/citología , Adulto , Trasplante de Células/efectos adversos , Método Doble Ciego , Femenino , Cabello/anatomía & histología , Cabello/crecimiento & desarrollo , Humanos , Masculino , Persona de Mediana Edad , Prueba de Estudio Conceptual , Trasplante Autólogo , Resultado del TratamientoRESUMEN
Dermal sheath (DS) is located at the outermost border of hair follicles, comprising the connective tissue sheath of these follicles; DS cells are known to contribute to hair cycling and follicle neogenesis. However, the mechanisms by which DS cells contribute to hair formation are currently unclear. We investigated the global transcriptional profile of human DS cells in early passaged culture, compared with those of human dermal papilla cells (DP cells) and dermal fibroblasts. Vascular related genes were highly expressed in DS cells, and expression of the multi-ligand receptor, CD36, was significantly higher in DS cells than in DP cells. Further analyses with whole-mount imaging technique showed that dense networks of blood capillaries were formed in the DS of human anagen hair follicles, whereas regression of blood capillaries was observed in telogen and catagen hair follicles. We found that CD36-expressing cells were present in populations of DS cells, but were rarely observed in populations of DP cells and fibroblasts. Furthermore, our results indicated that CD36-expressing DS cells may participate in angiogenesis. Therefore, we concluded that CD36-expressing DS cells may modulate blood capillaries in hair follicles, in association with hair cycling.
Asunto(s)
Antígenos CD36/genética , Dermis/metabolismo , Células Endoteliales/metabolismo , Fibroblastos/metabolismo , Folículo Piloso/metabolismo , Factor de Crecimiento de Hepatocito/genética , Antígenos CD36/metabolismo , Proliferación Celular , Técnicas de Cocultivo , Dermis/irrigación sanguínea , Dermis/citología , Células Endoteliales/citología , Fibroblastos/citología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Folículo Piloso/irrigación sanguínea , Folículo Piloso/citología , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Análisis por Micromatrices , Especificidad de Órganos , Cultivo Primario de CélulasRESUMEN
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
Cells that constitute the dermal papillae of hair follicles might be derived from the dermal sheath, the peribulbar component of which is the dermal sheath cup. The dermal sheath cup is thought to include the progenitor cells of the dermal papillae and possesses hair inductive potential; however, it has not yet been well characterized. This study investigated the gene expression profile of the intact dermal sheath cup, and identified dermal sheath cup signature genes, including extracellular matrix components and bone morphogenetic protein-binding molecules, as well as transforming frowth factor beta 1 as an upstream regulator. Among these, gremilin-2, a member of the bone morphogenetic protein antagonists, was found by in situ hybridization to be highly specific to the dermal sheath cup, implying that gremlin-2 is a key molecule contributing to maintenance of the properties of the dermal sheath cup.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Folículo Piloso/química , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcriptoma , Adulto , Anciano , Citocinas , Femenino , Redes Reguladoras de Genes , Humanos , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/genética , Persona de Mediana EdadRESUMEN
BACKGROUND: Autologous cell-based therapy using dermal sheath cup (DSC) cells was reported as a new treatment for male and female pattern hair loss. However, the mechanisms underlying its action remain unclear. OBJECTIVE: We investigated the mechanisms underlying the efficacy of DSC cells in cell-based therapy. METHODS: We conducted multivariate analysis to categorize individuals based on treatment response as responders and non-responders. The differentially expressed genes in DSC cells from the two groups were evaluated using bulk transcriptome, quantitative polymerase chain reaction, and single-cell transcriptome analyses. We performed live cell imaging combined with immunostaining to characterize the DSC subpopulation associated with responders. RESULTS: We identified nine and three genes as high efficacy (HE) and low efficacy (LE) marker genes, respectively. The HE subpopulations were enriched for cell migration-related genes in single-cell analysis. In contrast, the LE subpopulation was enriched for basement membrane and vasculature-related genes. Moreover, DSC cells in culture were immunocytochemically and morphologically heterogeneous, expressing characteristic factors. Furthermore, live cell imaging showed that DSC cells expressing integrin subunit alpha 6 (ITGA6), an HE subpopulation gene, had markedly higher mobility than those expressing the LE subpopulation genes collagen type IV or CD36. CONCLUSIONS: ITGA6-positive DSC cells, with superior migratory activity, may contribute to cell-based therapy by promoting cell migration into nearby hair follicles.
Asunto(s)
Alopecia , Células Epiteliales , Folículo Piloso , Femenino , Humanos , Masculino , Alopecia/terapia , Células Cultivadas , Folículo Piloso/metabolismo , Transcriptoma , Resultado del TratamientoRESUMEN
It is well known that dermal papilla cells (DPCs) play crucial roles in hair follicle induction. In this study, we examined whether Wnt/ß-catenin activation results in maintenance of the hair-inducing ability of human DPCs. Expression of DPC marker genes was maintained under Wnt/ß-catenin signalling stimulation by GSK-3ß inhibition. Furthermore, human DPCs showed constant hair induction when transplanted with murine epidermal cell fraction. Alu-positive human DPCs were essentially detected adjacent to the reconstructing epidermal structure positive for P-cadherin immunoreactivity. The transplanted human DPCs were abundant in the surrounding dermal sheath portion of the fully regenerated hair follicles. These results support the importance of Wnt/ß-catenin signalling in hair follicle induction. This study may provide valuable information to establish a culture method of human DPCs for cell-based therapy.
Asunto(s)
Dermis/citología , Dermis/metabolismo , Cabello/crecimiento & desarrollo , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Dermis/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta , Folículo Piloso/citología , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/metabolismo , Humanos , Ratones , Ratones Desnudos , Trasplante Heterólogo , Trasplantes , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
The aim of this study was to identify novel inducers of hair growth using gene expression profiling at various stages of hair-growth induction. First, we analyzed gene expression at the onset of hair growth in mice induced by cyclosporin A (CsA), a well-known hair-growth inducer, using DNA microarray analysis. The results unveiled genes involved in the step-by-step progression of hair growth, including increases in melanin biosynthesis and decreases in immune response at d 2 and the subsequent stimulation of cell proliferation at d 4, followed by the up-regulation of hair specific keratins at d 7 after CsA treatment. With the use of the connectivity map (Cmap), agents that had a similar "gene signature" to that of the profiles of CsA-treated mice were identified. Several agents, including CsA, were identified by the Cmap and were evaluated for hair induction activity in vivo. One of the proposed agents, fluphenazine (from the d 2 signature) actually induced hair growth in vivo (ED(50): 2 mM for single application), and the subsequent application of 5 mM iloprost (from the d 4 signature) significantly enhanced the hair-growth effect of fluphenazine. From these results, Cmap analysis was proven to be a useful method that connects gene expression profiles of complicated biological processes, such as hair-growth induction, to effective agents.
Asunto(s)
Descubrimiento de Drogas/métodos , Perfilación de la Expresión Génica , Folículo Piloso/efectos de los fármacos , Folículo Piloso/crecimiento & desarrollo , Animales , Ciclosporina/farmacología , Femenino , Folículo Piloso/metabolismo , Ratones , Ratones Endogámicos C3HRESUMEN
Epidermal lamellar granules transport various lipids, proteins, and protein inhibitors from the trans-Golgi network to the extracellular space, and play an important role in skin barrier formation. We elucidated the 3-dimensional structure of lamellar granules and the trans-Golgi network in normal human skin by focused ion beam scanning electron microscopy. Reconstructed focused ion beam scanning electron microscopy 3-dimensional images revealed that the overall lamellar granule structure changed from vesicular to reticular within the second layer of the stratum granulosum. Furthermore, the trans-Golgi network was well developed within this layer and spread through the cytoplasm with branched, tubular structures that connected to lamellar granules. Our study reveals the unique overall 3-dimensional structure of lamellar granules and the trans-Golgi network within the cells of the epidermis, and provides the basis for an understanding of the skin barrier formation.
Asunto(s)
Diferenciación Celular , Epidermis/fisiología , Queratinocitos/fisiología , Red trans-Golgi/ultraestructura , Adulto , Anciano , Epidermis/diagnóstico por imagen , Femenino , Humanos , Imagenología Tridimensional , Queratinocitos/ultraestructura , Masculino , Microscopía Electrónica de Rastreo , Persona de Mediana EdadRESUMEN
Adenosine upregulates the expression of vascular endothelial growth factor and fibroblast growth factor-7 in cultured dermal papilla cells. It has been shown that, in Japanese men, adenosine improves androgenetic alopecia due to the thickening of thin hair due to hair follicle miniaturization. To investigate the efficacy and safety of adenosine treatment to improve hair loss in women, 30 Japanese women with female pattern hair loss were recruited for this double-blind, randomized, placebo-controlled study. Volunteers used either 0.75% adenosine lotion or a placebo lotion topically twice daily for 12 months. Efficacy was evaluated by dermatologists and by investigators and in phototrichograms. As a result, adenosine was significantly superior to the placebo according to assessments by dermatologists and investigators and by self-assessments. Adenosine significantly increased the anagen hair growth rate and the thick hair rate. No side-effects were encountered during the trial. Adenosine improved hair loss in Japanese women by stimulating hair growth and by thickening hair shafts. Adenosine is useful for treating female pattern hair loss in women as well as androgenetic alopecia in men.
Asunto(s)
Adenosina/uso terapéutico , Alopecia/tratamiento farmacológico , Cabello/efectos de los fármacos , Adulto , Método Doble Ciego , Femenino , Cabello/crecimiento & desarrollo , Humanos , Persona de Mediana Edad , Adulto JovenRESUMEN
We have succeeded in culturing dermal papilla (DP) cells long term and developed new techniques that enhance their hair follicle-inducing efficiency in a patch assay. The outgrowing DP cells from mouse vibrissae were markedly stimulated by 10% fetal bovine serum-Dulbecco's modified essential medium that included fibroblast growth factor-2 (FGF-2). Moreover, the potency of proliferation was maintained during serial cultivations (more than 30 passages). We combined these established DP cells with epidermal cells and implanted them subcutaneously into athymic mice to examine their hair follicle-inducing ability. New hair follicles were induced by dissociated DP cells at earlier passages (under passage 4), but the cells from later passages could not induce follicles. We next aggregated the DP cells to form spheres and then injected them with epidermal cells. Unlike the dissociated DP cells, the spheres made from the later passaged cells (more than 10 passages) did induce new hair follicles. We examined several genes specific for DP of anagen follicles and confirmed that their expression level was elevated in the spheres compared with their expression level in adherent DP cells. These results suggest that FGF-2 is essential for dermal papilla cell culture and that sphere formation partially models the intact DP, resulting in hair follicle induction, even by later passaged cells.
Asunto(s)
Diferenciación Celular , Dermis/citología , Folículo Piloso/citología , Esferoides Celulares/citología , Esferoides Celulares/trasplante , Vibrisas/citología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Proliferación Celular , Células Cultivadas , Dermis/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Folículo Piloso/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Vibrisas/metabolismoRESUMEN
Adenosine is an effective treatment for androgenetic alopecia (AGA) in Japanese men and women. Adenosine exerts its effects by significantly increasing the proportion of thick hair. In this study, we assessed the clinical outcome of adenosine treatment for 6 months in 38 Caucasian men. The change in proportion of thick hair (≥60 µm) compared with baseline in the adenosine group was significantly higher than that in the placebo group (P < 0.0001). The change in vellus hair proportion (<40 µm) was significantly lower in the adenosine group than that in the placebo group (P = 0.0154). The change in hair density compared with baseline of the adenosine group was also significantly higher compared with that of the placebo group (P = 0.0470). No adverse effects due to treatment were noted during this study by dermatological evaluation. Adenosine is effective in increasing the proportion of thick hair in Caucasian men with AGA as well as in Japanese men and women.
Asunto(s)
Adenosina/administración & dosificación , Alopecia/tratamiento farmacológico , Cabello/efectos de los fármacos , Adenosina/efectos adversos , Administración Tópica , Adulto , Método Doble Ciego , Cabello/anatomía & histología , Cabello/crecimiento & desarrollo , Humanos , Japón , Masculino , Persona de Mediana Edad , Resultado del Tratamiento , Población BlancaAsunto(s)
Cisteína Endopeptidasas/metabolismo , Dermatitis Atópica/patología , Epidermis/enzimología , Estaciones del Año , Adolescente , Estudios de Casos y Controles , Niño , Preescolar , Cisteína Endopeptidasas/análisis , Epidermis/patología , Femenino , Voluntarios Sanos , Humanos , Japón , Masculino , Índice de Severidad de la EnfermedadRESUMEN
The hair cycle is a highly regulated process controlled by multiple factors. Systematic analysis of gene expression patterns in each stage of the hair cycle would provide information useful for understanding this complicated process. To identify genes associated with the hair cycle, we used DNA microarray hybridization to analyze sequential gene expression patterns in mouse skin following hair cycle synchronization by wax depilation. Messenger RNA levels in mouse skin at various times after depilation were compared with those prior to depilation (resting phase). According to their expression patterns, upregulated genes were categorized into four groups: early anagen, middle anagen, late anagen/early catagen, and middle/late catagen, and processes that take place in each stage were evaluated. We identified 12 new components that are specifically expressed in the hair follicle, 11 genes in anagen including carbonic anhydrase 6, cytokeratin 12, and matrix metalloproteinase-11 in catagen that were confirmed using in situ hybridization. The strategy used here allowed us to identify unknown genes or process previously not suspected to have a role in hair biology. These analyses will contribute to elucidating the mechanisms of hair cycle regulation and should lead to the identification of novel molecular targets for hair growth and/or depilation agents.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Folículo Piloso/metabolismo , Cabello/crecimiento & desarrollo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Animales , Femenino , Perfilación de la Expresión Génica , Folículo Piloso/química , Folículo Piloso/citología , Remoción del Cabello , Ratones , Ratones Endogámicos C57BL , Regulación hacia ArribaRESUMEN
BACKGROUND: Versican, a large chondroitin sulfate proteoglycan molecule, is implicated in the induction of hair morphogenesis, the initiation of hair regeneration, and the maintenance of hair growth in mouse species. In contrast, in human hair follicles, the distribution and the roles of versican remains obscure. OBJECTIVES: To elucidate the implication of versican in normal human hair growth. METHODS: Versican expression was examined by in situ hybridization (mRNA) and immunohistochemistry (protein). RESULTS: The results clearly showed specific versican gene expression in the dermal papilla of anagen, which apparently decreased in the dermal papilla of catagen hair follicles. No specific signal was detectable in telogen hair follicles. Consistent with ISH results, versican immunoreactivity was extended over the dermal papilla of anagen hair follicles, and again, this staining diminished in the catagen phase of human hair follicles. Interestingly, versican proteins were deposited outside K15-positive epithelial cells in the bulge throughout the hair cycle. Versican immunoreactivity in the dermal papilla was almost lost in vellus-like hair follicles affected by male pattern baldness. CONCLUSION: Specific expression of versican in the anagen hair follicles suggests its importance to maintain the normal growing phase of human as well as mouse.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/genética , Folículo Piloso/fisiología , Adulto , Alopecia/genética , Animales , Femenino , Regulación de la Expresión Génica , Folículo Piloso/citología , Folículo Piloso/crecimiento & desarrollo , Humanos , Inmunohistoquímica , Hibridación in Situ , Lectinas Tipo C , Masculino , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , VersicanosRESUMEN
To investigate the pathophysiologic role of matrix metalloproteinase 9 (MMP-9), we analyzed the mechanism of its transcriptional regulation in keratinocytes and in HT1080 fibrosarcoma cells in culture. The KRE-M9 element, which is located between the 12-O-tetradecanoyl-phorbol-13-acetate responsive element (TRE) and the transcription initiation site in the MMP-9 promoter, is essential for MMP-9 transcription in the absence of the TRE. The KRE-M9 binding protein, however, is shown to be a repressor of transcription rather than an activator; we found several times higher transcriptional activity when the KRE-M9 element was mutated. In contrast, activator protein 1 proteins (AP-1) are shown to activate transcription of MMP-9 by binding to the TRE, which is located adjacent to the KRE-M9 element. Moreover, we found that the KRE-M9 binding protein could serve as a differentiation repressing factor 1 (DRF-1) as shown by the decrease in levels of this protein with differentiation. In addition, the TRE binding protein is able to bind to the KRE-M9 to some extent. These results indicate that the coordinated modulation of MMP-9 transcription via the TRE and the KRE-M9 elements is important in epidermal and mesenchymal tissues. Our findings could facilitate consideration of the molecular mechanism in a variety of pathophysiologic conditions with which MMP-9 is involved.
Asunto(s)
Queratinocitos/fisiología , Metaloproteinasa 9 de la Matriz/genética , Carcinógenos , Diferenciación Celular , Línea Celular Tumoral , Núcleo Celular/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fibrosarcoma , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Queratinocitos/citología , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/fisiología , Factores de Transcripción del Factor Regulador X , Elementos de Respuesta/fisiología , Acetato de Tetradecanoilforbol , Factores de Transcripción , Transcripción Genética/fisiologíaRESUMEN
Recently, an automated mRNA in situ hybridization application was introduced for the Ventana Discovery instrument. The application was designed so that all necessary steps from baking through signal detection were completed within 1 day on the instrument. We applied this technology for visualizing the expression site of versican in formalin-fixed mouse skin paraffin tissue sections. Our focus of this study was to demonstrate the effects of protease digestion or heating pretreatment, termed cell conditioning, on the hybridization signal using a well characterized versican antisense riboprobe. Paraffin sections were automatically deparaffinized, fixed, and acid-treated. Then, the tissue sections were subjected to protease digestion alone (3 strengths), cell conditioning alone, or the combination of cell conditioning and protease digestion. Hybridization was performed with digoxigenin-labeled versican antisense probe (20 ng/slide) for 6 hours, and the signal was detected using a Nitro blue Tetrazolium chloride 5-Bromo-4-cloro-3-indolyl phosphate toluidine salt (NBT/BCLIP) substrate solution for 3 hours on the instrument. Cell conditioning alone did not produce any signal, whereas the highest strength of protease digestion produced noticeable background staining. However, when cell conditioning and mild protease digestion were combined, the signal for versican mRNA was clearly demonstrated in the hair papilla region. Thus, we demonstrated the effects of the cell conditioning step followed by mild protease digestion for enhancing the mRNA target staining compared with protease digestion or the cell conditioning step alone. We verified that the automated in situ hybridization process was applicable for formalin-fixed mouse skin paraffin sections and that the automated 1-day protocol is simple and reproducible. The precise control of automation allows fine tuning of temperature and enzyme dose to find the optimized assay condition for the signal to noise ratio and morphology.
Asunto(s)
Hibridación in Situ/métodos , ARN Mensajero/análisis , Piel/citología , Animales , Automatización , Proteoglicanos Tipo Condroitín Sulfato/análisis , Proteoglicanos Tipo Condroitín Sulfato/genética , Endopeptidasas/metabolismo , Formaldehído , Calor , Lectinas Tipo C , Ratones , Adhesión en Parafina , Fijación del Tejido , VersicanosRESUMEN
D-serine is an endogenous coagonist of the N-methyl-D-aspartate (NMDA)-type glutamate receptor in the central nervous system and its synthesis is catalyzed by serine racemase (SR). Recently, the NMDA receptor has been found to be expressed in keratinocytes (KCs) of the skin and involved in the regulation of KC growth and differentiation. However, the localization and role of SR in the skin remain unknown. Here, using SR-knockout (SR-KO) mice as the control, we demonstrated the localization of the SR protein in the granular and cornified layer of the epidermis of wild-type (WT) mice and its appearance in confluent WT KCs. We also demonstrated the existence of a mechanism for conversion of L-serine to D-serine in epidermal KCs. Furthermore, we found increased expression levels of genes involved in the differentiation of epidermal KCs in adult SR-KO mice, and alterations in the barrier function and ultrastructure of the epidermis in postnatal day 5 SR-KO mice. Our findings suggest that SR in the skin epidermis is involved in the differentiation of epidermal KCs and the formation of the skin barrier.