RESUMEN
Considerable global demand exists for the development of novel drugs for the treatment of alopecia. A recent report demonstrated that oxytocin promotes hair growth activity in human dermal papilla (DP) cells; however, its application in drugs or cosmetic products is challenging because rapid degradation and relatively large molecular weight prevent long-term topical administration on the scalp. Here, we examined cinnamic acid, a small molecule activator for oxytocin receptor (OXTR) expression. Treatment with cinnamic acid led to upregulation of OXTR and trichogenic gene expression in human DP cells. Furthermore, inhibition of OXTR with an antagonist, L-371,257, suppressed hair growth-related gene expression in DP cells. These findings suggest that cinnamic acid enhances the hair growth ability of DP cells via oxytocin signaling. Additionally, we tested the hair growth-promoting effects of cinnamic acid using hair follicle organoids in vitro and observed that cinnamic acid significantly promoted the growth of hair peg-like sprouting. These promising results may be useful for developing hair growth-promoting products targeting oxytocin.
Asunto(s)
Cinamatos , Folículo Piloso , Oxitocina , Humanos , Folículo Piloso/metabolismo , Oxitocina/farmacología , Oxitocina/metabolismo , Receptores de Oxitocina/genética , Receptores de Oxitocina/metabolismo , Células Cultivadas , Cabello , OrganoidesRESUMEN
Hair follicle morphogenesis during embryonic development is driven by the formation of hair follicle germs (HFGs) via interactions between epithelial and mesenchymal cells. Bioengineered HFGs are potential tissue grafts for hair regenerative medicine because they can replicate interactions and hair follicle morphogenesis after transplantation. However, a mass preparation approach for HFGs is necessary for clinical applications, given that thousands of de novo hair follicles are required to improve the appearance of a single patient with alopecia. In this study, we developed a microfluidics-based approach for the large-scale preparation of HFGs. A simple flow-focusing microfluidic device allowed collagen solutions containing epithelial and mesenchymal cells to flow and generate collagen microbeads with distinct Janus structures. During the 3 days of culture, the collagen beads contracted owing to cellular traction forces, resulting in collagen- and cell-dense HFGs. The transplantation of HFGs into nude mice resulted in highly efficient de novo hair follicle regeneration. This method provides a scalable and robust tissue graft preparation approach for hair regeneration.
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Folículo Piloso , Células Madre Mesenquimatosas , Humanos , Ratones , Animales , Folículo Piloso/trasplante , Ratones Desnudos , Colágeno , Dispositivos Laboratorio en un ChipRESUMEN
Exosomes are lipid bilayer vesicles, 30-200 nm in diameter, that are produced by cells and play essential roles in cell-cell communication. Exosomes have been studied in several medical fields including dermatology. Hair loss, a major disorder that affects people and sometimes causes mental stress, urgently requires more effective treatment. Because the growth and cycling of hair follicles are governed by interactions between hair follicle stem cells (HFSCs) and dermal papilla cells (DPCs), a better understanding of the mechanisms responsible for hair growth and cycling through exosomes may provide new insights into novel treatments for hair loss. In this review, we focused on the comprehensive knowledge and recent studies on exosomes in the field of hair development and regeneration. We classified exosomes of several cellular origins for the treatment of hair loss. Exosomes and their components, such as microRNAs, are promising drugs for effective hair loss treatment.
Asunto(s)
Dermis , Exosomas , Humanos , Folículo Piloso , Cabello , Células Cultivadas , Alopecia/terapia , RegeneraciónRESUMEN
Oxytocin (OXT) is a neuropeptide hormone termed "love hormone" produced and released during childbirth and lactation. It is also produced in response to skin stimulation (e.g., during hugging and massaging) and music therapy. The effects of OXT on various organs have been revealed in recent years; however, the relationship between hair follicles and OXT remains unclear. In this study, we examined the effects of OXT on dermal papilla (DP) cells that control hair growth by secreting growth/regression signals. Gene expression analysis revealed that DP signature markers were significantly upregulated in DP cells treated with OXT. In addition, we tested the hair growth-promoting effects of OXT using in vitro hair follicle organoids. OXT promoted the growth of hair peg-like sprouting by upregulating the expression of growth-promoting factors, including genes encoding vascular endothelial growth factor A (VEGFA). This study highlights the positive effects of OXT in hair follicles and may assist in the development of new treatments for alopecia.
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Dermis , Oxitocina , Femenino , Humanos , Dermis/metabolismo , Oxitocina/farmacología , Oxitocina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Células Cultivadas , Folículo Piloso/metabolismo , CabelloRESUMEN
Hair regenerative medicine must involve practical procedures, such as cryopreservation of tissue grafts. This can aid in evaluating tissue safety and quality, as well as transportation to a clinic and multiple transplants. Hair follicle germs (HFGs), identified during in vivo development, are considered effective tissue grafts for hair regenerative medicine. However, to the best of our knowledge, methods for cryopreserving HFGs have not been explored yet. This study investigated the efficacy of slow vitrification methods for freezing HFGs. Cryoprotectants such as dimethyl sulfoxide (DMSO) and carboxylated poly-l-lysine were used for vitrification. The results indicate that DMSO vitrification yielded the most efficient de novo hair regeneration in mouse skin, comparable to that of non-cryoprotected HFGs. A microfinger was fabricated to scale up the cryopreservation method, considering that thousands of tissue grafts were required per patient in clinical practice. The microfinger can be used for a series of processes, holding the HFG, replacing it with a cryopreservation solution, freezing it in liquid nitrogen, thawing it in a warm medium, and transplanting it into the skin. Although de novo hair regeneration by HFGs cryopreserved using microfingers was reduced by approximately 20 % compared to those cryopreserved using flat plates for fertilized eggs, it exceeded 50 %. These findings demonstrate that vitrification with DMSO and microfingers could be a useful approach for the cryopreservation of tissue grafts in hair regenerative medicine for hair loss.
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Dimetilsulfóxido , Folículo Piloso , Ratones , Animales , Medicina Regenerativa/métodos , Criopreservación/métodos , Congelación , Crioprotectores/farmacologíaRESUMEN
Photocrosslinkable gelatin has attracted increasing interest in the field of biofabrication, with the most studied and widely used photocrosslinkable gelatin being gelatin methacrylate (GelMa). However, the 3D fabrication of GelMa has presented several limitations and challenges, primarily due to its slow crosslinking speed. It is generally known that acryl-based functional groups have faster reaction kinetics than methacryl-base groups. However, gelatin acrylamide (GelAc) has not been widely investigated, largely due to its increased complexity of synthesis relative to GelMA. In this study, we developed a novel synthesis method for GelAc. By varying the reaction ratio of reagents, GelAc with a degree of substitution from 20% to 95% was produced. The UV crosslinking properties of GelAc was studied, demonstrating significantly faster crosslinking kinetics than GelMa, especially at lower concentrations and low photoinitiator concentrations. The swelling ratio and mechanical properties of the crosslinked GelAc hydrogel were also characterized, and biocompatibility experiments conducted via both surface seeding and hydrogel encapsulation of cells, with good cell viability observed. The application of GelAc for 3D biofabrication was demonstrated by 3D printing. GelAc can be a useful material for the fabrication of 3D conduits for tissue engineering applications.
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Gelatina , Ingeniería de Tejidos , Ingeniería de Tejidos/métodos , Hidrogeles , Impresión Tridimensional , Acrilamidas , Metacrilatos , Andamios del TejidoRESUMEN
In vitro models of human hair follicle-like tissue could be fundamental tools to better understand hair follicle morphogenesis and hair drug screening. During prenatal development and postnatal cyclic hair regeneration, hair follicle morphogenesis is triggered by reciprocal interactions and the organization of the epithelial and mesenchymal cell populations. Given this mechanism, we developed an approach to induce hair peg-like sprouting in organoid cultures composed of epithelial and mesenchymal cells. Human fetal/adult epithelial and mesenchymal cells were cultured in a medium supplemented with a low concentration of either Matrigel or collagen I. These extracellular matrices significantly enhanced the self-organization capabilities of the epithelial and mesenchymal cells, resulting in spherical aggregation and subsequent hair peg-like sprouting. The length of the hair peg sprouting and associated gene expression significantly increased in the presence of a well-known hair drug, minoxidil. This approach may be beneficial for testing hair growth-promoting drug candidates.
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Folículo Piloso , Células Madre Mesenquimatosas , Adulto , Embarazo , Femenino , Humanos , Cabello , Colágeno Tipo I , OrganoidesRESUMEN
Hair regeneration using hair follicle stem cells (HFSCs) and dermal papilla cells is a promising approach for the treatment of alopecia. One of the challenges faced in this approach is the quantitative expansion of HFSCs while maintaining their hair induction capacity. In this study, HFSC expansion was achieved through the formation of uniform-diameter cell aggregates that were subsequently encapsulated in Matrigel. We designed a microwell array device, wherein mouse HFSCs were seeded, allowed to form loosely packed aggregates for an hour, and then embedded in Matrigel. Quantitative analysis revealed a 20-fold increase in HFSC number in 2 weeks through this culture device. Gene expression of trichogenic stem cell markers in the device-grown cells showed a significant increase compared with that of typical flat substrate Matrigel suspension culture cells. These microwell array-cultured HFSCs mixed with freshly isolated embryonic mesenchymal cells indicated vigorous hair regeneration on the skin of nude mice. Furthermore, we examined the feasibility of this approach for the expansion of human HFSCs from androgenetic alopecia patients and found that the ratio of CD200+ cells was improved significantly in comparison with that of cells cultured in a typical culture dish or in a Matrigel suspension culture on a flat substrate. Therefore, the novel approach proposed in this study may be useful for HFSC expansion in hair regenerative medicine.
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Folículo Piloso , Células Madre , Ratones , Animales , Humanos , Ratones Desnudos , Células CultivadasRESUMEN
Dermal papilla cells (DPCs) play critical roles in hair follicle development, but the underlying mechanisms that contribute to hair regeneration have yet to be fully elucidated, particularly in terms of alterations in androgenetic alopecia patients. In this study, we demonstrated that hypoxia-inducible factor-1α (HIF-1α) is suppressed in scalp tissues of androgenetic alopecia patients and potentially associated with hair follicle development. Using RT-qPCR and western blot, we found that mRNA and protein levels of trichogenic genes, LEF1 and versican (VCAN), were attenuated in HIF-1α knockdown DPCs. Under an in vivo mimicked environment in a three-dimensional spheroid culture, HIF-1α-suppressed DPCs downregulated the expression of hair induction-related genes. Finally, treatment with a HIF-1α activator resulted in the elevated expression of trichogenic genes in DPCs. This study highlights the importance of dermal HIF-1α expression in regulating trichogenic genes and provides a promising therapeutic target and a fundamental tissue engineering approach for hair loss treatment.
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Folículo Piloso , Subunidad alfa del Factor 1 Inducible por Hipoxia , Humanos , Folículo Piloso/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Alopecia/genética , Alopecia/metabolismo , Expresión Génica , Hipoxia/metabolismo , Células CultivadasRESUMEN
Hair regenerative medicine is a promising approach to treat hair loss. The replication of in vivo tissue configurations and microenvironments, such as hair follicle germs, has been studied to prepare tissue grafts for hair regenerative medicine. However, such approaches should be scalable, because a single patient with alopecia requires thousands of tissue grafts. In this paper, we propose an approach for the scalable and automated preparation of highly hair-inductive tissue grafts using a bioprinter. Two collagen droplets (2 µL each) containing mesenchymal and epithelial cells were placed adjacent to each other to fabricate hair-follicle-germ-like grafts. During three days of culture, the pairs of microgel beads were spontaneously contracted by cell traction forces, whereas the two cell types remained separated, where the densities of the cells and collagen were enriched more than 10 times. This approach allowed us to fabricate submillimeter objects printed with millimeter-order accuracy, facilitating scalable and automated tissue graft preparation. Because of mesenchymal-epithelial interactions, hair microgels (HMGs, i.e., collagen- and cell-enriched microgels) efficiently regenerate hair follicles and shafts when transplanted into the back skin of mice. However, the generated hair shafts mostly remain under the skin. Therefore, we printed microgel beads onto surgical suture guides arrayed on a stage. The microgel beads were contracted along with the suture guides in culture prior to transplantation. The guide-inserted HMGs significantly improved hair-shaft sprouting through the skin, owing to the control of the orientation of the HMGs transplanted into the skin. This approach is a promising strategy to advance hair regenerative medicine. STATEMENT OF SIGNIFICANCE: This study proposes an approach for the scalable and automated preparation of highly hair-inductive grafts using a bioprinter. Two collagen droplets containing mesenchymal and epithelial cells were placed adjacently. Cell traction forces caused the pairs of microgel beads to spontaneously contract in culture. Because of mesenchymal-epithelial interactions, hair microgels (HMGs) efficiently regenerated hair follicles on the back skin of mice. However, the generated hair shafts remained mostly beneath the skin. Therefore, we printed microgel beads onto surgical suture guides arrayed on a stage. The guide-inserted HMGs significantly improved hair-shaft sprouting through the skin owing to the control of the orientation of the HMGs in the skin. This approach represents a promising strategy for advancing hair regenerative medicine.
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Bioimpresión , Microgeles , Animales , Ratones , Folículo Piloso , Medicina Regenerativa , ColágenoRESUMEN
The transplantation of pre-vascularized bone grafts is a promising strategy to improve the efficacy of engraftment and bone regeneration. We propose a hydrogel microbead-based approach for preparing vascularized and high-density tissue grafts. Mesenchymal stem cell-encapsulated collagen microgels (2 µL), termed bone beads, were prepared through spontaneous constriction, which improved the density of the mesenchymal stem cells and collagen molecules by more than 15-fold from the initial day of culture. Constriction was attributed to cell-attractive forces and involved better osteogenic differentiation of mesenchymal stem cells than that of spheroids. This approach was scalable, and â¼2000 bone beads were prepared semi-automatically using a liquid dispenser and spinner flask. The mechanical stimuli in the spinner flask further improved the osteogenic differentiation of the mesenchymal stem cells in the bone beads compared with that in static culture. Vascular endothelial cells readily attach to and cover the surface of bone beads. The in vitro assembly of the endothelial cell-enveloped bone beads resulted in microchannel formation in the interspaces between the bone beads. Significant effects of endothelialization on in vivo bone regeneration were shown in rats with cranial bone defects. The use of endothelialized bone beads may be a scalable and robust approach for treating large bone defects. STATEMENT OF SIGNIFICANCE: A unique aspect of this study is that the hMSC-encapsulated collagen microgels were prepared through spontaneous constriction, leading to the enrichment of collagen and cell density. This constriction resulted in favorable microenvironments for the osteogenic differentiation of hMSCs, which is superior to conventional spheroid culture. The microgel beads were then enveloped with vascular endothelial cells and assembled to fabricate a tissue graft with vasculature in the interspaces among the beads. The significant effects of endothelialization on in vivo bone regeneration were clearly demonstrated in rats with cranial bone defects. We believe that microgel beads covered with vascular endothelial cells provide a promising approach for engineering better tissue grafts for bone-regenerative medicine.
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Microgeles , Medicina Regenerativa , Ratas , Animales , Osteogénesis , Células Endoteliales , Ingeniería de Tejidos/métodos , Colágeno/farmacología , Diferenciación Celular , Regeneración ÓseaRESUMEN
During embryonic development, reciprocal interactions between epidermal and mesenchymal layers trigger hair follicle morphogenesis. This study revealed that microenvironmental reprogramming via control over these interactions enabled hair follicle induction in vitro. A key approach is to modulate spatial distributions of epithelial and mesenchymal cells in their spontaneous organization. The de novo hair follicles with typical morphological features emerged in aggregates of the two cell types, termed hair follicloids, and hair shafts sprouted with near 100% efficiency in vitro. The hair shaft length reached ~3 mm in culture. Typical trichogenic signaling pathways were up-regulated in hair follicloids. Owing to replication of hair follicle morphogenesis in vitro, melanosome production and transportation were also monitored in the hair bulb region. This in vitro hair follicle model might be valuable for better understanding hair follicle induction, evaluating hair growth and inhibition of hair growth by drugs, and modeling gray hairs in a well-defined environment.
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Hair follicle morphogenesis is triggered by epithelial-mesenchymal interactions. Several approaches have been developed for preparing hair follicle organoids using epithelial and mesenchymal cells; however, the current understanding of the relevance of in vitro spontaneous organization processes to hair regeneration is limited. In the present study, we used Y27632, a rho-associated kinase inhibitor, to investigate the effects of manipulation of cell sorting on hair regeneration in vitro. Dissociated hair follicle-inducible epithelial and mesenchymal cells were cultured in Y27632-containing media in 96-well plates or polydimethylsiloxane microarray plates. We found that Y27632 supplementation modulated the spatial distribution of epithelial and mesenchymal cells from a dumbbell shape to a core-shell configuration via a spontaneous organization process. New hair follicles with typical morphological features emerged in the Y27632-treated core-shell-shaped aggregates, and hair shafts sprouted with approximately 100% efficiency in vitro. Gene chip analysis and pathway-inhibition experiments revealed that the phosphatidylinositol-3-kinase/protein kinase B- and Ras-signaling pathways were involved in hair-like sprouting in the Y27632-treated hair follicle organoids. Our findings enhance the understanding of hair follicle organogenesis and the development of hair follicle organoids.
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Folículo PilosoRESUMEN
We describe a protocol for a live-cell luciferase assay system for continuously monitoring fibroblast growth factor (FGF) signal disruption in human-induced pluripotent stem cells (iPSCs). Signal disrupting effects of chemicals are used as an indicator to evaluate toxicity. The assay is reliably predictive of the effects of limb malformation chemicals (AUC = 0.93). The current approach is limited to FGF signal disruption, and combinations with other types of signaling will be required to detect the effects of different toxicants. For complete details on the use and execution of this protocol, please refer to Kanno et al. (2022a).
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Células Madre Pluripotentes Inducidas , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Luciferasas/genética , Transducción de SeñalRESUMEN
Repellent coatings are critical for the development of biomedical and analytical devices to prevent nonspecific protein and cell adhesion. In this study, prevelex (polyampholytes containing phosphate and amine units) was synthesized for the fine coating of microdevices for cell culture. The dip-coating of the prevelex on hydrophobic substrates altered their surfaces to be highly hydrophilic and electrically neutral. The range of prebake temperature (50-150 °C) after dip-coating was moderate and within a preferable range to treat typical materials for cell culture such as polystyrene and polydimethylsiloxane. Scanning electron microscopy revealed a conformal and ultra-thin film coating on the micro/nano structures. When compared with poly(2-hydroxyethyl methacrylate) and poly(2-methacryloyloxyethyl phosphorylcholine), prevelex exhibited better characteristics for coating on microwell array devices, thereby facilitating the formation of spheroids with uniform diameters using various cell types. Furthermore, to examine cellular functionalities, mouse embryonic epithelial and mesenchymal cells were seeded in a prevelex-coated microwell array device. The two types of cells formed hair follicle germ-like aggregates in the device. The aggregates were then transplanted to generate de novo hair follicles in nude mice. The coating material provided a robust and fine coating approach for the preparation of non-fouling surfaces for tissue engineering and biomedical applications.
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Materiales Biocompatibles Revestidos , Animales , Adhesión Celular , Materiales Biocompatibles Revestidos/química , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Ratones Desnudos , Microscopía Electrónica de RastreoRESUMEN
Dermal papilla cells (DPCs), which play a central role in the regulation of hair follicle development and hair growth, are among the most promising cell sources for hair regenerative medicine. However, a critical issue in the use of DPCs is the immediate loss of hair inducing functions in typical two-dimensional (2D) culture. We have previously demonstrated that when DPCs are encapsulated in drops of collagen gel (named hair beads, HBs), the density of collagen and cells is concentrated >10-fold during 3 d of culture through the spontaneous constriction of the drops, leading to efficient hair follicle regeneration upon transplantation. However, the mechanisms responsible for the activation of the hair-inducing functions of DPCs have been poorly elucidated. Here, transcriptome comparisons of human DPCs in HB culture and in typical 2D culture revealed that the phosphoinositide 3-kinase and Akt (PI3K/Akt) signaling pathway was significantly upregulated in HB culture. Inhibition of the PI3K/Akt signaling pathway decreased the hair-inducing capability of DPCs in HBs, while the activation of the PI3K/Akt signaling pathway using an activator improved trichogenous gene expression of DPCs in 2D culture. These results suggest that the PI3K/Akt signaling pathway is crucial for the maintenance and restoration of hair inductivity of DPCs. HB culture and/or activators of the PI3K/Akt signaling pathway could be a promising strategy for preparing DPCs for hair regenerative medicine.
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Folículo Piloso , Fosfatidilinositol 3-Quinasa , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Proliferación Celular , Células Cultivadas , Colágeno , Cabello , Folículo Piloso/citología , Humanos , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
The number of man-made chemicals has increased exponentially recently, and exposure to some of them can induce fetal malformations. Because complex and precisely programmed signaling pathways play important roles in developmental processes, their disruption by external chemicals often triggers developmental toxicity. However, highly accurate and high-throughput screening assays for potential developmental toxicants are currently lacking. In this study, we propose a reporter assay that utilizes human-induced pluripotent stem cells (iPSCs) to detect changes in fibroblast growth factor signaling, which is essential for limb morphogenesis. The dynamics of this signaling after exposure to a chemical were integrated to estimate the degree of signaling disruption, which afforded a good prediction of the capacity of chemicals listed in the ECVAM International Validation Study that induce limb malformations. This study presents an initial report of a human iPSC-based signaling disruption assay, which could be useful for the screening of potential developmental toxicants.
RESUMEN
The number of man-made chemicals has increased rapidly in recent decades, with certain chemicals potentially causing malformations in fetuses. Although the toxicities of chemicals have been tested in animals, chemicals that are not teratogenic in rodents can cause severe malformations in humans, owing to the differences in the susceptibility to the teratogenicity of chemicals among species. One possible cause of such species differences, other than pharmacokinetics, could be the difference in sensitivity to such chemicals at the cellular level. Therefore, a human cell-based high-throughput assay system is needed for detecting potential teratogenic chemicals. In this study, we proposed a signal reporter assay using human induced pluripotent stem cells (iPSCs). Because developmental processes are governed by highly intricate and precisely programmed signaling pathways, external chemical-induced disruption of these pathways often triggers developmental toxicities. The reporter assay using hiPSCs was used to detect changes in the fibroblast growth factor (FGF) signaling pathway, a pathway essential for limb morphogenesis. The method was based on monitoring and time-accumulation of the signal disruption over time, rather than the classical endpoint detection of the signal disruption. This approach was useful for detecting signal disruptions caused by the malformation chemicals listed in the ICH S5 guideline, including thalidomide. The human iPSC-based signal disruption assay could be a promising tool for the initial screening of developmental toxicants.
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Células Madre Pluripotentes Inducidas , Animales , Bioensayo , Factores de Crecimiento de Fibroblastos/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Teratógenos/toxicidadRESUMEN
Hair follicle dermal papilla cells (DPCs) are specialized mesenchymal cells that play pivotal roles in hair formation, growth, and cycles, and they are considered as a cell source in hair regenerative medicine. Rodent dermal papilla cells have been shown to induce de novo hair follicle generation in the skin of recipients following transplantation, suggesting that dermal papilla cells can reprogram epidermal microenvironments. However, human DPCs (hDPCs) lose their ability to generate de novo hair follicles under conventional culture methods. We investigated the effects of electrical stimulation (ES) on hDPCs to restore the depressed trichogenic activity. We demonstrated that ES with a polypyrrole (PPy)-modified electrode upregulated trichogenic gene expression in hDPCs in vitro, and the activated cells when transplanted into mice generated double the number of hairs compared to that without the ES. Using specific inhibitors, we revealed that the mechanisms behind the electrical activation are associated with voltage-gated ion channels. Further, ES can be adapted for hDPCs from a patient with androgenic alopecia. Thus, this approach is potentially beneficial in preparing hDPCs for hair regenerative medicine.
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Polímeros , Medicina Regenerativa , Animales , Células Cultivadas , Estimulación Eléctrica , Humanos , Ratones , PirrolesRESUMEN
Bhas 42 cell transformation assay (CTA) has been used to estimate the carcinogenic potential of chemicals by exposing Bhas 42 cells to carcinogenic stimuli to form colonies, referred to as transformed foci, on the confluent monolayer. Transformed foci are classified and quantified by trained experts using morphological criteria. Although the assay has been certified by international validation studies and issued as a guidance document by OECD, this classification process is laborious, time consuming, and subjective. We propose using deep neural network to classify foci more rapidly and objectively. To obtain datasets, Bhas 42 CTA was conducted with a potent tumor promotor, 12-O-tetradecanoylphorbol-13-acetate, and focus images were classified by experts (1405 images in total). The labeled focus images were augmented with random image processing and used to train a convolutional neural network (CNN). The trained CNN exhibited an area under the curve score of 0.95 on a test dataset significantly outperforming conventional classifiers by beginners of focus judgment. The generalization performance of unknown chemicals was assessed by applying CNN to other tumor promotors exhibiting an area under the curve score of 0.87. The CNN-based approach could support the assay for carcinogenicity as a fundamental tool in focus scoring.