RESUMEN
The skin of patients with an extensive deep burn injury is repaired by a process that leaves a hypertrophic scar without sweat glands and therefore loses the function of perspiration. The aim of this study was to identify whether the key factors related to sweat gland development could directly reprogram fibroblasts into sweat gland-like cells. After introducing the NF-κB and Lef-1 genes into fibroblasts, we found that stably transfected fibroblasts expressed specific markers of sweat glands, including CEA, CK7, CK14 and CK19, both at the protein and mRNA levels. The immunofluorescence staining also showed positive expression of CEA, CK7, CK14 and CK19 in induced fibroblasts, but there were no positive cells in the control groups. The expression of Shh and Cyclin D1, downstream genes of NF-κB and Lef-1, were also significantly increased during regeneration. The induced fibroblasts were implanted into an animal model. Twenty days later, iodine-starch perspiration tests showed that 7 out of the 10 cell-treated paws were positive for perspiration, with a distinctive black point-like area appearing in the center of the paw. Contralateral paws tested negative. Histological examination of skin biopsies from experimental and control paws revealed that sweat glands were fully reconstructed in the test paws, with integral, secretory and ductal portions, but were not present in the control paws. This is the first report of successful reprogramming of fibroblasts into sweat gland-like cells, which will provide a new cell source for sweat gland regeneration in patients with extensive deep burns.
Asunto(s)
Transdiferenciación Celular , Técnicas de Reprogramación Celular , Reprogramación Celular , Fibroblastos/metabolismo , Glándulas Sudoríparas/metabolismo , Animales , Antígeno Carcinoembrionario/genética , Antígeno Carcinoembrionario/metabolismo , Células Cultivadas , Ciclina D1/genética , Ciclina D1/metabolismo , Fibroblastos/trasplante , Regulación del Desarrollo de la Expresión Génica , Marcadores Genéticos , Supervivencia de Injerto , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Xenoinjertos , Humanos , Queratinas/genética , Queratinas/metabolismo , Factor de Unión 1 al Potenciador Linfoide/genética , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , FN-kappa B/genética , FN-kappa B/metabolismo , Fenotipo , ARN Mensajero/metabolismo , Glándulas Sudoríparas/citología , Glándulas Sudoríparas/trasplante , Sudoración , Factores de Tiempo , TransfecciónRESUMEN
Adult bone-marrow-derived mesenchymal stem cells (MSCs) are well-established as having the capacity to differentiate into cells with mesodermal, ectodermal, and endodermal characteristics and can leave their niche to home toward and engraft within foreign tissues. To investigate whether adult MSCs contribute to the repair of skin appendages after injury, BrdU-labeled MSCs were co-cultured with heat-shocked confluent sweat gland cells (SGCs) in vitro and later intravenously injected into full-thickness skin wounds in rats. When adult MSCs were co-cultured with heat-shocked SGCs, a subset of adult MSCs differentiated into SGCs, the percentage of differentiation being enhanced by epidermal growth factor and the injured microenviroment, but weakened by PD98059. The ERK (extracellular signal-regulated kinase) pathway, especially pERK, was involved in the phenotype conversion of human MSCs into human SGC. Labeled MSCs were noted in hair follicles, sebaceous glands, blood vessels, and dermis in full-thickness wounds, and the incorporated cells in hair follicles and sebaceous glands were also positive for pan-cytokeratin. After wound healing, some labeled MSCs returned to the bone marrow, whereas other were retained in the dermis. We conclude that adult MSCs have the capacity to dock at specific sites, to contribute to wound healing of skin appendages, and to home toward marrow, and that engraftment of bone-marrow-derived cells is a functional event.
Asunto(s)
Células de la Médula Ósea/citología , Células Madre Mesenquimatosas/fisiología , Piel/anatomía & histología , Glándulas Sudoríparas/citología , Cicatrización de Heridas/fisiología , Adolescente , Adulto , Animales , Biomarcadores/metabolismo , Linaje de la Célula , Células Cultivadas , Técnicas de Cocultivo , Inhibidores Enzimáticos/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Flavonoides/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunohistoquímica , Indoles/metabolismo , Ratas , Ratas Wistar , Glándulas Sudoríparas/cirugía , Glándulas Sudoríparas/trasplante , Sales de Tetrazolio/metabolismo , Trasplante HeterólogoRESUMEN
Contact with sweat gland acini causes sympathetic neurons to switch from a catecholaminergic to a cholinergic phenotype during development and following experimental manipulations. Substantial reductions of cholinergic innervation have been shown in the sweat glands of ageing rats and humans. Using in oculo transplantation, we have now studied whether sweat gland target tissues retain the capacity to regulate changes in the phenotype of sympathetic neurons observed in maturity and old age, including a switch from catecholaminergic to cholinergic characters. Markers have been used which indicate changes in nerve fibre morphology (the pan-neuronal marker, PGP9.5) as well as neurotransmitter expression (acetylcholinesterase (AChE), vasocative intestinal polypeptide (VIP) and tyrosine hydroxylase (TH). Sweat glands from young and old donor rats became reinnervated by an organotypic pattern of cholinergic host nerves. Surgical sympathectomy demonstrated that these cholinergic nerve fibres originate from sympathetic neurons of the host superior cervical ganglion (SCG). Retrograde tracing combined with staining for VIP (a marker associated with cholinergic phenotype in neurons supplying sweat glands) showed that SCG neurons projecting to irises with sweat gland implants may be induced to express VIP. We hypothesise that these neurons have been switched from their normal catecholaminergic phenotype to a cholinergic one by contact with the sweat gland implants. Transplants from old donors attracted a density of reinnervation by young host nerves which was appropriate to the age of the donor, thus old sweat glands received a significantly reduced density of innervation compared to young glands. Despite the reduced density of innervation, there was no obvious difference in the ability of young and old implants to induce the switch to a cholinergic phenotype, suggesting that different mechanisms regulate nerve growth and neurotransmitter phenotype.
Asunto(s)
Fibras Adrenérgicas/fisiología , Envejecimiento/fisiología , Fibras Colinérgicas/fisiología , Estilbamidinas , Glándulas Sudoríparas/inervación , Glándulas Sudoríparas/trasplante , Sistema Nervioso Simpático/fisiología , Fibras Adrenérgicas/química , Animales , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Regeneración Nerviosa/genética , Regeneración Nerviosa/fisiología , Fenotipo , Ratas , Ratas Sprague-Dawley , Simpatectomía , Sistema Nervioso Simpático/citología , Sistema Nervioso Simpático/cirugía , Péptido Intestinal Vasoactivo/análisis , Péptido Intestinal Vasoactivo/genéticaRESUMEN
Contact with sweat gland acini causes sympathetic neurons to switch from a catecholaminergic to a cholinergic phenotype during development and following experimental manipulations. Substantial reductions of cholinergic innervation have been shown in the sweat glands of ageing rats and humans. Using in oculo transplantation, we have now studied whether sweat gland target tissues retain the capacity to regulate changes in the phenotype of sympathetic neurons observed in maturity and old age, including a switch from catecholaminergic to cholinergic characters. Markers have been used which indicate changes in nerve fibre morphology (the pan-neuronal marker, PGP9.5) as well as neurotransmitter expression (acetylcholinesterase (AChE), vasocative intestinal polypeptide (VIP) and tyrosine hydroxylase (TH)). Sweat glands from young and old donor rats became reinnervated by an organotypic pattern of cholinergic host nerves. Surgical sympathectomy demonstrated that these cholinergic nerve fibres originate from sympathetic neurons of the host superior cervical ganglion (SCG). Retrograde tracing combined with staining for VIP (a marker associated with cholinergic phenotype in neurons supplying sweat glands) showed that SCG neurons projecting to irises with sweat gland implants may be induced to express VIP. We hypothesise that these neurons have been switched from their normal catecholaminergic phenotype to a cholinergic one by contact with the sweat gland implants. Transplants from old donors attracted a density of reinnervation by young host nerves which was appropriate to the age of the donor, thus old sweat glands received a significantly reduced density of innervation compared to young glands. Despite the reduced density of innervation, there was no obvious difference in the ability of young and old implants to induce the switch to a cholinergic phenotype, suggesting that different mechanisms regulate nerve growth and neurotransmitter phenotype.
Asunto(s)
Fibras Adrenérgicas/fisiología , Envejecimiento/fisiología , Fibras Colinérgicas/fisiología , Estilbamidinas , Glándulas Sudoríparas/trasplante , Fibras Adrenérgicas/química , Animales , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Regeneración Nerviosa/genética , Regeneración Nerviosa/fisiología , Fenotipo , Ratas , Ratas Sprague-Dawley , Ganglio Cervical Superior/fisiología , Glándulas Sudoríparas/inervación , Simpatectomía , Péptido Intestinal Vasoactivo/análisis , Péptido Intestinal Vasoactivo/genéticaRESUMEN
While the majority of sympathetic neurons are noradrenergic, a minority population are cholinergic. At least one population of cholinergic sympathetic neurons arises during development by a target-dependent conversion from an initial noradrenergic phenotype. Evidence for retrograde specification has been obtained from transplantation studies in which sympathetic neurons that normally express a noradrenergic phenotype throughout life were induced to innervate sweat glands, a target normally innervated by cholinergic sympathetic neurons. This was accomplished by transplanting footpad skin containing sweat gland primordia from early postnatal donor rats to the hairy skin region of host rats. The sympathetic neurons innervating the novel target decreased their expression of noradrenergic traits and developed choline acetyltransferase (ChAT) activity. In addition, many sweat gland-associated fibers acquired acetylcholinesterase (AChE) staining and VIP immunoreactivity. These studies indicate that sympathetic neurons in vivo alter their neurotransmitter phenotype in response to novel environmental signals and that sweat glands play a critical role in the cholinergic and peptidergic differentiation of the sympathetic neurons that innervate them. The sweat gland-derived cholinergic differentiation factor is distinct from leukemia inhibitory factor and ciliary neurotrophic factor, two well-characterized cytokines that alter the neurotransmitter properties of cultured sympathetic neurons in a similar fashion. Recent studies indicate that anterograde signalling is also important for the establishment of functional synapses in this system. We have found that the production of cholinergic differentiation activity by sweat glands requires sympathetic innervation, and the acquisition and maintenance of secretory competence by sweat glands depends upon functional cholinergic innervation.
Asunto(s)
Neuronas/fisiología , Neurotransmisores/fisiología , Glándulas Sudoríparas/trasplante , Sistema Nervioso Simpático/fisiología , Animales , Fenotipo , Transducción de Señal , Sistema Nervioso Simpático/citología , Sinapsis/fisiologíaRESUMEN
Using a double-antibody immunoperoxidase technique, we demonstrated human prolactin-like material in some cells of the secretory coil and in luminal duct cells of sweat glands of human skin which had been carried as grafts on mice for 32-35 weeks. It therefore seems likely that a population of cells in the secretory coil synthesizes prolactin and can be considered as diffuse peripheral endocrine cells. The prolactin may function locally to regulate sweat electrolyte concentration.