Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 38
Filtrar
1.
Cell Physiol Biochem ; 55(4): 387-399, 2021 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-34214388

RESUMEN

BACKGROUND/AIMS: The use of skin-derived stem cells and stem cells of other origins in regenerative medicine requires knowledge of stem cell fate after transplantation. In order to achieve non-invasive long-term imaging and tracking of transplanted stem cells in preclinical studies, a non-toxic, efficient labeling technique that does not alter stem cell characteristics must be used. Our aim was to investigate a method for such a long-term cell-compatible cell tracer using nanoparticles. METHODS: Nanotechnology, in particular the use of quantum dots (QDs), offers great advantages for this crucial requirement. In this study, we used nanocrystals coated with a specific target peptide that enables delivery into the cytoplasm of cells, resulting in an intense and stable fluorescent labeling. We analyzed the influence of biocompatible CdSe/ZnS-QDs on epidermal stem cells (EpiSCs) isolated from adult human skin. Thereby we analyzed on QD loading, cell proliferation including QD transfer to descendent daughter cells as well as the influence on the differentiation potential of stem cells after QD labeling. RESULTS: FACS analysis revealed a dose-dependent QD incorporation into the cells. Thereby, a high initial concentration of nanocrystals resulted in a more stable long-term labeling. QD labeled cells showed normal viability and unchanged ability to proliferate. The spread of QDs during cell division was monitored by time lapse microscopy and two modes of QD distribution could be observed. Daughter cells either received an equal amount of QDs after cell division, which led to a homogenously faded fluorescence signal, or there was an uneven transmission of QDs, which led to unchanged labeling of one cell and a complete loss of the fluorescence signal of the other cell. The spontaneous differentiation potential remained unaffected after QD exposure, since skin-derived EpiSCs showed an unchanged protein and gene expression profile. CONCLUSION: In summary, we can conclude that QDs offer a successful, non-invasive and efficient labeling technique for EpiSCs, which makes their in vitro and in vivo use in skin regeneration and wound healing models traceable. Nevertheless, the uneven transmission of QDs should not be disregarded and the extent and frequency should be investigated in further studies.


Asunto(s)
Citometría de Flujo , Puntos Cuánticos/química , Piel/citología , Células Madre/citología , Humanos
2.
Adv Exp Med Biol ; 1169: 55-62, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31487018

RESUMEN

Sweat glands play an important role in skin physiology and are an integral part of the natural skin barrier. In order to maintain functionality throughout life, sweat glands make use of several types of stem cells. This chapter focuses on the classification of different types of stem cells found in the sweat gland and their physiological roles. First, sweat gland formation during skin maturation is addressed in order to give an overview of sweat gland origin and formation in vivo. Then, different kinds of adult sweat gland stem cells are introduced and classified between different potency levels and corresponding physiological roles. Finally, the importance of these cell sources for future developments, including applications in wound healing and cosmetics research, is discussed.


Asunto(s)
Células Madre , Glándulas Sudoríparas , Humanos , Piel/citología , Piel/crecimiento & desarrollo , Células Madre/citología , Glándulas Sudoríparas/citología , Cicatrización de Heridas
3.
Cell Physiol Biochem ; 27(1): 1-12, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21325816

RESUMEN

BACKGROUND/AIMS: Cellular models are an interesting tool to study human heart diseases. To date, research groups mainly focus on mouse models, but important murine physiology is different from human characteristics. Recently, scientists found that the electrophysiology of fish cardiomyocytes largely resembles that of humans. So far, cardiomyocyte models were generated using differentiation medium, were stimulated electrically or, when contracting spontaneously, only did so over a short time period. We established an in vitro spontaneously, long-term beating heart model generated from rainbow trout, with the potential to be used as a new human heart model system because of its electrophysiology. METHODS: Spontaneously contracting 3D cell layers from rainbow trout were generated in vitro and analyzed using PCR and immunochemistry. Further, electrophysiology was measured via intra - and extracellular recordings. RESULTS: Contracting cardiomyogenic aggregates were generated without differentiation medium and were beating autonomously for more than one month. Electrophysiological measurements exhibit that the action potential properties of fish cardiomyocytes in part resemble the characteristics of human cardiomyocytes. The sensitivity of the beating cell aggregates to drugs could also be confirmed. CONCLUSION: Spontaneously contracting cardiomyogenic cell aggregates from rainbow trout generated in vitro are suitable for human heart research and pharmacology.


Asunto(s)
Miocitos Cardíacos/fisiología , Agonistas Adrenérgicos beta/farmacología , Animales , Antiarrítmicos/farmacología , Biomarcadores/metabolismo , Células Cultivadas , Cromanos/farmacología , Corazón/fisiología , Humanos , Isoproterenol/farmacología , Modelos Biológicos , Contracción Miocárdica/fisiología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Oncorhynchus mykiss , Canales de Potasio/química , Canales de Potasio/metabolismo , Pirrolidinas/farmacología , Receptores Adrenérgicos beta/química , Receptores Adrenérgicos beta/metabolismo
4.
J Clin Med ; 10(16)2021 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-34441782

RESUMEN

The use of in vitro systems to investigate the process of corneal wound healing offers the opportunity to reduce animal pain inflicted during in vivo experimentation. This study aimed to establish an easy-to-handle ex vivo organ culture model with porcine corneas for the evaluation and modulation of epithelial wound healing. Cultured free-floating cornea disks with a punch defect were observed by stereomicroscopic photo documentation. We analysed the effects of different cell culture media and investigated the impact of different wound sizes as well as the role of the limbus. Modulation of the wound healing process was carried out with the cytostatic agent Mitomycin C. The wound area calculation revealed that after three days over 90% of the lesion was healed. As analysed with TUNEL and lactate dehydrogenase assay, the culture conditions were cell protecting and preserved the viability of the corneal tissue. Wound healing rates differ dependent on the culture medium used. Mitomycin C hampered wound healing in a concentration-dependent manner. The porcine cornea ex vivo culture ideally mimics the in vivo situation and allows investigations of cellular behaviour in the course of wound healing. The effect of substances can be studied, as we have documented for a mitosis inhibitor. This model might aid in toxicological studies as well as in the evaluation of drug efficacy and could offer a platform for therapeutic approaches based on regenerative medicine.

5.
J Vis Exp ; (172)2021 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-34251366

RESUMEN

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing. This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 µm thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling. OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.


Asunto(s)
Neoplasias Pancreáticas , Microambiente Tumoral , Perfilación de la Expresión Génica , Humanos , Técnicas de Cultivo de Órganos , Proteómica
6.
Exp Dermatol ; 19(6): 549-55, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20100195

RESUMEN

Human skin-derived Nestin+ cells serve as a convenient source for autologous, adult, pluripotent progenitor cells that offer new therapeutic possibilities in cell-based regenerative medicine. However, the isolation of human Nestin+ cells has tended to be of very low efficiency and to produce highly variable cell yields. Here we report a standardised protocol that facilitates the isolation and enrichment of Nestin+ progenitor cells from enzymatically digested adult human scalp dermis. The use of distinct media like Dulbecco's modified Eagle medium supplemented with foetal bovine serum or, alternatively, serum-free, supplemented neural stem cell medium greatly affected cell morphology, proliferation and differentiation (e.g. towards a neural versus mesenchymal phenotype). Finally, Nestin+ cells were isolated from a heterogeneous dermis-derived progenitor cell population, which proliferates within clones or floating microspheres under defined serum-free culture conditions. Supplementation of the medium with epidermal growth factor and basic fibroblast growth factor as well as coating with fibronectin allowed the highest enrichment level of Nestin+ progenitors and differentiation towards neural fate. These methodological advances should greatly facilitate the isolation, culture and targeted differentiation of primary, adult human scalp skin dermis-derived Nestin+ cells.


Asunto(s)
Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Separación Celular/métodos , Dermis/citología , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Actinas/metabolismo , Adulto , Células Madre Adultas/efectos de los fármacos , Anciano , Proliferación Celular/efectos de los fármacos , Medios de Cultivo/farmacología , Medio de Cultivo Libre de Suero/farmacología , Femenino , Fibronectinas/farmacología , Expresión Génica/genética , Humanos , Masculino , Mesodermo/citología , Persona de Mediana Edad , Nestina , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Neuronas/metabolismo
7.
Exp Dermatol ; 19(4): 313-24, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20158518

RESUMEN

Fish skin is a multi-purpose tissue that serves numerous vital functions including chemical and physical protection, sensory activity, behavioural purposes or hormone metabolism. Further, it is an important first-line defense system against pathogens, as fish are continuously exposed to multiple microbial challenges in their aquatic habitat. Fish skin excels in highly developed antimicrobial features, many of which have been preserved throughout evolution, and infection defense principles employed by piscine skin are still operative in human skin. This review argues that it is both rewarding and important for investigative dermatologists to revive their interest in fish skin biology, as it provides insights into numerous fundamental issues that are of major relevance to mammalian skin. The basic molecular insights provided by zebrafish in vivo-genomics for genetic, regeneration and melanoma research, the complex antimicrobial defense systems of fish skin and the molecular controls of melanocyte stem cells are just some of the fascinating examples that illustrate the multiple potential uses of fish skin models in investigative dermatology. We synthesize the essentials of fish skin biology and highlight selected aspects that are of particular comparative interest to basic and clinically applied human skin research.


Asunto(s)
Dermatología , Peces/fisiología , Fenómenos Fisiológicos de la Piel , Piel , Animales , Dermatología/métodos , Peces/microbiología , Humanos , Modelos Animales , Piel/citología , Piel/inmunología , Piel/microbiología
8.
Methods Mol Biol ; 1993: 139-148, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31148084

RESUMEN

The interaction of peripheral nerves with different cells of the skin is a relevant aspect of many physiological processes including nociception, temperature control, and wound healing. Here we describe a protocol for the setup of an indirect co-culture system of peripheral nerve cells and sweat gland-derived stem cells, which can be used to quantify neurite outgrowth.


Asunto(s)
Técnicas de Cocultivo/métodos , Regeneración Nerviosa , Nervios Periféricos/citología , Células Madre/fisiología , Glándulas Sudoríparas/citología , Separación Celular , Humanos , Nervios Periféricos/fisiología , Glándulas Sudoríparas/fisiología , Cicatrización de Heridas
9.
Arch Dermatol Res ; 311(4): 325-330, 2019 May.
Artículo en Inglés | MEDLINE | ID: mdl-30798352

RESUMEN

The combination of an aging population and an increasing prevalence of diseases associated with impaired-wound healing, including obesity, peripheral vascular disease and diabetes, is likely to result in a dramatic increase in the incidence and prevalence of chronic skin wounds. Indeed, systemic reviews are now not only trying to establish both the prevalence and the often under-estimated socio-economic costs of chronic skin wounds, but most importantly are addressing the impact that chronic wounds have on quality of life. Given the clear need for novel approaches to the management of chronic skin ulceration, ideally developed and tested in the human system in a manner that can be rapidly translated into clinical practice, we examined the effects of multipotent primary human nestin+ progenitor cells on human wound healing in an ex vivo model. Human sweat gland-derived nestin+ cells demonstrated the capacity to significantly promote two key wound healing parameters, i.e., both reepithelialisation and angiogenesis in experimentally wounded, organ-cultured human skin. The current data further support the use of full-thickness human skin wound-healing models ex vivo to pre-clinically test wound healing-promoting candidate agents. Whilst larger studies are required to substantiate a firm "proof-of-concept," our preliminary studies encourage further efforts to systemically determine the potential of cell-based regenerative medicine strategies in general, and the use of skin appendage-associated human nestin+ cells in particular, as novel treatment strategies for chronic skin ulceration.


Asunto(s)
Terapia Biológica/métodos , Úlcera Cutánea/terapia , Piel/patología , Células Madre/fisiología , Células del Estroma/fisiología , Glándulas Sudoríparas/citología , Adulto , Células Cultivadas , Regeneración Tisular Dirigida , Humanos , Neovascularización Fisiológica , Nestina/metabolismo , Técnicas de Cultivo de Órganos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Calidad de Vida , Repitelización , Cicatrización de Heridas
10.
Eur J Cell Biol ; 87(1): 39-46, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17881083

RESUMEN

Multiple tissue niches in the human body are now recognised to harbour stem cells. Here, we have asked how different adult stem cell populations, isolated from two ontogenetically distinct human organs (skin, pancreas), actually are with respect to a panel of standard markers/characteristics. Here we show that an easily accessible adult human tissue such as skin may serve as a convenient source of adult stem cell-like populations that share markers with stem cells derived from an internal, exocrine organ. Surprisingly, both, human pancreas- and skin-derived stem/progenitor cells demonstrate differentiation patterns across lineage boundaries into cell types of ectoderm (e.g. PGP 9.5+ and GFAP+), mesoderm (e.g. alpha-SMA+) and entoderm (e.g. amylase+ and albumin+). This intriguing differentiation capability warrants systemic follow-up, since it raises the theoretical possibility that an adult human skin-derived progenitor cell population could be envisioned for possible application in cell replacement therapies.


Asunto(s)
Páncreas/citología , Piel/citología , Células Madre/citología , Antígenos de Diferenciación/metabolismo , Diferenciación Celular/fisiología , Femenino , Humanos , Persona de Mediana Edad , Especificidad de Órganos/fisiología , Páncreas/metabolismo , Piel/metabolismo , Trasplante de Células Madre , Células Madre/metabolismo
11.
Stem Cells Int ; 2018: 4623615, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29765411

RESUMEN

The human skin fulfills important barrier, sensory, and immune functions-all of which contribute significantly to health and organism integrity. Widespread skin damage requires immediate treatment and coverage because massive skin loss fosters the invasion of pathogens, causes critical fluid loss, and may ultimately lead to death. Since the skin is a highly immunocompetent organ, autologous transplants are the only viable approach to permanently close a widespread skin wound. Despite the development of tissue-saving autologous transplantation techniques such as mesh and Meek grafts, treatment options for extensive skin damage remain severely limited. Yet, the skin is also a rich source of stem and progenitor cells. These cells promote wound healing under physiological conditions and are potential sources for tissue engineering approaches aiming to augment transplantable tissue by generating cultured epidermal autografts (CEAs). Here, we review autologous tissue engineering strategies as well as transplantation products based on skin-derived stem cells. We further provide an overview of clinical trial activities in the field and discuss relevant translational and clinical challenges associated with the use of these products.

12.
Eur J Cell Biol ; 86(7): 355-76, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17576022

RESUMEN

The discovery of epithelial stem cells (eSCs) in the bulge region of the outer root sheath of hair follicles in mice and man has encouraged research into utilizing the hair follicle as a therapeutic source of stem cells (SCs) for regenerative medicine, and has called attention to the hair follicle as a highly instructive model system for SC biology. Under physiological circumstances, bulge eSCs serve as cell pool for the cyclic regeneration of the anagen hair bulb, while they can also regenerate the sebaceous gland and the epidermis after injury. More recently, melanocyte SCs, nestin+, mesenchymal and additional, as yet ill-defined "stem cell" populations, have also been identified in or immediately adjacent to the hair follicle epithelium, including in the specialized hair follicle mesenchyme (connective tissue sheath), which is crucial to wound healing. Thus the hair follicle and its adjacent tissue environment contain unipotent, multipotent, and possibly even pluripotent SC populations of different developmental origin. It provides an ideal model system for the study of central issues in SC biology such as plasticity and SC niches, and for the identification of reliable, specific SC markers, which distinguish them from their immediate progeny (e.g. transient amplifying cells). The current review attempts to provide some guidance in this growing maze of hair follicle-associated SCs and their progeny, critically reviews potential or claimed hair follicle SC markers, highlights related differences between murine and human hair follicles, and defines major unanswered questions in this rapidly advancing field.


Asunto(s)
Folículo Piloso/citología , Células Madre/citología , Animales , Linaje de la Célula , Folículo Piloso/metabolismo , Humanos , Células Madre Neoplásicas/citología , Pigmentación , Medicina Regenerativa
13.
Ann Anat ; 188(6): 503-17, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17140143

RESUMEN

Cells isolated from pancreas have a remarkable potential for self-renewal and multilineage differentiation. We here present a comprehensive characterisation of stem/progenitor cells derived from exocrine parts of the adult rat pancreas. Using purified cells from either single colonies or even single-cell clones, we specifically demonstrate: (i) the cells contain the typical stem/progenitor cell markers alkaline phophatase, SSEA-1, Oct-4, CD9, Nestin, Pax6, CD44, a-Fetoprotein and Brachyury, demonstrated by immunocytochemistry and RT-PCR; (ii) the cells have the potential to differentiate into lineages of all three germ layers in vitro; (iii) a clonal analysis revealed that even cell lines derived from a single cell have stem/progenitor cell properties such as self-renewal and spontaneous differentiation into various cell lineages; (iv) the cells have the propensity to form three-dimensional, teratoma-like structures in vitro, which contain cells of different lineages; and (v) external stimuli can activate the generation of certain cell types. For instance, cells treated with retinoic acid show an increased expression of alpha-smooth muscle actin. These results suggest that exocrine glands, such as pancreas may be a potential source of adult stem/progenitor cells, suitable for cell therapy of degenerative diseases.


Asunto(s)
Páncreas/citología , Células Madre/citología , Teratoma/patología , Actinas/biosíntesis , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Clonales , Masculino , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/efectos de los fármacos , Tretinoina/farmacología
14.
Anat Embryol (Berl) ; 210(3): 175-85, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16211392

RESUMEN

Pluripotent embryonic stem (ES) cells cultivated as cellular aggregates, so called embryoid bodies (EBs), differentiate spontaneously into different cell types of all three germ layers in vitro resembling processes of cellular differentiation during embryonic development. Regarding chondrogenic differentiation, murine ES cells differentiate into progenitor cells, which form pre-cartilaginous condensations in the EB-outgrowths and express marker molecules characteristic for mesenchymal cell types such as Sox5 and Sox6. Later, mature chondrocytes appear which express collagen type II, and the collagen fibers show a typical morphology as demonstrated by electron-microscopical analysis. These mature chondrogenic cells are organized in cartilage nodules and produce large amounts of extracellular proteoglycans as revealed by staining with cupromeronic blue. Finally, cells organized in nodules express collagen type X, indicating the hypertrophic stage. In conclusion, differentiation of murine ES cells into chondrocytes proceeds from the undifferentiated stem cell via progenitor cells up to mature chondrogenic cells, which then undergo hypertrophy. Furthermore, because the ES-cell-derived chondrocytes did not express elastin, a marker for elastic cartilage tissue, we suggest the cartilage nodules to resemble hyaline cartilage tissue.


Asunto(s)
Cartílago/embriología , Condrocitos/citología , Embrión de Mamíferos/citología , Ratones/embriología , Células Madre Pluripotentes/citología , Animales , Biomarcadores/metabolismo , Cartílago/citología , Diferenciación Celular , Línea Celular , Condrocitos/metabolismo , Colágeno Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Matriz Extracelular/ultraestructura , Proteínas del Grupo de Alta Movilidad/metabolismo , Hibridación Fluorescente in Situ , Microscopía Confocal , Microscopía Electrónica de Transmisión , Proteínas Nucleares/metabolismo , Células Madre Pluripotentes/metabolismo , Factores de Transcripción SOXD , Factores de Transcripción/metabolismo
15.
PLoS One ; 10(11): e0142907, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26565617

RESUMEN

High numbers of adult stem cells are still required to improve the formation of new vessels in scaffolds to accelerate dermal regeneration. Recent data indicate a benefit for vascularization capacity by stimulating stem cells with lipopolysaccharide (LPS). In this study, stem cells derived from human skin (SDSC) were activated with LPS and seeded in a commercially available dermal substitute to examine vascularization in vivo. Besides, in vitro assays were performed to evaluate angiogenic factor release and tube formation ability. Results showed that LPS-activated SDSC significantly enhanced vascularization of the scaffolds, compared to unstimulated stem cells in vivo. Further, in vitro assays confirmed higher secretion rates of proangiogenic as well as proinflammatoric factors in the presence of LPS-activated SDSC. Our results suggest that combining activated stem cells and a dermal substitute is a promising option to enhance vascularization in scaffold-mediated dermal regeneration.


Asunto(s)
Regeneración/fisiología , Fenómenos Fisiológicos de la Piel , Piel/irrigación sanguínea , Piel/patología , Células Madre/citología , Ingeniería de Tejidos/métodos , Animales , Biopsia , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Colágeno/química , Medios de Cultivo Condicionados/química , Humanos , Inflamación , Lipopolisacáridos/química , Ratones , Ratones Desnudos , Neovascularización Fisiológica , Piel/citología , Piel Artificial , Andamios del Tejido , Cicatrización de Heridas
16.
Invest Ophthalmol Vis Sci ; 56(8): 4350-63, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26176872

RESUMEN

PURPOSE: Aqueous tear deficiency due to lacrimal gland insufficiency is one of the major causes of dry eye disease. In severe cases, such as Sjogren's syndrome, Stevens-Johnson syndrome, or ocular cicatricial pemphigoid, therapy with artificial tears is often insufficient to relieve severe discomfort, prevent progressive ocular surface disease, or enable visual rehabilitation by corneal transplantation. Cell or organ generation from stem cells, resulting in tear-like secretion, presents an option as a suitable alternative treatment. To obtain deeper insights into lacrimal gland stem cells we analyzed murine lacrimal glands for markers of pluripotency, self-renewal, and differentiation. METHODS: A special, patented technique with mechanical and enzymatic digestion was used to generate high numbers of cells in vitro from murine lacrimal glands. These presumptive "murine lacrimal gland stem cells" ("mLGSCs") can be propagated as monolayer cultures over multiple passages. By means of RT-PCR, Western blot, and immunohistochemistry, markers of pluripotency and differentiation were demonstrated. Hanging drop culture was used to build organoid bodies from mLGSCs to investigate their spontaneous differentiation in three-dimensional culture with histology, immunohistochemistry, and transmission electron microscopy methods. RESULTS: Isolated mLGSCs were cultured over more than 65 passages. Murine lacrimal gland stem cells expressed markers of pluripotency such as Nanog, Sox2, Kruppel-like factor 4 (Klf4), as well as early-lineage markers of all three germ layers. Three-dimensional culture of these cells revealed their ability to differentiate into various cell types. CONCLUSIONS: Our results suggest that mLGSCs were isolated and cultured successfully. These cells have the ability to differentiate into all three germ layers. The results provide further insights into lacrimal gland stem cell physiology for engineering of a lacrimal gland construct to treat severe cases of tear deficiency in the future.


Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Síndromes de Ojo Seco/terapia , Aparato Lagrimal/ultraestructura , Células Madre/ultraestructura , Lágrimas/metabolismo , Animales , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Síndromes de Ojo Seco/metabolismo , Síndromes de Ojo Seco/patología , Inmunohistoquímica , Factor 4 Similar a Kruppel , Aparato Lagrimal/metabolismo , Ratones , Microscopía Electrónica de Transmisión
17.
Int J Biochem Cell Biol ; 36(7): 1306-18, 2004 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15109574

RESUMEN

The biological relevance of vigilin a ubiquitous multi (KH)-domain protein is still barely understood. Investigations over the last years, however, provided evidence for a possible involvement of vigilin in the nucleo-cytoplasmic transport of tRNA and in the subsequent association of tRNA with ribosomes. We therefore investigated the potential association of vigilin with 80S ribosomes. Immunostaining, gel filtration, westernblot analysis of polyribosomes and high salt treatment of 80S ribosomes isolated from fresh human placenta were applied to analyze the possible association of vigilin with ribosomes. Overlay assays were performed to examine whether vigilin is capable of binding to ribosomal proteins. Immunostaining of HEp-2 cells, gel filtration of a cytoplasmic extract of HEp-2 cells and westernblot analysis of isolated 80S ribosomes clearly demonstrate that vigilin is bond to the ribosomal complex. Vigilin detaches from the ribosomal complex under the influence of high salt concentrations. We present data that radioactively labeled human vigilin interacts directly with a subset of ribosomal proteins from both subunits. We were able to narrow down the putative binding region to the C-terminal domain by using vigilin mutant constructs. Therefore our results provide strong evidence that vigilin is bond to the ribosomal complex and underline the hypothesis that vigilin might be involved in the link between tRNA-export and the channeled tRNA-cycle on ribosomes.


Asunto(s)
Proteínas Portadoras/análisis , Proteínas Portadoras/metabolismo , Polirribosomas/metabolismo , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Portadoras/genética , Línea Celular , Nucléolo Celular/ultraestructura , Femenino , Expresión Génica , Humanos , Placenta/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/genética , Ribosomas/química , Eliminación de Secuencia/genética
18.
Int J Mol Med ; 12(1): 35-43, 2003 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12792806

RESUMEN

Vigilin, a member of the KH protein family, is exceptional among these proteins as it contains 14 KH domains in consecutive order. Vigilin is present in the nucleus and the cytoplasm of all eucaryotic cells studied so far and has apparently high affinity to tRNA and mRNA. There is circumstantial evidence that vigilin expression parallels high translational activity as demonstrated for pancreatic cells in vitro and in vivo as well as for carcinoma cell lines. On a molecular level we have recently demonstrated that vigilin promotes in vitro the export of tRNA from the nucleus to the translational machinery in the cytoplasm and may hence function as an intercompartimental conveyor. In the present study we show that exposure to a vigilin antisense oligo DNA (VAOD) expectedly resulted in a decrease of vigilin-expression, and was concomitant to lower amylase- and trypsin synthesis in freshly isolated pancreatic cells. In addition, carcinoma cells reacted with an increased mortality under exposure to VAOD giving further support for the notion that vigilin participates in cellular life-sustaining processes such as protein translation.


Asunto(s)
Carcinoma/tratamiento farmacológico , Proteínas Portadoras , ADN sin Sentido/farmacología , Páncreas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas de Unión al ARN/genética , Animales , Humanos , Immunoblotting , Masculino , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley
19.
Stem Cell Res Ther ; 4(4): 78, 2013 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-23835213

RESUMEN

INTRODUCTION: Nestin-expressing cells isolated from different human tissues reveal self-renewal capacity and a multilineage differentiation potential. In particular, adult stem/progenitor cell populations from exocrine glands such as the pancreas, salivary gland and sweat gland are characterized by prominent nestin expression. Interestingly, human mammary gland histological examinations also demonstrated the existence of nestin-positive cells in the ductal compartments. Within the scope of our previous work we wonder whether an isolation of nestin-positive cell populations from human mammary gland biopsies is possible and what characteristics they have in vitro. Cell populations from both sexes were propagated and subjected to a comparison with other gland-derived cell populations. METHODS: Human mammary tissue biopsies were mechanically and enzymatically treated, and the isolated acini structures were observed with time-lapse microscopy to track adherently outgrowing cells. The proliferation potential of the cell population was assessed by performing growth curves. On the gene and protein levels we investigated the expression of stem cell markers as well as markers indicating multilineage differentiation. RESULTS: We succeeded in establishing proliferating cell populations from breast tissue biopsies of both sexes. Our results display several similarities to the glandular stem cell populations from other exocrine glands. Beside their proliferation capacity during in vitro culture, the obtained cell populations are characterized by their prominent nestin expression. The cells share surface proteins commonly expressed on adult stem cells. We demonstrated the expression of stem cell-related genes like Oct4, Sox2, KLF4 and Nanog, and confirmed multipotent differentiation capacity by detecting transcripts expressed in endodermal, mesodermal and ectodermal cell types. CONCLUSION: With this study we present an efficient procedure for isolation and propagation of nestin-positive stem cells obtained from male and female breast tissue, which is frequently available. The established multipotent cell populations could be easily expanded in vitro and thus hold promise for cell-based therapies and personalized medicine.


Asunto(s)
Glándulas Mamarias Humanas/patología , Nestina/metabolismo , Células Madre/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Femenino , Humanos , Factor 4 Similar a Kruppel , Masculino , Células Madre/citología , Donantes de Tejidos
20.
J Invest Dermatol ; 133(5): 1140-9, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23407389

RESUMEN

Mammalian and fish skin share protective activities against environments that are rich in infectious agents. Fish epidermis is endowed with an extrinsic barrier consisting of a mucus layer and antimicrobial peptides (AMPs). These operate together as a protective chemical shield. As these AMPs are evolutionarily well preserved and also found in higher vertebrate skin (including human epidermis), fish skin offers a unique opportunity to study the origins of innate antimicrobial defense systems. Furthermore, the broad spectrum of fish mucus antimicrobial activities renders piscine AMPs interesting to investigative dermatology, as these may become exploitable for various indications in clinical dermatology. Therefore, this article aims at casting light on fish mucus, the evolutionary relationship between human and fish AMPs, and the latter's antibacterial, antifungal, and even antiviral activities. Moreover, we develop dermatological lessons from, and sketch potential future clinical applications of, fish mucus and piscine AMPs.


Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Epidermis/metabolismo , Peces/metabolismo , Animales , Epidermis/inmunología , Peces/inmunología , Humanos , Inmunidad Innata/fisiología , Modelos Animales , Moco/inmunología , Moco/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA