RESUMO
Adipose tissue-derived stem cells (ASCs) can be obtained from different adipose tissue sources within the body. It is an abundant cell pool, easily accessible, suitable for cultivation and expansion in vitro and preparation for therapeutic approaches. Amongst these therapeutic approaches are tissue engineering and nervous system disorders such as spinal cord injuries. For such treatment, ASCs have to be reliably differentiated in to the neuronal direction. Therefore, we investigated the neural differentiation potential of ASCs using protocols with neurogenic inductors such as valproic acid and forskolin, while dog brain tissue served as control. Morphological changes could already be noticed 1 h after neuronal induction. Gene expression analysis revealed that the neuronal markers nestin and ßIII-tubulin as well as MAP2 were expressed after induction of neuronal differentiation. Additionally, the expression of the neurotrophic factors NGF, BDNF and GDNF was determined. Some of the neuronal markers and neurotrophic factors were already expressed in undifferentiated cells. Our findings point out that ASCs can reliably be differentiated into the neuronal lineage; therefore, these cells are a suitable cell source for cell transplantation in disorders of the central nervous system. Follow-up studies would show the clinical benefit of these cells after transplantation.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/veterinária , Células-Tronco Mesenquimais/citologia , Doenças Neurodegenerativas/terapia , Neurônios/citologia , Traumatismos da Medula Espinal/terapia , Tecido Adiposo/citologia , Animais , Biomarcadores/análise , Encéfalo/citologia , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Diferenciação Celular/fisiologia , Cães , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Proteínas Associadas aos Microtúbulos/biossíntese , Fator de Crescimento Neural/biossíntese , Nestina/biossíntese , Doenças Neurodegenerativas/veterinária , Traumatismos da Medula Espinal/veterinária , Tubulina (Proteína)/biossínteseRESUMO
Samples of low modulus beta-type Ti40Nb and cp2-Ti were chemically treated with 98% H2 SO4 + 30% H2 O2 (vol. ratio 1:1) solution. Surface analytical studies conducted with HR-SEM, AFM, and XPS identified a characteristic nanoroughness of the alloy surface related with a network of nanopits of â¼25 nm diameter. This is very similar to that obtained for cp2-Ti. The treatment enhances the oxide layer growth compared to mechanically ground states and causes a strong enrichment of Nb2 O5 relative to TiO2 on the alloy surface. The in vitro analyses clearly indicated that the chemical treatment accelerates the adhesion and spreading of human mesenchymal stromal cells (hMSC), increases the metabolic activity, and the enzyme activity of tissue non-specific alkaline phosphatase (TNAP). Surface structures which were generated mimic the cytoplasmic projections of the cells on the nanoscale. Those effects are more pronounced for the Ti40Nb alloy than for cp2-Ti. The relation between alloy surface topography and chemistry and cell functions is discussed.
Assuntos
Materiais Biocompatíveis/química , Células-Tronco Mesenquimais/fisiologia , Nióbio/química , Titânio/química , Fosfatase Alcalina/metabolismo , Ligas/química , Adesão Celular/fisiologia , Humanos , Teste de Materiais , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Nanotecnologia , Próteses e Implantes , Propriedades de SuperfícieRESUMO
Amniotic fluid contains heterogeneous cell types and has become an interesting source for obtaining fetal stem cells. These stem cells have a high proliferative capacity and a good differentiation potential and may thus be suitable for regenerative medicine. As there is increasing evidence, that these stem cells are also able to be directed into the neural lineage, in our study we investigated the neuronal and glial differentiation potential of these cells, so that they may also be applied to cure degenerative diseases of the retina. Mesenchymal stem cells were isolated from routine prenatal amniocentesis at 15 to 18 weeks of pregnancy of human amniotic fluid and expanded in the cell culture. Cells were cultivated according to standard procedures for mesenchymal stem cells and were differentiated along the neural lineage using various protocols. Furthermore, it was also tried to direct them into cell types of the retina as well as into endothelial cells. Cells of more than 72 amniotic fluid samples were collected and characterized. While after induction neural-like phenotypes could actually be detected, which was confirmed using neural marker proteins such as GFAP and ßIII tubulina further differentiation into retinal like cells could not reliably be shown. These data suggest that amniotic fluid derived cells are an interesting cell source, which may also give rise to neural-like cells. However, a more specific differentiation into neuronal and glial cells could not unequivocally be shown, so that further investigations have to becarried out.
RESUMO
Mesenchymal stem cells are regarded as common cellular precursors of the musculoskeletal tissue and are responsible for tissue regeneration in the course of musculoskeletal disorders. In equine veterinary medicine extracorporeal shock wave therapy (ESWT) is used to optimize healing processes of bone, tendon and cartilage. Nevertheless, little is known about the effects of the shock waves on cells and tissues. Thus, the aim of this study was to investigate the influence of focused ESWT on the viability, proliferation, and differentiation capacity of adipose tissue-derived mesenchymal stem cells (ASCs) and to explore its effects on gap junctional communication and the activation of signalling cascades associated with cell proliferation and differentiation. ASCs were treated with different pulses of focused ESWT. Treated cells showed increased proliferation and expression of Cx43, as detected by means of qRT-PCR, histological staining, immunocytochemistry and western blot. At the same time, cells responded to ESWT by significant activation (phosphorylation) of Erk1/2, detected in western blots. No significant effects on the differentiation potential of the ASCs were evident. Taken together, the present results show significant effects of shock waves on stem cells in vitro.
RESUMO
Mesenchymal stem cells have become extremely interesting for regenerative medicine and tissue engineering in the horse. Stem cell therapy has been proven to be a powerful and successful instrument, in particular for the healing of tendon lesions. We pre-differentiated equine adipose-tissue-derived stem cells (ASCs) in a collagen I gel scaffold by applying tensile strain, growth differentiation factors (GDFs) and various oxygen tensions in order to determine the optimal conditions for in vitro differentiation toward the tenogenic lineage. We compared the influence of 3% versus 21% oxygen tension, the use of GDF 5, GDF 6 and GDF 7 and the application of uniaxial tensile strain versus no mechanical stimulation on differentiation results as evaluated by cell morphology and by the expression of the tendon-relevant genes collagen I, collagen III, cartilage oligomeric matrix protein and scleraxis. The best results were obtained with an oxygen tension of 21%, tensile stimulation and supplementation with GDF 5 or GDF 7. This approach raises the hope that the in vivo application of pre-differentiated stem cells will improve healing and recovery time in comparison with treatment involving undifferentiated stem cells.
Assuntos
Tecido Adiposo/citologia , Diferenciação Celular/efeitos dos fármacos , Fatores de Diferenciação de Crescimento/farmacologia , Oxigênio/farmacologia , Células-Tronco/citologia , Tendões/citologia , Resistência à Tração/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Reatores Biológicos , Comunicação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Colágeno Tipo I/farmacologia , Géis , Regulação da Expressão Gênica/efeitos dos fármacos , Cavalos , Imuno-Histoquímica , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Células-Tronco/efeitos dos fármacos , Células-Tronco/ultraestrutura , Alicerces TeciduaisRESUMO
Amniotic fluid (AF) has become an interesting source of fetal stem cells. However, AF contains heterogeneous and multiple, partially differentiated cell types. After isolation from the amniotic fluid, cells were characterized regarding their morphology and growth dynamics. They were sorted by magnetic associated cell sorting using the surface marker CD 117. In order to show stem cell characteristics such as pluripotency and to evaluate a possible therapeutic application of these cells, AF fluid-derived stem cells were differentiated along the adipogenic, osteogenic, and chondrogenic as well as the neuronal lineage under hypoxic conditions. Our findings reveal that magnetic associated cell sorting (MACS) does not markedly influence growth characteristics as demonstrated by the generation doubling time. There was, however, an effect regarding an altered adipogenic, osteogenic, and chondrogenic differentiation capacity in the selected cell fraction. In contrast, in the unselected cell population neuronal differentiation is enhanced.
RESUMO
Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-ß1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-ß1 (5 ng/ml) or fish collagen (0.5 mg/ml) for a 3 week period. After the 3 weeks in vitro differentiation, RT-PCR and histological staining for proteoglycan synthesis and type II collagen were performed to evaluate the degree of chondrogenic differentiation and the formation of cartilaginous extracellular matrix (ECM). The differentiation of ADSCs induced by TGF-ß1 showed a high expression of glycosaminoglycan (GAG). Histological analysis of cultures stimulated by hydrolyzed fish collagen demonstrated an even higher GAG expression than cultures stimulated under standard conditions by TGF-ß1. The expression of cartilage-specific type II collagen and Sox9 was about the same in both stimulated cultures. In this study, chondrogenesis was as effectively induced by hydrolyzed fish collagen as it was successfully induced by TGF-ß1. These findings demonstrated that hydrolyzed fish collagen alone has the potential to induce and maintain ADSCs-derived chondrogenesis. These results support the application of ADSCs in equine veterinary tissue engineering, especially for cartilage repair.
Assuntos
Tecido Adiposo/citologia , Diferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Células Estromais/metabolismo , Animais , Condrócitos/ultraestrutura , Colágeno/farmacologia , Colágeno Tipo II/biossíntese , Colágeno Tipo II/ultraestrutura , Matriz Extracelular/metabolismo , Peixes , Cavalos , Hidrolisados de Proteína/farmacologia , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
NADPH-diaphorase (NADPH-d) staining of the bovine olfactory epithelium was compared with the immunohistochemical localization of nitric oxide synthase (NOS), soluble guanylyl cyclase, and cGMP (cyclic guanosine 3',5'-monophosphate). Out of the three isoforms, only the inducible NOS (NOS-II) was found at the epithelial surface correlating with the strong labelling for NADPH-d. In contrast, light diaphorase staining associated with deeper epithelial regions did not coincide with any NOS immunoreactivity. As there is overlapping expression of NOS-II, soluble guanylyl cyclase and cGMP at the luminal surface morphologically occupied by dendritic knobs of olfactory receptor neurons and microvillar endings of supporting cells, the nitric oxide (NO)/cGMP pathway is likely to be involved in modulating the odour signals during olfactory transduction.
Assuntos
NADPH Desidrogenase/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Mucosa Olfatória/enzimologia , Animais , Bovinos , GMP Cíclico/metabolismo , Dendritos/enzimologia , Guanilato Ciclase/metabolismo , Imuno-Histoquímica , Óxido Nítrico/metabolismo , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/enzimologia , Neurônios Receptores Olfatórios/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Guanilil Ciclase SolúvelRESUMO
Cell-based therapy using adult mesenchymal stem cells (MSCs) has already been the subject of clinical trials, but for further development and optimization the distribution and integration of the engrafted cells into host tissues have to be monitored. Today, for this purpose magnetic resonance imaging (MRI) is the most suitable technique, and micron-sized iron oxide particles (MPIOs) used for labeling are favorable due to their low detection limit. However, constitutional data concerning labeling efficiency, cell viability, and function are lacking. We demonstrate that cell viability and migratory potential of bone marrow mesenchymal stromal cells (BMSCs) are negatively correlated with incorporated MPIOs, presumably due to interference with the actin cytoskeleton. Nevertheless, labeling of BMSCs with low amounts of MPIOs results in maintained cellular function and sufficient contrast for in vivo observation of single cells by MRI in a rat glioma model. Conclusively, though careful titration is indicated, MPIOs are a promising tool for in vivo cell tracking and evaluation of cell-based therapies.
Assuntos
Movimento Celular , Meios de Contraste/análise , Compostos Férricos/análise , Imageamento por Ressonância Magnética , Transplante de Células-Tronco Mesenquimais , Nanopartículas Metálicas/análise , Animais , Células da Medula Óssea/citologia , Neoplasias Encefálicas/terapia , Sobrevivência Celular , Glioma/terapia , Masculino , Transplante de Neoplasias , Ratos , Ratos WistarRESUMO
Efficient transformation of primary human amniocytes by E1 gene functions of human adenovirus serotype 5 (Ad5) yield in stable cell lines, which exhibit morphological features of epithelial like cells. A thorough investigation using immunocytochemistry confirmed the expression of epithelial cell markers. The analysis also revealed the expression of neuronal and glial marker proteins, such as nestin, vimentin, A2B5 and GFAP. Using RT-PCR, transcripts of the neurotrophic factors nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), glial cell line derived neurotrophic factor (GDNF), and neurotrophin 3 (NT-3) could be detected. Neurotrophic factors could also be detected in the cell culture supernatants of transformed amniocytes. In line with previous experimental data on a human Ad5 E1-transformed embryonal kidney cell line (HEK-293), the results suggest a co-expression of epithelial and neuronal marker proteins in E1-transformed human amniotic fluid derived cells and thus a preferential transformation into neuronal-like cells.
Assuntos
Adenoviridae/genética , Âmnio/citologia , Células Epiteliais/citologia , Neurônios/citologia , Transformação Genética , Proteínas E1 de Adenovirus/genética , Âmnio/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem Celular Transformada , Células Cultivadas , Células Epiteliais/metabolismo , Vetores Genéticos/genética , Células HeLa , Humanos , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Iron oxide particles are especially suited for cell tracking experiments due to their extraordinarily molar relaxivity as compared with other paramagnetic nuclei. We have compared different iron oxide particles (Sinerem, Endorem and magnetic microspheres) for their suitability to label embryonic stem cells (D3 cell line). In addition to detectability thresholds, particular attention has been paid to the evaluation of long-term stability of the labelling procedure (up to 4 weeks) as well as to toxic and other adverse effects on cell viability. Comparative studies were performed using neural progenitor cells (C17.2) and dendritic cells. The present study indicates strong dependence of the label efficiency and stability on the iron oxide particles and cell lines in use.
Assuntos
Meios de Contraste/farmacologia , Células Dendríticas/citologia , Células-Tronco Embrionárias/citologia , Compostos Férricos/farmacologia , Imageamento por Ressonância Magnética , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Meios de Contraste/efeitos adversos , Estabilidade de Medicamentos , Compostos Férricos/efeitos adversos , Camundongos , Fatores de TempoRESUMO
BACKGROUND: Retinitis pigmentosa belongs to a large group of degenerative diseases of the retina with a hereditary background. It involves loss of retinal photoreceptor cells and consequently peripheral vision. At present there are no satisfactory therapeutic options for this disease. Just recently the use of mesenchymal stem cells has been discussed as one therapeutical option for retinal degeneration, as they have been shown to differentiate into various cell types, including photoreceptor cells. In this article we wanted to investigate the potency of mesenchymal stem cells to induce rescue effects in an animal model for retinitis pigmentosa, the rhodopsin knockout mouse. METHODS: For the experiments, three experimental groups of 10 animals each were formed. The first group consisted of untreated rhodopsin knockout (rho(-/-)) animals used as controls. The second group consisted of rho(-/-) mice that had received an injection of mouse mesenchymal stem cells, which were transduced using an adenoviral vector containing the sequence for the green fluorescent protein (GFP) prior to transplantation. In the third sham group, animals received an injection of medium only. Thirty-five days after transplantation, GFP-expressing cells were detected in whole-mount preparations of the retinas as well as in cryostat sections. For the detection of rescue effects, semi-thin sections of eyes derived from all experimental groups were produced. Furthermore, rescue effects were also analysed ultrastructurally in ultrathin sections. RESULTS: Histological analysis revealed that after transplantation, cells morphologically integrated not only into the retinal pigment epithelium but also into layers of the neuroretina displaying neuronal and glial morphologies. Furthermore, significant rescue effects, as demonstrated by the occurrence of preserved photoreceptor cells, were detected. CONCLUSIONS: Our data indicate that mesenchymal stem cells can prolong photoreceptor survival in the rhodopsin knockout mouse, also providing evidence of a therapeutical benefit in retinitis pigmentosa.
Assuntos
Células da Medula Óssea/citologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células Fotorreceptoras de Vertebrados/fisiologia , Retinose Pigmentar/cirurgia , Rodopsina/genética , Adenoviridae/genética , Animais , Sobrevivência Celular , Modelos Animais de Doenças , Técnica Indireta de Fluorescência para Anticorpo , Inativação Gênica/fisiologia , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Knockout , Células Fotorreceptoras de Vertebrados/ultraestrutura , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , TransfecçãoRESUMO
In the enteric nervous system, nitric oxide (NO) is regarded as an important messenger for the non-adrenergic and non-cholinergic neurotransmission. Synthesized mainly by the constitutive nitric oxide synthase (NOS) isoforms NOS I and NOS III, this molecule exerts prejunctional inhibitory effects in the submucosal plexus as well as relaxation of enteric smooth muscles. In order to elucidate the role for NO during enteric development, we looked for the expression of all three NOS-isoforms in the enteric nervous system during mouse development from E8 to E20 using immunohistochemistry. Starting around midgestation, a transient expression of the NOS-II isoform during the very early development of enteric neurones was detected in parallel to that of HNK-1 exclusively in the myenteric plexus. Similar to findings for other neuronal systems, NOS-I and NOS III isoforms could be traced starting significantly later to increase toward the end of embryonic development when NOS II immunoreactivity faded and a strong expression of the vasointestinal peptide could be detected. In contrast to the NOSII expression, the constitutive isoforms can also be detected in the submucosal plexus. Altogether, these findings suggest NOS-II to be exclusively involved during early steps of enteric nervous system development. Absence of downstream signalling elements, such as sGC and cGMP both in neurons and in enteric muscle until the end of the second third of gestation, may indicate different effects executed by NO during development, expressed by Ca(2+) -dependent and Ca(2+) -independent NOS isoforms.
Assuntos
Sistema Nervoso Entérico/embriologia , Sistema Nervoso Entérico/enzimologia , Isoenzimas/metabolismo , Óxido Nítrico Sintase/metabolismo , Animais , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Feminino , Idade Gestacional , Isoenzimas/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , GravidezRESUMO
For the treatment of neurodegenerative disorders such as Parkinson's disease cell or gene therapeutical options are increasingly verified. For such approaches, neural stem cells or astrocytes are discussed as possible cell candidates. As also fetal retinal pigment epithelial cells have been successfully tested for such therapeutical options, we investigated the potential of iris pigment epithelial cells as an autologous source for future cell replacement therapies. Using the ELISA technique, we looked for the secretion of neurotrophic factors under basal and stimulated conditions by iris pigment epithelial cells (IPE) cells and compared them with the secretion of retinal pigment epithelial cells (RPE) cells. As iron plays a causative role in cell death during Parkinson's disease, the iron-binding capacity by IPE cells was investigated. Furthermore, we checked the integrative capacity of IPE cells after transplantation into the striatum of adult rats. Our data reveal that IPE cells produce and secrete a variety of neurotrophic factors which can be stimulated after treatment with cytokines. Following transplantation, the cells can be easily detected by their pigmentation, survive for at least 8 weeks and as shown by electron microscopy integrate within the host tissue. Moreover, cells can be transduced with high efficiency using a third generation adenoviral vector, making them promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases in a combined cell and gene therapeutical approach.
Assuntos
Células Epiteliais/transplante , Células Epiteliais/ultraestrutura , Iris/citologia , Epitélio Pigmentado Ocular/transplante , Epitélio Pigmentado Ocular/ultraestrutura , Animais , Diferenciação Celular , Movimento Celular , Separação Celular , Células Epiteliais/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Sobrevivência de Enxerto/fisiologia , Humanos , Ferro/metabolismo , Neostriado/citologia , Neostriado/cirurgia , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/metabolismo , Doenças Neurodegenerativas/terapia , Epitélio Pigmentado Ocular/metabolismo , Ratos , Ratos Long-Evans , Técnicas EstereotáxicasRESUMO
Conventional therapeutical approaches such as surgery, radiotherapy, or chemotherapy have been shown to be rather unsuccessful in the treatment of infiltrative growing tumors such as the malignant glioblastoma multiforme. Thus, new therapeutical strategies have to be developed that are suitable for inducing cell death also in migrating tumor cells. These new therapeutical stategies include cell and/or gene therapeutical approaches. We demonstrate that glial-restricted progenitor cells as well as embryonic stem cell-derived neural stem cells belong to cell populations applicable to such therapeutical concepts. Both cell types can be efficiently transduced using a third-generation high-capacity "gutless" adenoviral vector, and show a tropism for the F98 glioma cells by migrating towards a spheroid of F98 glioma cells with a tendency to form a barrier around the tumor spheroid in an in vitro tumor confrontation model. Moreover, in a migration assay, secretion products of glial-restricted precursor cells have shown a potency to inhibit the migratory activity of glioma cells in vitro. In vivo, F98 glioma cell-derived tumor formation in the right striatum resulted in migration of glial as well as neural precursor cells towards the tumor area when cotransplanted in the corpus callosum of the contralateral hemisphere. After arrival, both cell types surround the tumor mass and even invade the experimentally induced tumor. These data indicate that glial-restricted as well as embryonic stem cell-derived neural precursor cells are good candidates as carriers for an ex vivo gene therapeutical approach in tumor therapy.
Assuntos
Terapia Genética/métodos , Vetores Genéticos , Neoplasias/terapia , Neuroglia/fisiologia , Neurônios/fisiologia , Células-Tronco/fisiologia , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Transplante de Células , Células Cultivadas , Quimiotaxia/fisiologia , Embrião de Mamíferos/fisiologia , Masculino , Transplante de Neoplasias , Neoplasias/metabolismo , Neoplasias/patologia , Neuroglia/citologia , Neurônios/citologia , Ratos , Ratos Endogâmicos F344 , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Células-Tronco/citologiaRESUMO
Nitric oxide (NO), a cell-derived highly diffusible and unstable gas is regarded to be involved in inter- and intracellular communication in the nervous system. Based on findings about the expression of the inducible NO synthase (NOS) isoform during development of early mouse olfactory as well as vestibulocochlear receptor neurons, we intended to prove a general role of this isoform for neuronal differentiation. Using immunohistochemical techniques, an exclusive expression of the inducible NOS-II isoform in early post-mitotic neurons of the developing mouse cortex and retina can be detected. In a pharmacological approach using cultures of the mouse cortex as well as embryonic stem cell-derived neural precursor cells, we investigated the functional role of NO on initial neuronal differentiation. Effects of NOS inhibitors and NO donors on the morphological differentiation were correlated with developmentally regulated calcium current densities, focusing on the effects of the specific NOS-II inhibitor GW 274150. Furthermore, involvement of the soluble guanylate cyclase (sGC)/cGMP signaling cascade was pharmacologically investigated. Our data indicate that while a specific block of NOS-II provokes a clear inhibition of neurite outgrowth formation as well as a decrease of calcium current densities, the inverse is true for exogenous NO donation. In line with lacking immunoreactivity for the sGC and cGMP there are only minor effects of compounds manipulating the sGC/cGMP pathway, suggesting the downstream sGC/cGMP pathway not to be essential in these early differentiation steps.
Assuntos
Córtex Cerebral/enzimologia , Córtex Cerebral/crescimento & desenvolvimento , Neurônios/enzimologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Retina/enzimologia , Retina/crescimento & desenvolvimento , Animais , Cálcio/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , GMP Cíclico/metabolismo , Eletrofisiologia , Embrião de Mamíferos , Imuno-Histoquímica , Camundongos , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Retina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sulfetos/farmacologiaRESUMO
To investigate the ability of genetically modified astrocytes to integrate into adult rat brain, two spontaneously immortalized cell lines and the allogenic nontumorigenic glioma cell line F98 were transduced with a high-capacity adenoviral vector (HC-Adv) expressing the EGFP gene from the hCMV promoter. In organotypic slice cultures the transduced astrocytes were shown to integrate into the brain tissue. Following transplantation of the transduced astrocytes into the striatum of adult rats, the transplanted cells survived at least for 6 weeks, continuously expressed the EGFP transgene, in close neighborhood with cells of the recipient tissue executing their differentiation capacity along the glial lineage. Thus, HC-Adv transduced astrocytes are promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases.
Assuntos
Adenoviridae/genética , Astrócitos/transplante , Linhagem Celular Transformada/transplante , Transplante de Células/métodos , Vetores Genéticos/uso terapêutico , Sobrevivência de Enxerto/genética , Células Tumorais Cultivadas/transplante , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Diferenciação Celular/genética , Linhagem Celular Transformada/citologia , Linhagem Celular Transformada/metabolismo , Linhagem da Célula/genética , Tamanho Celular/genética , Sobrevivência Celular/genética , Transformação Celular Viral/genética , Transplante de Células/tendências , Células Cultivadas , Corpo Estriado/citologia , Corpo Estriado/crescimento & desenvolvimento , Corpo Estriado/transplante , Feto , Regulação Viral da Expressão Gênica/genética , Vetores Genéticos/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes , Regiões Promotoras Genéticas/genética , Ratos , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/metabolismoRESUMO
Expression of the enhanced green fluorescent protein (EGFP) under control of a thymidine kinase promoter/nestin second intron was specifically detected in nestin immunoreactive neural precursor cells after selection of murine embryonic stem (ES) cells in chemically defined medium. Allowing differentiation in vitro, the capacity of these cells to give rise to astroglia, oligodendroglia, and neurones was investigated. After intracerebral transplantation, long-lasting integration of precursor cells into the host tissue was observed, serving as a pool for successive neuronal and glial differentiation. EGFP expression by ES cell-derived neural precursor cells may be a valuable tool to optimize protocols for maintenance and expansion of these cells in vitro as well as in vivo after intracerebral transplantation. In addition, preparative fluorescence-activated cell sorting of EGFP-labeled neural precursor cells should be useful for standardization of a donor cell population for cell replacement therapies.
Assuntos
Embrião de Mamíferos/citologia , Transplante de Células-Tronco Hematopoéticas , Proteínas de Filamentos Intermediários/metabolismo , Proteínas Luminescentes/metabolismo , Proteínas do Tecido Nervoso , Animais , Diferenciação Celular , Linhagem Celular , Separação Celular , Relação Dose-Resposta a Droga , Citometria de Fluxo , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Camundongos , Microscopia de Fluorescência , Nestina , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Ratos Wistar , Fatores de Tempo , TransgenesRESUMO
In the present study the localization of heme oxygenase-2 (HO-2) in developing and mature olfactory epithelium of the bovine is investigated using immunohistochemistry and post embedding immunogold labelling. HO-2 immunoreactivity is first seen in epithelial cells localized along the luminal surface of the olfactory pit. Up to midgestation the number of HO-2 immunoreactive cells increases throughout all layers of the developing olfactory epithelium. From midgestation through adulthood immunostaining is restricted to perinuclear cytoplasm and axons of mature olfactory receptor neurons localized in intermediate epithelial regions. The temporal and spatial expression patterns of HO-2 immunohistochemistry support the notion that CO plays a role in neuronal differentiation while its presence in mature neurons might be functionally related to olfactory transduction.
Assuntos
Heme Oxigenase (Desciclizante)/análise , Mucosa Olfatória/embriologia , Mucosa Olfatória/enzimologia , Fatores Etários , Animais , Anticorpos , Antígenos CD57/análise , Antígenos CD57/imunologia , Bovinos , Feminino , Feto/enzimologia , Heme Oxigenase (Desciclizante)/imunologia , Masculino , Microscopia Imunoeletrônica , Mucosa Olfatória/ultraestrutura , Fosfopiruvato Hidratase/análise , Fosfopiruvato Hidratase/imunologiaRESUMO
According to the view of differentiation-related alterations in the glycosylation pattern of neurones, recent studies have shown development dependent expression patterns of lactoseries carbohydrate epitopes, CD15 and HNK-1, on olfactory receptor neurones in rats and chicks. In order to evaluate a general role for these epitopes during development of vertebrate olfactory receptor neurones, this investigation focuses on the situation in the mouse, bovine and Xenopus olfactory epithelium. In all three species CD15 expression was found on a subpopulation of morphologically mature receptor cells starting at the time of initial synaptogenesis. Whereas for bovine and Xenopus the timetable of HNK-1 expression is similar to that described for the chick, suggesting involvement in pathfinding, in the mouse HNK-1 is found on immature cells when mature CD15 positive receptor cells could already be discerned. By our results a role for CD15 during establishment of synaptic contacts and for HNK-1 during their formation is suggested.