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1.
Acta Neuropathol Commun ; 8(1): 13, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019610

RESUMO

Tau deposition in the brain is a pathological hallmark of many neurodegenerative disorders, including Alzheimer's disease (AD). During the course of these tauopathies, tau spreads throughout the brain via synaptically-connected pathways. Such propagation of pathology is thought to be mediated by tau species ("seeds") containing the microtubule binding region (MTBR) composed of either three repeat (3R) or four repeat (4R) isoforms. The tau MTBR also forms the core of the neuropathological filaments identified in AD brain and other tauopathies. Multiple approaches are being taken to limit tau pathology, including immunotherapy with anti-tau antibodies. Given its key structural role within fibrils, specifically targetting the MTBR with a therapeutic antibody to inhibit tau seeding and aggregation may be a promising strategy to provide disease-modifying treatment for AD and other tauopathies. Therefore, a monoclonal antibody generating campaign was initiated with focus on the MTBR. Herein we describe the pre-clinical generation and characterisation of E2814, a humanised, high affinity, IgG1 antibody recognising the tau MTBR. E2814 and its murine precursor, 7G6, as revealed by epitope mapping, are antibodies bi-epitopic for 4R and mono-epitopic for 3R tau isoforms because they bind to sequence motif HVPGG. Functionally, both antibodies inhibited tau aggregation in vitro. They also immunodepleted a variety of MTBR-containing tau protein species. In an in vivo model of tau seeding and transmission, attenuation of deposition of sarkosyl-insoluble tau in brain could also be observed in response to antibody treatment. In AD brain, E2814 bound different types of tau filaments as shown by immunogold labelling and recognised pathological tau structures by immunohistochemical staining. Tau fragments containing HVPGG epitopes were also found to be elevated in AD brain compared to PSP or control. Taken together, the data reported here have led to E2814 being proposed for clinical development.


Assuntos
Doença de Alzheimer/imunologia , Doença de Alzheimer/terapia , Anticorpos Monoclonais/imunologia , Imunização Passiva/métodos , Proteínas tau/genética , Proteínas tau/imunologia , Doença de Alzheimer/patologia , Animais , Anticorpos Monoclonais/farmacologia , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Masculino , Camundongos Transgênicos , Agregação Patológica de Proteínas/imunologia , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/farmacologia
2.
Stem Cell Reports ; 11(5): 1171-1184, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30344006

RESUMO

Parkinson disease (PD) is a progressive neurological disease caused by selective degeneration of dopaminergic (DA) neurons in the substantia nigra. Although most cases of PD are sporadic cases, familial PD provides a versatile research model for basic mechanistic insights into the pathogenesis of PD. In this study, we generated DA neurons from PARK2 patient-specific, isogenic PARK2 null and PARK6 patient-specific induced pluripotent stem cells and found that these neurons exhibited more apoptosis and greater susceptibility to rotenone-induced mitochondrial stress. From phenotypic screening with an FDA-approved drug library, one voltage-gated calcium channel antagonist, benidipine, was found to suppress rotenone-induced apoptosis. Furthermore, we demonstrated the dysregulation of calcium homeostasis and increased susceptibility to rotenone-induced stress in PD, which is prevented by T-type calcium channel knockdown or antagonists. These findings suggest that calcium homeostasis in DA neurons might be a useful target for developing new drugs for PD patients.


Assuntos
Canais de Cálcio Tipo T/metabolismo , Neurônios Dopaminérgicos/patologia , Mitocôndrias/metabolismo , Estresse Oxidativo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Neurônios Dopaminérgicos/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas , Mitocôndrias/efeitos dos fármacos , Modelos Biológicos , Crescimento Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Quinases/metabolismo , Rotenona/toxicidade , Ubiquitina-Proteína Ligases/metabolismo
3.
J Biomol Screen ; 21(10): 1054-1064, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28139961

RESUMO

Because neurons are difficult to obtain from humans, generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant screening systems for drug discovery. To examine the culture conditions leading to efficient differentiation of functional neural cells, we investigated the effects of oxygen stress (2% or 20% O2) and differentiation medium (DMEM/F12:Neurobasal-based [DN] or commercial [PhoenixSongs Biologicals; PS]) on the expression of genes related to neural differentiation, glutamate receptor function, and the formation of networks of neurons differentiated from hiPSCs (201B7) via long-term self-renewing neuroepithelial-like stem (lt-NES) cells. Expression of genes related to neural differentiation occurred more quickly in PS and/or 2% O2 than in DN and/or 20% O2, resulting in high responsiveness of neural cells to glutamate, N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and ( S)-3,5-dihydroxyphenylglycine (an agonist for mGluR1/5), as revealed by calcium imaging assays. NMDA receptors, AMPA receptors, mGluR1, and mGluR5 were functionally validated by using the specific antagonists MK-801, NBQX, JNJ16259685, and 2-methyl-6-(phenylethynyl)-pyridine, respectively. Multielectrode array analysis showed that spontaneous firing occurred earlier in cells cultured in 2% O2 than in 20% O2. Optimization of O2 tension and culture medium for neural differentiation of hiPSCs can efficiently generate physiologically relevant cells for screening systems.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Meios de Cultivo Condicionados/farmacologia , Maleato de Dizocilpina/química , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , N-Metilaspartato/química , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Oxigênio/metabolismo , Consumo de Oxigênio/genética
4.
Hum Mol Genet ; 24(17): 4879-900, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26056228

RESUMO

Leucine-rich repeat kinase 2 (LRRK2) is the causative molecule of the autosomal dominant hereditary form of Parkinson's disease (PD), PARK8, which was originally defined in a study of a Japanese family (the Sagamihara family) harboring the I2020T mutation in the kinase domain. Although a number of reported studies have focused on cell death mediated by mutant LRRK2, details of the pathogenetic effect of LRRK2 still remain to be elucidated. In the present study, to elucidate the mechanism of neurodegeneration in PD caused by LRRK2, we generated induced pluripotent stem cells (iPSC) derived from fibroblasts of PD patients with I2020T LRRK2 in the Sagamihara family. We found that I2020T mutant LRRK2 iPSC-derived neurons released less dopamine than control-iPSC-derived neurons. Furthermore, we demonstrated that patient iPSC-derived neurons had a lower phospho-AKT level than control-iPSC-derived neurons, and that the former showed an increased incidence of apoptosis relative to the controls. Interestingly, patient iPSC-derived neurons exhibited activation of glycogen synthase kinase-3ß (GSK-3ß) and high Tau phosphorylation. In addition, the postmortem brain of the patient from whom the iPSC had been established exhibited deposition of neurofibrillary tangles as well as increased Tau phosphorylation in neurons. These results suggest that I2020T LRRK2-iPSC could be a promising new tool for reproducing the pathology of PD in the brain caused by the I2020T mutation, and applicable as a model in studies of targeted therapeutics.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas tau/metabolismo , Animais , Apoptose/genética , Autofagia , Caspase 3/metabolismo , Linhagem Celular , Dopamina/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Neurônios/citologia , Estresse Oxidativo , Fosforilação
5.
J Biomol Screen ; 19(8): 1174-84, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24980597

RESUMO

The hippocampus is an important brain region that is involved in neurological disorders such as Alzheimer disease, schizophrenia, and epilepsy. Ionotropic glutamate receptors-namely,N-methyl-D-aspartate (NMDA) receptors (NMDARs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (AMPARs), and kainic acid (KA) receptors (KARs)-are well known to be involved in these diseases by mediating long-term potentiation, excitotoxicity, or both. To predict the therapeutic efficacy and neuronal toxicity of drug candidates acting on these receptors, physiologically relevant systems for assaying brain region-specific human neural cells are necessary. Here, we characterized the functional differentiation of human fetal hippocampus-derived neural stem/progenitor cells-namely, HIP-009 cells. Calcium rise assay demonstrated that, after a 4-week differentiation, the cells responded to NMDA (EC50= 7.5 ± 0.4 µM; n= 4), AMPA (EC50= 2.5 ± 0.1 µM; n= 3), or KA (EC50= 33.5 ± 1.1 µM; n= 3) in a concentration-dependent manner. An AMPA-evoked calcium rise was observed in the absence of the desensitization inhibitor cyclothiazide. In addition, the calcium rise induced by these agonists was inhibited by antagonists for each receptor-namely, MK-801 for NMDA stimulation (IC50= 0.6 ± 0.1 µM; n= 4) and NBQX for AMPA and KA stimulation (IC50= 0.7 ± 0.1 and 0.7 ± 0.03 µM, respectively; n= 3). The gene expression profile of differentiated HIP-009 cells was distinct from that of undifferentiated cells and closely resembled that of the human adult hippocampus. Our results show that HIP-009 cells are a unique tool for obtaining human hippocampal neural cells and are applicable to systems for assay of ionotropic glutamate receptors as a physiologically relevant in vitro model.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Hipocampo/citologia , Células-Tronco Neurais/efeitos dos fármacos , Receptores Ionotrópicos de Glutamato/metabolismo , Cálcio/metabolismo , Diferenciação Celular , Linhagem Celular , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Eletrofisiologia/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Ácido Caínico/farmacologia , N-Metilaspartato/farmacologia , Células-Tronco Neurais/metabolismo , Quinoxalinas/farmacologia , Receptores Ionotrópicos de Glutamato/análise , Receptores Ionotrópicos de Glutamato/antagonistas & inibidores , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia
6.
J Neurochem ; 129(3): 388-99, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24286589

RESUMO

Human induced pluripotent stem (iPS) cells obtained by reprogramming technology are a source of great hope, not only in terms of applications in regenerative medicine, such as cell transplantation therapy, but also for modeling human diseases and new drug development. In particular, the production of iPS cells from the somatic cells of patients with intractable diseases and their subsequent differentiation into cells at affected sites (e.g., neurons, cardiomyocytes, hepatocytes, and myocytes) has permitted the in vitro construction of disease models that contain patient-specific genetic information. For example, disease-specific iPS cells have been established from patients with neuropsychiatric disorders, including schizophrenia and autism, as well as from those with neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. A multi-omics analysis of neural cells originating from patient-derived iPS cells may thus enable investigators to elucidate the pathogenic mechanisms of neurological diseases that have heretofore been unknown. In addition, large-scale screening of chemical libraries with disease-specific iPS cells is currently underway and is expected to lead to new drug discovery. Accordingly, this review outlines the progress made via the use of patient-derived iPS cells toward the modeling of neurological disorders, the testing of existing drugs, and the discovery of new drugs. The production of human induced pluripotent stem (iPS) cells from the patients' somatic cells and their subsequent differentiation into specific cells have permitted the in vitro construction of disease models that contain patient-specific genetic information. Furthermore, innovations of gene-editing technologies on iPS cells are enabling new approaches for illuminating the pathogenic mechanisms of human diseases. In this review article, we outlined the current status of neurological diseases-specific iPS cell research and described recently obtained knowledge in the form of actual examples.


Assuntos
Células-Tronco Pluripotentes Induzidas , Modelos Neurológicos , Doenças do Sistema Nervoso , Humanos
7.
Mol Brain ; 6: 19, 2013 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-23639079

RESUMO

BACKGROUND: Dravet syndrome is a devastating infantile-onset epilepsy syndrome with cognitive deficits and autistic traits caused by genetic alterations in SCN1A gene encoding the α-subunit of the voltage-gated sodium channel Na(v)1.1. Disease modeling using patient-derived induced pluripotent stem cells (iPSCs) can be a powerful tool to reproduce this syndrome's human pathology. However, no such effort has been reported to date. We here report a cellular model for DS that utilizes patient-derived iPSCs. RESULTS: We generated iPSCs from a Dravet syndrome patient with a c.4933C>T substitution in SCN1A, which is predicted to result in truncation in the fourth homologous domain of the protein (p.R1645*). Neurons derived from these iPSCs were primarily GABAergic (>50%), although glutamatergic neurons were observed as a minor population (<1%). Current-clamp analyses revealed significant impairment in action potential generation when strong depolarizing currents were injected. CONCLUSIONS: Our results indicate a functional decline in Dravet neurons, especially in the GABAergic subtype, which supports previous findings in murine disease models, where loss-of-function in GABAergic inhibition appears to be a main driver in epileptogenesis. Our data indicate that patient-derived iPSCs may serve as a new and powerful research platform for genetic disorders, including the epilepsies.


Assuntos
Epilepsias Mioclônicas/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Potenciais de Ação , Adulto , Diferenciação Celular , Epilepsias Mioclônicas/fisiopatologia , Feminino , Genes Reporter/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lactente , Lentivirus/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Parvalbuminas/genética , Parvalbuminas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
8.
Methods Mol Biol ; 989: 193-215, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23483397

RESUMO

The discovery of human induced pluripotent stem cells (iPSCs) has provided a model system for studying early events during human development. Developmentally melanocytes originate from migratory neural crest cells that emerge from the neural plate during embryogenesis after a complex process of differentiation, proliferation, and migration out of the neural tube along defined pathways. In the adult, human melanocytes are located in the basal layer of the epidermis, hair follicles, uvea, inner ear, and meninges. In the epidermis, melanocytes produce melanin pigment that gives color to the skin as well as providing protection from ultraviolet light damage. In addition, melanocytes transfer melanin pigment to hair matrix keratinocytes during each hair cycle to maintain hair pigmentation. Characterization of mouse melanocyte stem cells (MELSCs) is more complete than for humans. MELSCs are located in the bulge region of hair follicles, where hair follicle stem cells (HFSCs) also reside. Recently, it has been demonstrated that HFSCs provide a functional nice for MELSCs. According to current cancer stem cell theory, melanomas are considered to evolve from MELSCs, although the exact mechanism remains to be elucidated fully. In humans, importantly, the lack of more specific markers of MELSCs, current understanding of the molecular regulations of melanocyte development remains incomplete. Recently, the generation of melanocytes from iPSCs has lead to some clarification of human melanocyte development in vitro. Utilization of iPSC-derived melanocytes may prove invaluable in further study of human melanocytic development and novel therapies for patients suffering with pigmentation disorders and melanoma.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Melanócitos/citologia , Animais , Células Cultivadas , Humanos , Imuno-Histoquímica , Melanoma/patologia , Camundongos , Crista Neural/citologia
9.
J Invest Dermatol ; 133(6): 1479-88, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23321923

RESUMO

Well-orchestrated epithelial-mesenchymal interactions are crucial for hair follicle (HF) morphogenesis. In this study, ectodermal precursor cells (EPCs) with the capacity to cross talk with hair-inductive dermal cells were generated from human induced pluripotent stem cells (hiPSCs) and assessed for HF-forming ability in vivo. EPCs derived from three hiPSC lines generated with 4 or 3 factors (POU5F1, SOX2, KLF4 +/- MYC) mostly expressed keratin 18, a marker of epithelial progenitors. When cocultured with human dermal papilla (DP) cells, a 4 factor 201B7 hiPSC-EPC line upregulated follicular keratinocyte (KC) markers more significantly than normal human adult KCs (NHKCs) and other hiPSC-EPC lines. DP cells preferentially increased DP biomarker expression in response to this line. Interestingly, 201B7 hiPSCs were shown to be ectodermal/epithelial prone, and the derived EPCs were putatively in a wingless-type MMTV integration site family (WNT)-activated state. Importantly, co-transplantation of 201B7 hiPSC-EPCs, but not NHKCs, with trichogenic mice dermal cells into immunodeficient mice resulted in HF formation. Human HF stem cell markers were detected in reconstituted HFs; however, a low frequency of human-derived cells implied that hiPSC-EPCs contributed to HF morphogenesis via direct repopulation and non-cell autonomous activities. The current study suggests a, to our knowledge, previously unrecognized advantage of using hiPSCs to enhance epithelial-mesenchymal interactions in HF bioengineering.


Assuntos
Derme/citologia , Ectoderma/citologia , Folículo Piloso/citologia , Queratinócitos/citologia , Células-Tronco Pluripotentes/citologia , Adulto , Animais , Animais Recém-Nascidos , Bioengenharia/métodos , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular , Técnicas de Cocultura , Derme/crescimento & desenvolvimento , Ectoderma/crescimento & desenvolvimento , Transição Epitelial-Mesenquimal/fisiologia , Folículo Piloso/crescimento & desenvolvimento , Humanos , Queratinócitos/metabolismo , Fator 4 Semelhante a Kruppel , Camundongos , Morfogênese/fisiologia , Células-Tronco Pluripotentes/metabolismo , Regeneração/fisiologia , Transplante de Pele/métodos , Via de Sinalização Wnt/fisiologia
10.
Mol Brain ; 5: 35, 2012 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-23039195

RESUMO

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease characterized by selective degeneration of dopaminergic neurons in the substantia nigra (SN). The familial form of PD, PARK2, is caused by mutations in the parkin gene. parkin-knockout mouse models show some abnormalities, but they do not fully recapitulate the pathophysiology of human PARK2. RESULTS: Here, we generated induced pluripotent stem cells (iPSCs) from two PARK2 patients. PARK2 iPSC-derived neurons showed increased oxidative stress and enhanced activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. iPSC-derived neurons, but not fibroblasts or iPSCs, exhibited abnormal mitochondrial morphology and impaired mitochondrial homeostasis. Although PARK2 patients rarely exhibit Lewy body (LB) formation with an accumulation of α-synuclein, α-synuclein accumulation was observed in the postmortem brain of one of the donor patients. This accumulation was also seen in the iPSC-derived neurons in the same patient. CONCLUSIONS: Thus, pathogenic changes in the brain of a PARK2 patient were recapitulated using iPSC technology. These novel findings reveal mechanistic insights into the onset of PARK2 and identify novel targets for drug screening and potential modified therapies for PD.


Assuntos
Encéfalo/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/patologia , Neurônios/metabolismo , Estresse Oxidativo , Doença de Parkinson/patologia , alfa-Sinucleína/metabolismo , Adulto , Idoso , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Corpos de Lewy/metabolismo , Corpos de Lewy/patologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Renovação Mitocondrial/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/ultraestrutura , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson/metabolismo , Mudanças Depois da Morte , Transdução de Sinais/efeitos dos fármacos
11.
Mol Brain ; 4: 33, 2011 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-21884595

RESUMO

Galectins are a 15 member family of carbohydrate-binding proteins that have been implicated in cancer, immunity, inflammation and development. While galectins are expressed in the central nervous system, little is known about their function in the adult brain. Previously we have shown that galectin-1 (gal-1) is expressed in the adult hippocampus, and, in particular, in putative neural stem cells in the subgranular zone. To evaluate how gal-1 might contribute to hippocampal memory function here we studied galectin-1 null mutant (gal-1-/-) mice. Compared to their wildtype littermate controls, gal-1-/- mice exhibited impaired spatial learning in the water maze and contextual fear learning. Interestingly, tone fear conditioning was normal in gal-1-/- mice suggesting that loss of gal-1 might especially impact hippocampal learning and memory. Furthermore, gal-1-/- mice exhibited normal motor function, emotion and sensory processing in a battery of other behavioral tests, suggesting that non-mnemonic performance deficits are unlikely to account for the spatial and contextual learning deficits. Together, these data reveal a role for galectin-carbohydrate signalling in hippocampal memory function.


Assuntos
Galectina 1/deficiência , Memória/fisiologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Comportamento Animal , Medo , Galectina 1/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neurônios/metabolismo , Neurônios/patologia
12.
PLoS One ; 6(1): e16182, 2011 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-21249204

RESUMO

Epidermal melanocytes play an important role in protecting the skin from UV rays, and their functional impairment results in pigment disorders. Additionally, melanomas are considered to arise from mutations that accumulate in melanocyte stem cells. The mechanisms underlying melanocyte differentiation and the defining characteristics of melanocyte stem cells in humans are, however, largely unknown. In the present study, we set out to generate melanocytes from human iPS cells in vitro, leading to a preliminary investigation of the mechanisms of human melanocyte differentiation. We generated iPS cell lines from human dermal fibroblasts using the Yamanaka factors (SOX2, OCT3/4, and KLF4, with or without c-MYC). These iPS cell lines were subsequently used to form embryoid bodies (EBs) and then differentiated into melanocytes via culture supplementation with Wnt3a, SCF, and ET-3. Seven weeks after inducing differentiation, pigmented cells expressing melanocyte markers such as MITF, tyrosinase, SILV, and TYRP1, were detected. Melanosomes were identified in these pigmented cells by electron microscopy, and global gene expression profiling of the pigmented cells showed a high similarity to that of human primary foreskin-derived melanocytes, suggesting the successful generation of melanocytes from iPS cells. This in vitro differentiation system should prove useful for understanding human melanocyte biology and revealing the mechanism of various pigment cell disorders, including melanoma.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/farmacologia , Células-Tronco Pluripotentes Induzidas/citologia , Melanócitos/citologia , Técnicas de Cultura de Células/métodos , Corpos Embrioides , Fibroblastos/citologia , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/farmacologia , Fator 3 de Transcrição de Octâmero/farmacologia , Fatores de Transcrição SOXB1/farmacologia , Pele/citologia
13.
Mol Brain ; 4: 7, 2011 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-21269521

RESUMO

BACKGROUND: In the adult mammalian brain, neural stem cells (NSCs) proliferate in the dentate gyrus (DG) of the hippocampus and generate new neurons throughout life. A multimodal protein, Galectin-1, is expressed in neural progenitor cells (NPCs) and implicated in the proliferation of the NPCs in the DG. However, little is known about its detailed expression profile in the NPCs and functions in adult neurogenesis in the DG. RESULTS: Our immunohistochemical and morphological analysis showed that Galectin-1 was expressed in the type 1 and 2a cells, which are putative NSCs, in the subgranular zone (SGZ) of the adult mouse DG. To study Galectin-1's function in adult hippocampal neurogenesis, we made galectin-1 knock-out mice on the C57BL6 background and characterized the effects on neurogenesis. In the SGZ of the galectin-1 knock-out mice, increased numbers of type 1 cells, DCX-positive immature progenitors, and NeuN-positive newborn neurons were observed. Using triple-labeling immunohistochemistry and morphological analyses, we found that the proliferation of the type-1 cells was increased in the SGZ of the galectin-1 knock-out mice, and we propose that this proliferation is the mechanism for the net increase in the adult neurogenesis in these knock-out mice DG. CONCLUSIONS: Galectin-1 is expressed in the neural stem cells and down-regulates neurogenesis in the adult hippocampus.


Assuntos
Galectina 1/metabolismo , Hipocampo/citologia , Hipocampo/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Animais , Proliferação de Células , Proteína Duplacortina , Regulação para Baixo , Galectina 1/genética , Imuno-Histoquímica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/classificação , Células-Tronco Neurais/citologia
14.
J Neurochem ; 113(6): 1516-24, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20367753

RESUMO

Neural stem cells (NSCs) proliferate and generate new neurons in the adult brain. A carbohydrate-binding protein (lectin), Galectin-1, is expressed in the NSCs in the subependymal zone (SEZ) of the adult mouse brain. The infusion and knockout of Galectin-1 in the SEZ results in an increase and decrease, respectively, of NSCs and subsequently born progenitor cells. The molecular mechanism of this effect, however, has been unknown. Previous studies outside the brain suggest that Galectin-1 binds to a carbohydrate structure of beta1 Integrin and modulates cell adhesion. Here, we studied the functional interaction between Galectin-1 and beta1 Integrin in the adult mouse SEZ. Beta1 Integrin was purified from adult SEZ tissue by binding to a Galectin-1 affinity column, and this binding depended on Galectin-1's carbohydrate-binding activity. In adult brain sections, Galectin-1-binding activity was detected on beta1 Integrin-expressing cells in the SEZ. Furthermore, in the adult SEZ, the simultaneous infusion of a beta1 Integrin-neutralizing antibody with Galectin-1 protein reversed the increasing effect of Galectin-1 on the number of adult neural progenitor cells (NPCs). Finally, intact beta1 Integrin was required for Galectin-1's function in NPC adhesion in vitro. These results suggest that the interaction between beta1 Integrin and Galectin-1 plays an important role in regulating the number of adult NPCs through mechanisms including cell adhesion.


Assuntos
Células-Tronco Adultas/fisiologia , Galectina 1/metabolismo , Integrina beta1/metabolismo , Neurônios/fisiologia , Animais , Anticorpos/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/citologia , Bromodesoxiuridina/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Cromatografia de Afinidade/métodos , Galectina 1/genética , Galectina 1/imunologia , Proteínas de Fluorescência Verde/genética , Integrina beta1/genética , Lactose/farmacologia , Camundongos , Neurônios/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Edulcorantes/farmacologia , Fatores de Tempo
15.
Brain Res ; 1244: 82-8, 2008 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-18848927

RESUMO

The spatiotemporal dynamics of the neurovascular response to brief acoustic stimuli were investigated in guinea pig primary auditory cortex. Neural activity and cortical tonotopic organization were measured with a voltage-sensitive dye (VSD) technique, whereas cerebral blood flow (CBF) response to neural stimulation was measured with laser-Doppler flowmetry (LDF). The acoustic stimulus was given as a wide band sound (click), which induced global activation or as one of two pure tones (1 kHz and 12 kHz), which induced distinct localizations in the auditory cortex. The VSD imaging showed that the sound-induced activation area varied dynamically, and that the spatial extent had peaks at 37+/-3 ms and 38+/-8 ms after the onset of stimulation during 1-kHz and 12-kHz tones, respectively. We observed that the average CBF response had a similar peak intensity irrespective of the type of stimuli: 16+/-9%, 18+/-11%, and 16+/-8% for click, 1-kHz, and 12-kHz tones, respectively. No significant differences in the CBF time course, time-to-onset (approximately 0.6 s), or time-to-peak (approximately 3.3 s) were found across the recording sites and stimulus types. These results showed that the CBF response measured with LDF produced a less specific spatial pattern relative to the neural map determined with VSD. The findings can be explained by the methodological limitations of LDF and/or neurovascular regulatory systems in the auditory cortex.


Assuntos
Estimulação Acústica/métodos , Córtex Auditivo/irrigação sanguínea , Circulação Cerebrovascular/fisiologia , Neurônios/fisiologia , Animais , Córtex Auditivo/citologia , Cobaias , Fluxometria por Laser-Doppler/métodos , Neurônios/citologia , Fluxo Sanguíneo Regional/fisiologia , Fatores de Tempo
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