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1.
Artigo em Inglês | MEDLINE | ID: mdl-34949141

RESUMO

Objective: Uniform data collection is fundamental for multicentre clinical trials. We aim to determine the variability, between ALS trial centers, in the prevalence of unexpected or implausible improvements in the revised ALS functional rating scale (ALSFRS-R) score, and its associations with individual patient and item characteristics.Methods: We used data from two multicentre studies to estimate the prevalence of an unexpected increase or implausible improvement in the ALSFRS-R score, defined as an increase of 5 points or more between two consecutive, monthly visits. For each patient with a 5-point or more increase, we evaluated the individual contribution of each ALSFRS-R item.Results: Longitudinal ALSFRS-R scores, originating from 114 trial centers enrolling a total of 1,240 patients, were analyzed. A 5-point or more increase in ALSFRS-R total score was found in 151 (12.2%) patients, with prevalence per study center ranging from 0% to 83%. Bulbar onset, faster disease progression at enrollment, and a lower ALSFRS-R score at baseline were associated with a sudden 5-point or more increase in the ALSFRS-R total score. ALSFRS-R items 2 (saliva), 9 (stairs), 10 (dyspnea), and 11 (orthopnea) were the primary drivers when a 5-point or more increase occurred.Conclusions: Sudden 5-point or more increases in ALSFRS-R total scores between two consecutive visits are relatively common. These sudden increases were not found to occur with equal frequency in trial centers; which underscores the need for amending existing standard operating procedures toward a universal version and monitoring of data quality during the study, in multicentre research.

2.
Neurology ; 97(11): 528-536, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34315786

RESUMO

Development of effective treatments for amyotrophic lateral sclerosis (ALS) has been hampered by disease heterogeneity, a limited understanding of underlying pathophysiology, and methodologic design challenges. We have evaluated 2 major themes in the design of pivotal, phase 3 clinical trials for ALS-(1) patient selection and (2) analytical strategy-and discussed potential solutions with the European Medicines Agency. Several design considerations were assessed using data from 5 placebo-controlled clinical trials (n = 988), 4 population-based cohorts (n = 5,100), and 2,436 placebo-allocated patients from the Pooled Resource Open-Access ALS Clinical Trials (PRO-ACT) database. The validity of each proposed design modification was confirmed by means of simulation and illustrated for a hypothetical setting. Compared to classical trial design, the proposed design modifications reduce the sample size by 30.5% and placebo exposure time by 35.4%. By making use of prognostic survival models, one creates a potential to include a larger proportion of the population and maximize generalizability. We propose a flexible design framework that naturally adapts the trial duration when inaccurate assumptions are made at the design stage, such as enrollment or survival rate. In case of futility, the follow-up time is shortened and patient exposure to ineffective treatments or placebo is minimized. For diseases such as ALS, optimizing the use of resources, widening eligibility criteria, and minimizing exposure to futile treatments and placebo is critical to the development of effective treatments. Our proposed design modifications could circumvent important pitfalls and may serve as a blueprint for future clinical trials in this population.


Assuntos
Esclerose Amiotrófica Lateral/terapia , Projetos de Pesquisa , Ensaios Clínicos como Assunto , Progressão da Doença , Humanos , Seleção de Pacientes , Fatores de Risco
3.
Nat Commun ; 7: 13283, 2016 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-27796305

RESUMO

The study of amyotrophic lateral sclerosis (ALS) and potential interventions would be facilitated if motor axon degeneration could be more readily visualized. Here we demonstrate that stimulated Raman scattering (SRS) microscopy could be used to sensitively monitor peripheral nerve degeneration in ALS mouse models and ALS autopsy materials. Three-dimensional imaging of pre-symptomatic SOD1 mouse models and data processing by a correlation-based algorithm revealed that significant degeneration of peripheral nerves could be detected coincidentally with the earliest detectable signs of muscle denervation and preceded physiologically measurable motor function decline. We also found that peripheral degeneration was an early event in FUS as well as C9ORF72 repeat expansion models of ALS, and that serial imaging allowed long-term observation of disease progression and drug effects in living animals. Our study demonstrates that SRS imaging is a sensitive and quantitative means of measuring disease progression, greatly facilitating future studies of disease mechanisms and candidate therapeutics.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Degeneração Neural/patologia , Nervos Periféricos/patologia , Análise Espectral Raman , Algoritmos , Animais , Antibacterianos , Artefatos , Simulação por Computador , Progressão da Doença , Eletromiografia , Feminino , Humanos , Imageamento Tridimensional , Lipídeos/química , Masculino , Camundongos , Camundongos Transgênicos , Minociclina/química , Neurônios Motores/patologia , Bainha de Mielina/química , Nervo Isquiático/patologia , Superóxido Dismutase-1/genética , Transgenes
4.
Mol Biol Cell ; 25(17): 2571-8, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25009283

RESUMO

Mutations in the RNA-binding protein FUS have been shown to cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We investigate whether mutant FUS protein in ALS patient-derived fibroblasts affects normal FUS functions in the nucleus. We investigated fibroblasts from two ALS patients possessing different FUS mutations and a normal control. Fibroblasts from these patients have their nuclear FUS protein trapped in SDS-resistant aggregates. Genome-wide analysis reveals an inappropriate accumulation of Ser-2 phosphorylation on RNA polymerase II (RNA Pol II) near the transcription start sites of 625 genes for ALS patient cells and after small interfering RNA (siRNA) knockdown of FUS in normal fibroblasts. Furthermore, both the presence of mutant FUS protein and siRNA knockdown of wild-type FUS correlate with altered distribution of RNA Pol II within fibroblast nuclei. A loss of FUS function in orchestrating Ser-2 phosphorylation of the CTD of RNA Pol II is detectable in ALS patient-derived fibroblasts expressing mutant FUS protein, even when the FUS protein remains largely nuclear. A likely explanation for this loss of function is the aggregation of FUS protein in nuclei. Thus our results suggest a specific mechanism by which mutant FUS can have biological consequences other than by the formation of cytoplasmic aggregates.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Núcleo Celular/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Esclerose Amiotrófica Lateral/genética , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Fosforilação , Agregados Proteicos , Interferência de RNA , RNA Polimerase II/metabolismo , Proteína FUS de Ligação a RNA/antagonistas & inibidores , Proteína FUS de Ligação a RNA/genética , Sítio de Iniciação de Transcrição
5.
Cell Rep ; 7(1): 1-11, 2014 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-24703839

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor nervous system. We show using multielectrode array and patch-clamp recordings that hyperexcitability detected by clinical neurophysiological studies of ALS patients is recapitulated in induced pluripotent stem cell-derived motor neurons from ALS patients harboring superoxide dismutase 1 (SOD1), C9orf72, and fused-in-sarcoma mutations. Motor neurons produced from a genetically corrected but otherwise isogenic SOD1(+/+) stem cell line do not display the hyperexcitability phenotype. SOD1(A4V/+) ALS patient-derived motor neurons have reduced delayed-rectifier potassium current amplitudes relative to control-derived motor neurons, a deficit that may underlie their hyperexcitability. The Kv7 channel activator retigabine both blocks the hyperexcitability and improves motor neuron survival in vitro when tested in SOD1 mutant ALS cases. Therefore, electrophysiological characterization of human stem cell-derived neurons can reveal disease-related mechanisms and identify therapeutic candidates.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Neurônios Motores/patologia , Potenciais de Ação/fisiologia , Esclerose Amiotrófica Lateral/enzimologia , Esclerose Amiotrófica Lateral/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Neurônios Motores/enzimologia , Neurônios Motores/metabolismo , Mutação , Técnicas de Patch-Clamp , Fenótipo , Superóxido Dismutase/genética , Superóxido Dismutase-1
6.
Neuron ; 81(3): 536-543, 2014 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-24507191

RESUMO

The RNA-binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP-43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Transporte Axonal/genética , Proteínas de Ligação a DNA/genética , Neurônios Motores/metabolismo , Mutação/genética , RNA Mensageiro/metabolismo , Esclerose Amiotrófica Lateral/genética , Animais , Animais Geneticamente Modificados , Células Cultivadas , Córtex Cerebral/citologia , Drosophila , Proteínas de Drosophila/genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Luminescentes/genética , Camundongos , Mitocôndrias/metabolismo , Neurônios Motores/ultraestrutura , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
7.
Neuron ; 70(4): 626-44, 2011 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-21609821

RESUMO

Among the disciplines of medicine, the study of neurological disorders is particularly challenging. The fundamental inaccessibility of the human neural types affected by disease prevents their isolation for in vitro studies of degenerative mechanisms or for drug screening efforts. However, the ability to reprogram readily accessible tissue from patients into pluripotent stem (iPS) cells may now provide a general solution to this shortage of human neurons. Gradually improving methods for directing the differentiation of patient-specific stem cells has enabled the production of several neural cell types affected by disease. Furthermore, initial studies with stem cell lines derived from individuals with pediatric, monogenic disorders have validated the stem cell approach to disease modeling, allowing relevant neural phenotypes to be observed and studied. Whether iPS cell-derived neurons will always faithfully recapitulate the same degenerative processes observed in patients and serve as platforms for drug discovery relevant to common late-onset diseases remains to be determined.


Assuntos
Modelos Teóricos , Doenças do Sistema Nervoso/cirurgia , Transplante de Células-Tronco/métodos , Células-Tronco , Animais , Diferenciação Celular/fisiologia , Humanos , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/patologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/transplante , Células-Tronco/citologia , Células-Tronco/fisiologia
8.
Arch Neurol ; 62(2): 223-9, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15710850

RESUMO

BACKGROUND: Replacement of neurons and glia by transplantation has been proposed as a therapy for neurodegenerative diseases, including amyotrophic lateral sclerosis. This strategy requires using human motor neuronal progenitor cells or xenografts of animal cells, but there is little evidence that xenografted neuronal cells can survive in spinal cord despite immunosuppression. OBJECTIVE: To clarify the mechanisms responsible for the death of xenografted neurons in spinal cord. METHODS: Cells from an immortalized, neuronally committed, human embryonic spinal cord-derived cell line (HSP1) that expresses motor neuronal properties in vitro were transplanted into adult rat spinal cord. The rats were killed at intervals up to 8 weeks and serial sections through the graft sites were processed for immunofluorescence using primary antibodies against human nuclear and mitochondrial antigens, microtubule-associated protein 2, TUJ1, CD5, natural killer cells, and activated microglia-macrophages, caspase-3 and caspase-9. RESULTS: Grafted cells did not migrate and underwent partial differentiation along a neuronal pathway. They were rejected after 4 weeks despite cyclosporine immunosuppression. Cells died by apoptosis via the cytochrome c/caspase-9/caspase-3 pathway. The host response included natural killer cells and activated microglia-macrophages but few T cells. CONCLUSIONS: Intraspinal neuronal xenotransplantation failed because of apoptotic cell death. Neither T cells nor the spinal cord environment, which favors gliogenesis, are likely to have been responsible, but natural killer cells may have been involved.


Assuntos
Neurônios/fisiologia , Neurônios/transplante , Medula Espinal/citologia , Transplante de Células-Tronco/métodos , Animais , Autoantígenos/metabolismo , Antígenos CD5/metabolismo , Caspase 3 , Caspase 9 , Caspases/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase , Doxiciclina/farmacologia , Humanos , Imuno-Histoquímica/métodos , Indóis , Antígeno Ki-67/metabolismo , Células Matadoras Naturais/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/metabolismo , Neurônios/efeitos dos fármacos , Ratos , Transplante Heterólogo/métodos , Tubulina (Proteína)/metabolismo
9.
J Neurotrauma ; 22(1): 138-56, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15665609

RESUMO

Encapsulation of cells has the potential to provide a protective barrier against host immune cell interactions after grafting. Previously we have shown that alginate encapsulated BDNF-producing fibroblasts (Fb/BDNF) survived for one month in culture, made bioactive neurotrophins, survived transplantation into the injured spinal cord in the absence of immune suppression, and provided a permissive environment for host axon growth. We extend these studies by examining the effects of grafting encapsulated Fb/BDNF into a subtotal cervical hemisection on recovery of forelimb and hindlimb function and axonal growth in the absence of immune suppression. Grafting of encapsulated Fb/BDNF resulted in partial recovery of forelimb usage in a test of vertical exploration and of hindlimb function while crossing a horizontal rope. Recovery was significantly greater compared to animals that received unencapsulated Fb/BDNF without immune suppression, but similar to that of immune suppressed animals receiving unencapsulated Fb/BDNF. Immunocytochemical examination revealed neurofilament (RT-97), 5-HT, CGRP and GAP-43 containing axons surrounding encapsulated Fb/BDNF within the injury site, indicating axonal growth. BDA labeling however showed no evidence of regeneration of rubrospinal axons in recipients of encapsulated Fb/BDNF, presumably because the amounts of BDNF available from the encapsulated grafts are substantially less than those provided by the much larger numbers of Fb/BDNF grafted in a gelfoam matrix in the presence of immune suppression. These results suggest that plasticity elicited by the BDNF released from the encapsulated cells contributed to reorganization that led to behavioral recovery in these animals and that the behavioral recovery could proceed in the absence of rubrospinal tract regeneration. Alginate encapsulation is therefore a feasible strategy for delivery of therapeutic products produced by non-autologous engineered fibroblasts and provides an environment suitable for recovery of lost function in the injured spinal cord.


Assuntos
Alginatos , Materiais Biocompatíveis , Transplante de Células/métodos , Fibroblastos/transplante , Ácido Glucurônico , Rejeição de Enxerto/prevenção & controle , Ácidos Hexurônicos , Traumatismos da Medula Espinal/cirurgia , Animais , Axônios/fisiologia , Cápsulas , Técnicas de Cultura de Células , Vértebras Cervicais , Feminino , Membro Anterior/fisiologia , Membro Posterior/fisiologia , Regeneração Nervosa/fisiologia , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Fatores de Tempo
10.
Dev Biol ; 276(1): 31-46, 2004 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-15531362

RESUMO

The precise lineage between neural stem cells and mature astrocytes remains poorly defined. To examine astrocyte development, we have characterized glial precursors from neural tissue derived from early embryonic ages. We show that CD44 identifies an astrocyte-restricted precursor cell (ARP) that is committed to generating astrocytes in vitro and in vivo in both rodent and human tissue. CD44+ cells arise later in development than neuronal-restricted precursors (NRPs) or tripotential glial-restricted precursors (GRPs). ARPs are distinguished from GRP and NRP cells by their antigenic profile and differentiation ability. ARPs can be generated from GRP cells in mass or clonal cultures and in vivo after transplantation, suggesting a sequential differentiation of neuroepithelial stem cells (NEPs) to GRPs to ARPs and then to astrocytes. The properties of ARPs are different from other astrocyte precursors described previously in their expression of CD44 and S-100beta and absence of other lineage markers. Using a CD44 misexpression transgenic mouse model (CNP-CD44 mouse), we show that CD44 overexpression in vivo and in vitro decreases the number of mature glia and increases the number of O4+/GFAP+ cells tenfold. Misexpression of CD44 in culture inhibits oligodendrocytes and arrests cells at the precursor state. In summary, our data provide strong evidence for the existence of a CD44+ ARP in the developing nervous system.


Assuntos
Astrócitos/metabolismo , Linhagem da Célula , Receptores de Hialuronatos/metabolismo , Neuroglia/citologia , Células-Tronco/citologia , Animais , Animais Geneticamente Modificados , Astrócitos/citologia , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Células Cultivadas , Células Clonais , Feminino , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Receptores de Hialuronatos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Células-Tronco/metabolismo
11.
Glia ; 45(1): 1-16, 2004 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-14648541

RESUMO

Glial-restricted precursor (GRP) cells are among a number of candidate cells for transplantation repair of CNS injury. The isolation and characterization of these cells in vitro have been described previously, but their in vivo properties are not well understood. We examined the fate and migration of grafted fetal GRP cells harvested from alkaline phosphatase-expressing transgenic rats into intact and injured spinal cord. Transplanted GRP cells survived for at least 6 weeks and differentiated along astrocytic and oligodendrocytic but not neuronal lineages. Cells grafted into the intact spinal cord exhibited robust migration along longitudinal white matter tracts and by 6 weeks migrated more than 15 mm. In contrast, migration of GRP cells in the gray matter was very limited. We then examined the phenotypic properties of proliferating endogenous precursors in response to injury by BrdU labeling. The predominant proliferating population seen after injury consisted of GRP-like cells with Nkx2.2/olig2 phenotype. Incorporation of BrdU by endogenous cells suggests that the environment provides proliferation signals and is permissive to glial precursor survival. To test if exogenous GRP cells would respond similarly, we transplanted GRP cells into a lateral funiculus injury. GRP cells survived and differentiated along glial lineages and migrated along white matter tracts in the injured spinal cord. Directed homing toward the lesion was not seen and there was no significant bias in differentiation between cells transplanted into injured and uninjured spinal cord. GRP cell transplants may therefore provide a cellular transplant that can respond to appropriate endogenous cues to produce therapeutic molecules and new glial cells after injury.


Assuntos
Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Neuroglia/transplante , Medula Espinal/transplante , Animais , Animais Geneticamente Modificados , Sobrevivência Celular/fisiologia , Feminino , Fibras Nervosas Mielinizadas/fisiologia , Neuroglia/citologia , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Medula Espinal/citologia , Traumatismos da Medula Espinal/cirurgia , Transplantes
12.
Exp Neurol ; 177(2): 360-75, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12429183

RESUMO

Multipotent neural stem cells (NSCs) have the potential to differentiate into neuronal and glial cells and are therefore candidates for cell replacement after CNS injury. Their phenotypic fate in vivo is dependent on the engraftment site, suggesting that the environment exerts differential effects on neuronal and glial lineages. In particular, when grafted into the adult spinal cord, NSCs are restricted to the glial lineage, indicating that the host spinal cord environment is not permissive for neuronal differentiation. To identify the stage at which neuronal differentiation is inhibited we examined the survival, differentiation, and integration of neuronal restricted precursor (NRP) cells, derived from the embryonic spinal cord of transgenic alkaline phosphatase rats, after transplantation into the adult spinal cord. We found that grafted NRP cells differentiate into mature neurons, survive for at least 1 month, appear to integrate within the host spinal cord, and extend processes in both the gray and white matter. Conversely, grafted glial restricted precursor cells did not differentiate into neurons. We did not observe glial differentiation from the grafted NRP cells, indicating that they retained their neuronal restricted properties in vivo. We conclude that the adult nonneurogenic CNS environment does not support the transition of multipotential NSCs to the neuronal commitment stage, but does allow the survival, maturation, and integration of NRP cells.


Assuntos
Diferenciação Celular/fisiologia , Neurônios/citologia , Medula Espinal/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Fosfatase Alcalina/genética , Animais , Animais Geneticamente Modificados , Antígenos de Diferenciação/biossíntese , Linhagem da Célula/fisiologia , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Sobrevivência de Enxerto/fisiologia , Imuno-Histoquímica , Neuritos/ultraestrutura , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/metabolismo , Fenótipo , Ratos , Ratos Endogâmicos F344 , Medula Espinal/embriologia , Células-Tronco/metabolismo
13.
Glia ; 38(1): 65-79, 2002 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-11921204

RESUMO

Neuroepithelial stem cells (NEPs), glial-restricted precursors (GRPs), and neuron-restricted precursors (NRPs) are present during early differentiation of the spinal cord and can be identified by cell surface markers. In this article, we describe the properties of GRP cells that have been immortalized using a regulatable v-myc retrovirus construct. Immortalized GRP cells can be maintained in an undifferentiated dividing state for long periods and can be induced to differentiate into two types of astrocytes and into oligodendrocytes in culture. A clonal cell line prepared from immortalized GRP cells, termed GRIP-1, was also shown to retain the properties of a glial-restricted tripotential precursor. Transplantation of green fluorescent protein (GFP)-labeled subclones of the immortalized cells into the adult CNS demonstrates that this cell line can also participate in the in vivo development of astrocytes and oligodendrocytes. Late passages of the immortalized cells undergo limited transdifferentiation into neurons as assessed by expression of multiple neuronal markers. The availability of a conditionally immortalized cell line obviates the difficulties of obtaining a large and homogeneous population of GRPs that can be used for studying the mechanism and signals for glial cell differentiation as well as their application in transplantation protocols.


Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Neuroglia/citologia , Medula Espinal/citologia , Medula Espinal/embriologia , Células-Tronco/citologia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Transplante de Tecido Encefálico/métodos , Técnicas de Cultura de Células/métodos , Linhagem Celular Transformada , Feminino , Feto , Genes myc/genética , Vetores Genéticos/genética , Sobrevivência de Enxerto/genética , Substâncias de Crescimento/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Medula Espinal/cirurgia , Células-Tronco/metabolismo
14.
Exp Neurol ; 174(1): 48-57, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11869033

RESUMO

Multipotent stem cells and more developmentally restricted precursors have previously been isolated from the developing nervous system and their properties analyzed by culture assays in vitro and by transplantation in vivo. However, the variety of labeling techniques that have been used to identify grafted cells in vivo have been unsatisfactory. In this article we describe the characteristics of cells isolated from a transgenic rat in which the marker gene human placental alkaline phosphatase (hPAP) is linked to the ubiquitously active R26 gene promoter. We show that hPAP is readily detected in embryonic neuroepithelial stem cells, neuronal-restricted precursor cells, and glial-restricted precursor cells. Transgene expression is robust and can be detected by both immunocytochemistry and histochemistry. Furthermore, the levels of hPAP on the cell surface are sufficient for live cell labeling and fluorescence-activated cell sorting. Expression of hPAP is stable in isolated cells in culture and in cells transplanted into the spinal cord for at least 1 month. We submit that cells isolated from this transgenic rat will be valuable for studies of neural development and regeneration.


Assuntos
Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Neurônios/metabolismo , Neurônios/transplante , Medula Espinal/citologia , Animais , Animais Geneticamente Modificados , Sobrevivência Celular/fisiologia , Células Cultivadas , Transplante de Tecido Fetal , Citometria de Fluxo , Expressão Gênica/fisiologia , Genes Reporter/genética , Sobrevivência de Enxerto/fisiologia , Humanos , Imuno-Histoquímica , Neurônios/citologia , Ratos , Medula Espinal/fisiologia , Transplante de Células-Tronco , Células-Tronco/citologia , Transgenes/fisiologia
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