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1.
Sci Rep ; 5: 9205, 2015 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-25777362

RESUMO

Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript, we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics. We extend this concept to making lineage reporters using a C-terminal targeting strategy to endogenous genes that express in a lineage-specific fashion. Furthermore, we demonstrate that we can develop a master cell line strategy and then use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. Equally important we show that this recombination strategy allows targeting at progenitor cell stages, further increasing the utility of the platform system. The results in concert provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays.


Assuntos
Marcação de Genes , Genes Reporter , Células-Tronco Pluripotentes Induzidas/metabolismo , Linhagem Celular , Linhagem da Célula/genética , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 19 , Expressão Gênica , Técnicas de Introdução de Genes/métodos , Ordem dos Genes , Marcação de Genes/métodos , Loci Gênicos , Vetores Genéticos/genética , Humanos , Reprodutibilidade dos Testes
2.
Methods Mol Biol ; 1210: 79-90, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25173162

RESUMO

The neural crest cells give rise to neurons and glia in the peripheral nervous system, which is an important component of the nervous system. Here we developed a scalable process of inducing neural crest stem cells (NCSCs) from hESCs/iPSCs by a combination of growth factors in medium conditioned on stromal cells, and showed that NCSCs could be purified by p75 using FACS. In vitro-expanded NCSCs were able to differentiate into neurons and glia (Schwann cells) of the peripheral nervous system (PNS) as well as mesenchymal derivatives.


Assuntos
Diferenciação Celular , Crista Neural/citologia , Células-Tronco Neurais/citologia , Células-Tronco Pluripotentes/citologia , Adipogenia , Técnicas de Cultura de Células , Células-Tronco Embrionárias/citologia , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Osteogênese , Células de Schwann/citologia
3.
J Biomol Screen ; 19(1): 32-43, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24019252

RESUMO

Rapid and effective drug discovery for neurodegenerative disease is currently impeded by an inability to source primary neural cells for high-throughput and phenotypic screens. This limitation can be addressed through the use of pluripotent stem cells (PSCs), which can be derived from patient-specific samples and differentiated to neural cells for use in identifying novel compounds for the treatment of neurodegenerative diseases. We have developed an efficient protocol to culture pure populations of neurons, as confirmed by gene expression analysis, in the 96-well format necessary for screens. These differentiated neurons were subjected to viability assays to illustrate their potential in future high-throughput screens. We have also shown that organelles such as nuclei and mitochondria could be live-labeled and visualized through fluorescence, suggesting that we should be able to monitor subcellular phenotypic changes. Neurons derived from a green fluorescent protein-expressing reporter line of PSCs were live-imaged to assess markers of neuronal maturation such as neurite length and co-cultured with astrocytes to demonstrate further maturation. These studies confirm that PSC-derived neurons can be used effectively in viability and functional assays and pave the way for high-throughput screens on neurons derived from patients with neurodegenerative disorders.


Assuntos
Diferenciação Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Neurais/citologia , Neurônios/citologia , Neurônios/metabolismo , Células-Tronco Pluripotentes/citologia , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Técnicas de Cocultura , Descoberta de Drogas/métodos , Expressão Gênica , Perfilação da Expressão Gênica , Genes Reporter , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Neurais/metabolismo , Neurônios/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo
4.
Mol Cell Proteomics ; 13(1): 311-28, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24173317

RESUMO

Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important in cellular signaling and recognition, and their function and activity are frequently regulated by post-translational modifications such as phosphorylation and glycosylation. To obtain information about membrane-associated proteins and their modified amino acids potentially involved in changes of hESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well as changes in phosphorylation and sialylation between hESCs and NSCs. Combining proteomics and modification specific proteomics we identified a total of 5105 proteins whereof 57% contained transmembrane domains or signal peptides. The enrichment strategy yielded a total of 10,087 phosphorylated peptides in which 78% of phosphopeptides were identified with ≥99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development. In the latter group of proteins, we could identify potential NSC markers as Crumbs 2 and several novel proteins. A motif analysis of the altered phosphosites showed a sequence consensus motif (R-X-XpS/T) significantly up-regulated in NSC. This motif is among other kinases recognized by the calmodulin-dependent protein kinase-2, emphasizing a possible importance of this kinase for this cell stage. Collectively, this data represent the most diverse set of post-translational modifications reported for hESCs and NSCs. This study revealed potential markers to distinguish NSCs from hESCs and will contribute to improve our understanding on the differentiation process.


Assuntos
Diferenciação Celular/genética , Proteínas de Membrana/biossíntese , Fosfoproteínas/biossíntese , Proteômica , Células-Tronco Embrionárias/metabolismo , Glicopeptídeos/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Transdução de Sinais/genética
5.
Stem Cells Dev ; 23(4): 406-20, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24074155

RESUMO

Recent advances in human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) biology enable generation of dopaminergic neurons for potential therapy and drug screening. However, our current understanding of molecular and cellular signaling that controls human dopaminergic development and function is limited. Here, we report on a whole genome analysis of gene expression during dopaminergic differentiation of human ESC/iPSC using Illumina bead microarrays. We generated a transcriptome data set containing the expression levels of 28,688 unique transcripts by profiling five lines (three ESC and two iPSC lines) at four stages of differentiation: (1) undifferentiated ESC/iPSC, (2) neural stem cells, (3) dopaminergic precursors, and (4) dopaminergic neurons. This data set provides comprehensive information about genes expressed at each stage of differentiation. Our data indicate that distinct pathways are activated during neural and dopaminergic neuronal differentiation. For example, WNT, sonic hedgehog (SHH), and cAMP signaling pathways were found over-represented in dopaminergic populations by gene enrichment and pathway analysis, and their role was confirmed by perturbation analyses using RNAi (small interfering RNA of SHH and WNT) or small molecule [dibutyryl cyclic AMP (dcAMP)]. In summary, whole genome profiling of dopaminergic differentiation enables systematic analysis of genes/pathways, networks, and cellular/molecular processes that control cell fate decisions. Such analyses will serve as the foundation for better understanding of dopaminergic development, function, and development of future stem cell-based therapies.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Células-Tronco Embrionárias/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Transcriptoma , Sítios de Ligação , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica , Genes Mitocondriais , Genoma Humano , Humanos , Células-Tronco Neurais/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Fatores de Transcrição/fisiologia
6.
Curr Protoc Stem Cell Biol ; Chapter 2: Unit2D.12, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22872425

RESUMO

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are potentially an unlimited cell source for cell replacement therapy and personalized medicine. Before hESC- and iPSC-based therapy can be moved from bench to bedside, however, it is essential to establish protocols for generating therapeutically relevant cells, like dopaminergic neurons in defined conditions that are suitable for scalable good manufacturing practice (GMP)-compliant protocols. Here, the derivation and differentiation of functional dopaminergic neurons from hESCs and iPSCs under xeno-free defined conditions are described. These protocols have been validated in multiple hESC and iPSC lines.


Assuntos
Técnicas de Cultura de Células/métodos , Neurônios Dopaminérgicos/transplante , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Diferenciação Celular , Linhagem Celular , Criopreservação , Dissecação , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Embrionárias/metabolismo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Xenobióticos
7.
Stem Cells Dev ; 21(2): 191-205, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-21699412

RESUMO

Lineage reporters of human embryonic stem cell (hESC) lines are useful for differentiation studies and drug screening. Previously, we created reporter lines driven by an elongation factor 1 alpha (EF1α) promoter at a chromosome 13q32.3 locus in the hESC line WA09 and an abnormal hESC line BG01V in a site-specific manner. Expression of reporters in these lines was maintained in long-term culture at undifferentiated state. However, when these cells were differentiated into specific lineages, reduction in reporter expression was observed, indicating transgene silencing. To develop an efficient and reliable genetic engineering strategy in hESCs, we used chromatin insulator elements to flank single-copy transgenes and integrated the combined expression constructs via PhiC31/R4 integrase-mediated recombination technology to the chromosome 13 locus precisely. Two copies of cHS4 double-insulator sequences were placed adjacent to both 5' and 3' of the promoter reporter constructs. The green fluorescent protein (GFP) gene was driven by EF1α or CMV early enhancer/chicken ß actin (CAG) promoter. In the engineered hESC lines, for both insulated CAG-GFP and EF1α-GFP, constitutive expression at the chromosome 13 locus was maintained during prolonged culture and in directed differentiation assays toward diverse types of neurons, pancreatic endoderm, and mesodermal progeny. In particular, described here is the first normal hESC fluorescent reporter line that robustly expresses GFP in both the undifferentiated state and throughout dopaminergic lineage differentiation. The dual strategy of utilizing insulator sequences and integration at the constitutive chromosome 13 locus ensures appropriate transgene expression. This is a valuable tool for lineage development study, gain- and loss-of-function experiments, and human disease modeling using hESCs.


Assuntos
Cromatina/genética , Células-Tronco Embrionárias , Engenharia Genética/métodos , Elementos Isolantes/genética , Recombinação Genética , Diferenciação Celular , Linhagem Celular Transformada , Linhagem da Célula , Cromossomos Humanos Par 13 , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Genes Reporter , Loci Gênicos , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Humanos , Integrases/genética , Integrases/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Regiões Promotoras Genéticas , Transgenes
8.
J Cell Sci ; 124(Pt 3): 348-58, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21242311

RESUMO

Here, we have investigated mitochondrial biology and energy metabolism in human embryonic stem cells (hESCs) and hESC-derived neural stem cells (NSCs). Although stem cells collectively in vivo might be expected to rely primarily on anaerobic glycolysis for ATP supply, to minimise production of reactive oxygen species, we show that in vitro this is not so: hESCs generate an estimated 77% of their ATP through oxidative phosphorylation. Upon differentiation of hESCs into NSCs, oxidative phosphorylation declines both in absolute rate and in importance relative to glycolysis. A bias towards ATP supply from oxidative phosphorylation in hESCs is consistent with the expression levels of the mitochondrial gene regulators peroxisome-proliferator-activated receptor γ coactivator (PGC)-1α, PGC-1ß and receptor-interacting protein 140 (RIP140) in hESCs when compared with a panel of differentiated cell types. Analysis of the ATP demand showed that the slower ATP turnover in NSCs was associated with a slower rate of most energy-demanding processes but occurred without a reduction in the cellular growth rate. This mismatch is probably explained by a higher rate of macromolecule secretion in hESCs, on the basis of evidence from electron microscopy and an analysis of conditioned media. Taken together, our developmental model provides an understanding of the metabolic transition from hESCs to more quiescent somatic cell types, and supports important roles for mitochondria and secretion in hESC biology.


Assuntos
Trifosfato de Adenosina/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias , Mitocôndrias , Células-Tronco Neurais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Proliferação de Células , Meios de Cultivo Condicionados , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/ultraestrutura , Metabolismo Energético , Proteínas de Choque Térmico/metabolismo , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/ultraestrutura , Proteínas Nucleares/metabolismo , Proteína 1 de Interação com Receptor Nuclear , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas de Ligação a RNA , Fatores de Transcrição/metabolismo
9.
Stem Cells ; 28(10): 1893-904, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20715183

RESUMO

Human induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells represent a promising unlimited cell source for generating patient-specific cells for biomedical research and personalized medicine. As a first step, critical to clinical applications, we attempted to develop defined culture conditions to expand and differentiate human iPSCs into functional progeny such as dopaminergic neurons for treating or modeling Parkinson's disease (PD). We used a completely defined (xeno-free) system that we previously developed for efficient generation of authentic dopaminergic neurons from human embryonic stem cells (hESCs), and applied it to iPSCs. First, we adapted two human iPSC lines derived from different somatic cell types for the defined expansion medium and showed that the iPSCs grew similarly as hESCs in the same medium regarding pluripotency and genomic stability. Second, by using these two independent adapted iPSC lines, we showed that the process of differentiation into committed neural stem cells (NSCs) and subsequently into dopaminergic neurons was also similar to hESCs. Importantly, iPSC-derived dopaminergic neurons were functional as they survived and improved behavioral deficits in 6-hydroxydopamine-leasioned rats after transplantation. In addition, iPSC-derived NSCs and neurons could be efficiently transduced by a baculoviral vector delivering episomal DNA for future gene function study and disease modeling using iPSCs. We also performed genome-wide microarray comparisons between iPSCs and hESCs, and we derived NSC and dopaminergic neurons. Our data revealed overall similarity and visible differences at a molecular level. Efficient generation of functional dopaminergic neurons under defined conditions will facilitate research and applications using PD patient-specific iPSCs.


Assuntos
Dopamina/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Neurônios/metabolismo , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase
10.
Stem Cells Dev ; 19(1): 71-82, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19788370

RESUMO

Stromal cells have been used to induce dopaminergic differentiation of mouse, primate, and human embryonic stem cells (hESCs), but the mechanism that governs this induction is unknown. In this manuscript, we show that medium conditioned by the stromal cell line PA6 (PA6-CM) can induce dopaminergic differentiation in neural stem cells (NSCs) derived from hESCs but not directly from hESCs, indicating that soluble factors produced by PA6 cells act at the NSC stage to specify a dopaminergic fate. To identify such soluble factors, we analyzed the transcriptomes of PA6 cells, NSCs, and dopaminergic populations induced by PA6-CM from hESC-derived NSCs. We focused our analysis on growth factors expressed by PA6 and receptors expressed by NSCs, and generated a list of growth factors and receptors that are differentially expressed. Some of the growth factor/receptor pairs are categorized into the Shh, Wnt5A, TGFbeta, and IGF pathways. The expression of genes activated by these pathways in dopaminergic populations was analyzed to confirm that these signals were likely candidates for specifying dopaminergic fate. Results were verified for Shh by using perturbation agents such as cyclopamine to show that Shh is indeed one of the active agents in PA6-CM, and by showing that Shh and FGF8 can substitute for PA6-CM at the NSC induction stage. We conclude that PA6-CM can induce dopaminergic differentiation in hESCs in a stage-specific manner. Shh is likely an important soluble dopaminergic inducing factor secreted by stromal cells and acts after the neural fate determination.


Assuntos
Diferenciação Celular , Dopamina/metabolismo , Células-Tronco Embrionárias/fisiologia , Proteínas Hedgehog/fisiologia , Neurônios/fisiologia , Células Estromais/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Fase de Clivagem do Zigoto/metabolismo , Fase de Clivagem do Zigoto/fisiologia , Técnicas de Cocultura , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Indução Embrionária/genética , Indução Embrionária/fisiologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Neurogênese/genética , Neurogênese/fisiologia , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Células Estromais/fisiologia
11.
J Neurosci ; 29(50): 15703-12, 2009 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-20016085

RESUMO

Multiple recent reports implicate amyloid precursor protein (APP) signaling in the pathogenesis of Alzheimer's disease, but the APP-dependent signaling network involved has not been defined. Here, we report a novel consensus sequence for interaction with the PDZ-1 and PDZ-2 domains of the APP-interacting proteins Mint1, Mint2, and Mint3 (X11alpha, X11beta, and X11gamma), and multiple novel interactors for these proteins, with the finding that transcriptional coactivators are highly represented among these interactors. Furthermore, we show that Mint3 interaction with a set of the transcriptional coactivators leads to nuclear localization and transactivation, whereas interaction of the same set with Mint1 or Mint2 prevents nuclear localization and transactivation. These results define new mediators of the signal transduction network mediated by APP.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Precursor de Proteína beta-Amiloide/metabolismo , Caderinas/fisiologia , Proteínas de Transporte/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/química , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/fisiologia , Caderinas/química , Proteínas de Transporte/química , Linhagem Celular , Ensaios de Triagem em Larga Escala , Humanos , Proteínas do Tecido Nervoso/química , Domínios PDZ/fisiologia , Ligação Proteica/fisiologia , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Ativação Transcricional/fisiologia
12.
PLoS One ; 4(9): e7155, 2009 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-19774075

RESUMO

BACKGROUND: We have previously described fundamental differences in the biology of stem cells as compared to other dividing cell populations. We reasoned therefore that a differential screen using US Food and Drug Administration (FDA)-approved compounds may identify either selective survival factors or specific toxins and may be useful for the therapeutically-driven manufacturing of cells in vitro and possibly in vivo. METHODOLOGY/PRINCIPAL FINDINGS: In this study we report on optimized methods for feeder-free culture of hESCs and hESC-derived neural stem cells (NSCs) to facilitate automated screening. We show that we are able to measure ATP as an indicator of metabolic activity in an automated screening assay. With this optimized platform we screened a collection of FDA-approved drugs to identify compounds that have differential toxicity to hESCs and their neural derivatives. Nine compounds were identified to be specifically toxic for NSCs to a greater extent than for hESCs. Six of these initial hits were retested and verified by large-scale cell culture to determine dose-responsive NSC toxicity. One of the compounds retested, amiodarone HCL, was further tested for possible effects on postmitotic neurons, a likely target for transplant therapy. Amiodarone HCL was found to be selectively toxic to NSCs but not to differentiated neurons or glial cells. Treated and untreated NSCs and neurons were then interrogated with global gene expression analysis to explore the mechanisms of action of amiodarone HCl. The gene expression analysis suggests that activation of cell-type specific cationic channels may underlie the toxicity of the drug. CONCLUSIONS/SIGNIFICANCE: In conclusion, we have developed a screening strategy that allows us to rapidly identify clinically approved drugs for use in a Chemistry, Manufacture and Control protocol that can be safely used to deplete unwanted contaminating precursor cells from a differentiated cell product. Our results also suggest that such a strategy is rich in the potential of identifying lineage specific reagents and provides additional evidence for the utility of stem cells in screening and discovery paradigms.


Assuntos
Dopamina/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Embrionárias/citologia , Neurônios/citologia , Neurônios/metabolismo , Células-Tronco/citologia , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Amiodarona/farmacologia , Astrócitos/citologia , Automação , Cátions , Células Cultivadas , Colágeno/química , Técnicas de Química Combinatória , Combinação de Medicamentos , Laminina/química , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Proteoglicanas/química
13.
PLoS One ; 4(7): e6233, 2009 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-19597550

RESUMO

BACKGROUND: Human embryonic stem cells (hESCs) may provide an invaluable resource for regenerative medicine. To move hESCs towards the clinic it is important that cells with therapeutic potential be reproducibly generated under completely defined conditions. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a four-step scalable process that is readily transferable to a Good Manufacture Practice (GMP) facility for the production of functional dopaminergic neurons from hESCs for potential clinical uses. We show that each of the steps (propagation of ESC-->generation of neural stem cells (NSC)-->induction of dopaminergic precursors-->maturation of dopaminergic neurons) could utilize xeno-free defined media and substrate, and that cells could be stored at intermediate stages in the process without losing their functional ability. Neurons generated by this process expressed midbrain and A9 dopaminergic markers and could be transplanted at an appropriate time point in development to survive after transplant. CONCLUSIONS/SIGNIFICANCE: hESCs and NSCs can be maintained in xeno-free defined media for a prolonged period of time while retaining their ability to differentiate into authentic dopaminergic neurons. Our defined medium system provides a path to a scalable GMP-applicable process of generation of dopaminergic neurons from hESCs for therapeutic applications, and a ready source of large numbers of neurons for potential screening applications.


Assuntos
Dopamina/metabolismo , Células-Tronco Embrionárias/citologia , Neurônios/citologia , Animais , Células Cultivadas , Meios de Cultura Livres de Soro , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Neurônios/metabolismo , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley
14.
Neurosurg Clin N Am ; 20(2): 211-8, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19555884

RESUMO

Transplantation of neural cells for the treatment of neurologic disorders has garnered much attention and considerable enthusiasm from patients and physicians alike. Cell-based therapies have been proposed for a wide range of central nervous system pathologies ranging from stroke and trauma to demyelinating disorders and neurodegenerative diseases. Notably, cell transplantation for Parkinson disease (PD) has become even more attractive with the rapid advances in derivation of dopaminergic neurons from human embryonic stem cells. This article briefly reviews some of the relevant issues regarding the transplantation of cells for treatment of PD and hypothesizes how interventional MRI may be useful to optimize the surgical delivery of cells for PD and other central nervous system disorders.


Assuntos
Transplante de Células/métodos , Imageamento por Ressonância Magnética/métodos , Procedimentos Neurocirúrgicos/métodos , Doença de Parkinson/terapia , Transplante de Células/tendências , Dopamina/fisiologia , Células-Tronco Embrionárias , Humanos , Imageamento por Ressonância Magnética/tendências , Neurônios/transplante , Procedimentos Neurocirúrgicos/tendências
15.
BMC Genomics ; 8: 478, 2007 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-18162134

RESUMO

BACKGROUND: Much of our current knowledge of the molecular expression profile of human embryonic stem cells (hESCs) is based on transcriptional approaches. These analyses are only partly predictive of protein expression however, and do not shed light on post-translational regulation, leaving a large gap in our knowledge of the biology of pluripotent stem cells. RESULTS: Here we describe the use of two large-scale western blot assays to identify over 600 proteins expressed in undifferentiated hESCs, and highlight over 40 examples of multiple gel mobility variants, which are suspected protein isoforms and/or post-translational modifications. Twenty-two phosphorylation events in cell signaling molecules, as well as potential new markers of undifferentiated hESCs were also identified. We confirmed the expression of a subset of the identified proteins by immunofluorescence and correlated the expression of transcript and protein for key molecules in active signaling pathways in hESCs. These analyses also indicated that hESCs exhibit several features of polarized epithelia, including expression of tight junction proteins. CONCLUSION: Our approach complements proteomic and transcriptional analysis to provide unique information on human pluripotent stem cells, and is a framework for the continued analyses of self-renewal.


Assuntos
Células-Tronco Embrionárias/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Western Blotting , Imunofluorescência , Humanos , Imuno-Histoquímica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ocludina , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/classificação , Proteoma/genética , Transcrição Gênica , Proteína da Zônula de Oclusão-1
16.
PLoS One ; 2(9): e953, 2007 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-17895993

RESUMO

Specific protein associations define the wiring of protein interaction networks and thus control the organization and functioning of the cell as a whole. Peptide recognition by PDZ and other protein interaction domains represents one of the best-studied classes of specific protein associations. However, a mechanistic understanding of the relationship between selectivity and promiscuity commonly observed in the interactions mediated by peptide recognition modules as well as its functional meaning remain elusive. To address these questions in a comprehensive manner, two large populations of artificial and natural peptide ligands of six archetypal PDZ domains from the synaptic proteins PSD95 and SAP97 were generated by target-assisted iterative screening (TAIS) of combinatorial peptide libraries and by synthesis of proteomic fragments, correspondingly. A comparative statistical analysis of affinity-ranked artificial and natural ligands yielded a comprehensive picture of known and novel PDZ ligand specificity determinants, revealing a hitherto unappreciated combination of specificity and adaptive plasticity inherent to PDZ domain recognition. We propose a reconceptualization of the PDZ domain in terms of a complex adaptive system representing a flexible compromise between the rigid order of exquisite specificity and the chaos of unselective promiscuity, which has evolved to mediate two mutually contradictory properties required of such higher order sub-cellular organizations as synapses, cell junctions, and others--organizational structure and organizational plasticity/adaptability. The generalization of this reconceptualization in regard to other protein interaction modules and specific protein associations is consistent with the image of the cell as a complex adaptive macromolecular system as opposed to clockwork.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Domínios PDZ/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína 1 Homóloga a Discs-Large , Proteína 4 Homóloga a Disks-Large , Ensaio de Imunoadsorção Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Domínios PDZ/genética , Biblioteca de Peptídeos , Ligação Proteica , Mapeamento de Interação de Proteínas
17.
Anal Bioanal Chem ; 376(7): 1006-13, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12677339

RESUMO

We describe the development of a process for the genome-wide mapping of interactions between protein domains and peptide ligands entirely based on high-throughput biochip technologies. A phage library displaying protein domains from a randomly fragmented and cloned cDNA library will be "panned" on an array of synthetic peptide ligands. After multiplexed affinity enrichment, peptide-specific phage populations will be automatically eluted, propagated, labelled and identified by hybridisation to a DNA microarray. Peptide arrays are synthesized in situ by SPOT synthesis on a planar substrate. By utilizing a commercially available library of human brain cDNA plus a set of distinct model domains cloned into T7-phage, we could show that a single panning round on an array of known peptide ligands for these model domains synthesized on a cellulose membrane can yield an enrichment of better than a factor of 1,000. This is sufficient to detect peptide-specific enrichment of Cy3(post-panning) against Cy5(pre-panning)-labelled phage DNA inserts on a cDNA microarray. Thus, the proof-of-principle of our approach could be successfully demonstrated and first interaction data are being collected.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos , Proteoma/química , Sequência de Aminoácidos , Automação , Bacteriófago T7/genética , Bacteriófago lambda/genética , Mapeamento de Epitopos/métodos , Epitopos/análise , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Nanotecnologia , Biblioteca de Peptídeos , Análise Serial de Proteínas/métodos , Reprodutibilidade dos Testes
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