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1.
Sci Rep ; 9(1): 11434, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391476

RESUMO

The highly conserved SNARE protein SEC22B mediates diverse and critical functions, including phagocytosis, cell growth, autophagy, and protein secretion. However, these characterizations have thus far been limited to in vitro work. Here, we expand our understanding of the role Sec22b plays in vivo. We utilized Cre-Lox mice to delete Sec22b in three tissue compartments. With a germline deletion of Sec22b, we observed embryonic death at E8.5. Hematopoietic/endothelial cell deletion of Sec22b also resulted in in utero death. Notably, mice with Sec22b deletion in CD11c-expressing cells of the hematopoietic system survive to adulthood. These data demonstrate Sec22b contributes to early embryogenesis through activity both in hematopoietic/endothelial tissues as well as in other tissues yet to be defined.

2.
BMC Bioinformatics ; 20(Suppl 5): 180, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31272389

RESUMO

BACKGROUND: Stem cells and stem cell lines are widely used in biomedical research. The Cell Ontology (CL) and Cell Line Ontology (CLO) are two community-based OBO Foundry ontologies in the domains of in vivo cells and in vitro cell line cells, respectively. RESULTS: To support standardized stem cell investigations, we have developed an Ontology for Stem Cell Investigations (OSCI). OSCI imports stem cell and cell line terms from CL and CLO, and investigation-related terms from existing ontologies. A novel focus of OSCI is its application in representing metadata types associated with various stem cell investigations. We also applied OSCI to systematically categorize experimental variables in an induced pluripotent stem cell line cell study related to bipolar disorder. In addition, we used a semi-automated literature mining approach to identify over 200 stem cell gene markers. The relations between these genes and stem cells are modeled and represented in OSCI. CONCLUSIONS: OSCI standardizes stem cells found in vivo and in vitro and in various stem cell investigation processes and entities. The presented use cases demonstrate the utility of OSCI in iPSC studies and literature mining related to bipolar disorder.


Assuntos
Ontologias Biológicas , Pesquisa Biomédica/normas , Animais , Humanos , Células-Tronco
4.
J Cell Biol ; 216(12): 3981-3990, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29021220

RESUMO

Human pluripotent stem cells (hPSCs) self-organize into apicobasally polarized cysts, reminiscent of the lumenal epiblast stage, providing a model to explore key morphogenic processes in early human embryos. Here, we show that apical polarization begins on the interior of single hPSCs through the dynamic formation of a highly organized perinuclear apicosome structure. The membrane surrounding the apicosome is enriched in apical markers and displays microvilli and a primary cilium; its lumenal space is rich in Ca2+ Time-lapse imaging of isolated hPSCs reveals that the apicosome forms de novo in interphase, retains its structure during mitosis, is asymmetrically inherited after mitosis, and relocates to the recently formed cytokinetic plane, where it establishes a fully polarized lumen. In a multicellular aggregate of hPSCs, intracellular apicosomes from multiple cells are trafficked to generate a common lumenal cavity. Thus, the apicosome is a unique preassembled apical structure that can be rapidly used in single or clustered hPSCs to initiate self-organized apical polarization and lumenogenesis.


Assuntos
Citocinese , Camadas Germinativas/ultraestrutura , Morfogênese/genética , Células-Tronco Pluripotentes/ultraestrutura , Actinas/genética , Actinas/metabolismo , Biomarcadores/metabolismo , Cálcio/metabolismo , Calnexina/genética , Calnexina/metabolismo , Linhagem Celular , Polaridade Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Expressão Gênica , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Humanos , Interfase , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Mitose , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Análise de Célula Única , Imagem com Lapso de Tempo
5.
Stem Cell Res ; 17(2): 238-247, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27591480

RESUMO

Over-expression of the early neural inducer, Noggin, in nestin positive subventricular zone (SVZ), neural stem cells (NSC) promotes proliferation and neuronal differentiation of neural progenitors and inhibits the expression of a CNS-enriched microRNA-410 (miR-410) (Morell et al., 2015). When expressed in neurospheres derived from the adult SVZ, miR-410 inhibits neuronal and oligodendrocyte differentiation, and promotes astrocyte differentiation. miR-410 also reverses the increase in neuronal differentiation and decreased astroglial differentiation caused by Noggin over-expression. Conversely, inhibition of miR-410 activity promotes neuronal and decreases astroglial differentiation of NSC. Using computer prediction algorithms and luciferase reporter assays we identified multiple neurogenic genes including Elavl4 as downstream targets of miR-410 via the canonical miRNA-3'UTR interaction. Over-expression of Elavl4 transcripts without the endogenous 3'UTR rescued the decrease in neuronal differentiation caused by miR-410 overexpression. Interestingly, we also observed that miR-410 affected neurite morphology; over-expression of miR-410 resulted in the formation of short, unbranched neurites. We conclude that miR-410 expression provides a new link between BMP signaling and the crucial lineage choice of adult neural stem cells via its ability to bind and control the expression of neurogenic gene transcripts.


Assuntos
Ventrículos Laterais/citologia , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Proteína Semelhante a ELAV 4/antagonistas & inibidores , Proteína Semelhante a ELAV 4/genética , Proteína Semelhante a ELAV 4/metabolismo , Imuno-Histoquímica , Ventrículos Laterais/metabolismo , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Nestina/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Oligodendroglia/citologia , Oligodendroglia/metabolismo
6.
Stem Cells Transl Med ; 5(12): 1595-1606, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27465073

RESUMO

: The establishment of an abundant source of autologous cardiac progenitor cells would represent a major advance toward eventual clinical translation of regenerative medicine strategies in children with prenatally diagnosed congenital heart disease. In support of this concept, we sought to examine whether functional, transgene-free human cardiomyocytes (CMs) with potential for patient-specific and autologous applications could be reliably generated following routine amniocentesis. Under institutional review board approval, amniotic fluid specimens (8-10 ml) at 20 weeks gestation were expanded and reprogrammed toward pluripotency using nonintegrating Sendai virus (SeV) expressing OCT4, SOX2, cMYC, and KLF4. Following exposure of these induced pluripotent stem cells to cardiogenic differentiation conditions, spontaneously beating amniotic fluid-derived cardiomyocytes (AF-CMs) were successfully generated with high efficiency. After 6 weeks, quantitative gene expression revealed a mixed population of differentiated atrial, ventricular, and nodal AF-CMs, as demonstrated by upregulation of multiple cardiac markers, including MYH6, MYL7, TNNT2, TTN, and HCN4, which were comparable to levels expressed by neonatal dermal fibroblast-derived CM controls. AF-CMs had a normal karyotype and demonstrated loss of NANOG, OCT4, and the SeV transgene. Functional characterization of SIRPA+ AF-CMs showed a higher spontaneous beat frequency in comparison with dermal fibroblast controls but revealed normal calcium transients and appropriate chronotropic responses after ß-adrenergic agonist stimulation. Taken together, these data suggest that somatic cells present within human amniotic fluid can be used to generate a highly scalable source of functional, transgene-free, autologous CMs before a child is born. This approach may be ideally suited for patients with prenatally diagnosed cardiac anomalies. SIGNIFICANCE: This study presents transgene-free human amniotic fluid-derived cardiomyocytes (AF-CMs) for potential therapy in tissue engineering and regenerative medicine applications. Using 8-10 ml of amniotic fluid harvested at 20 weeks gestation from normal pregnancies, a mixed population of atrial, ventricular, and nodal AF-CMs were reliably generated after Sendai virus reprogramming toward pluripotency. Functional characterization of purified populations of beating AF-CMs revealed normal calcium transients and appropriate chronotropic responses after ß-adrenergic agonist stimulation in comparison with dermal fibroblast controls. Because AF-CMs can be generated in fewer than 16 weeks, this approach may be ideally suited for eventual clinical translation at birth in children with prenatally diagnosed cardiac anomalies.


Assuntos
Líquido Amniótico/citologia , Reprogramação Celular , Miócitos Cardíacos/citologia , Diferenciação Celular , Linhagem da Célula , Regulação da Expressão Gênica , Vetores Genéticos/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Miócitos Cardíacos/metabolismo , Vírus Sendai/metabolismo , Pele/citologia , Transgenes
7.
Mol Cell Neurosci ; 73: 63-83, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26608002

RESUMO

Bipolar disorder (BP) is a chronic neuropsychiatric condition characterized by pathological fluctuations in mood from mania to depression. Adoption, twin and family studies have consistently identified a significant hereditary component to BP, yet there is no clear genetic event or consistent neuropathology. BP has been suggested to have a developmental origin, although this hypothesis has been difficult to test since there are no viable neurons or glial cells to analyze, and research has relied largely on postmortem brain, behavioral and imaging studies, or has examined proxy tissues including saliva, olfactory epithelium and blood cells. Neurodevelopmental factors, particularly pathways related to nervous system development, cell migration, extracellular matrix, H3K4 methylation, and calcium signaling have been identified in large gene expression and GWAS studies as altered in BP. Recent advances in stem cell biology, particularly the ability to reprogram adult somatic tissues to a pluripotent state, now make it possible to interrogate these pathways in viable cell models. A number of induced pluripotent stem cell (iPSC) lines from BP patient and healthy control (C) individuals have been derived in several laboratories, and their ability to form cortical neurons examined. Early studies suggest differences in activity, calcium signaling, blocks to neuronal differentiation, and changes in neuronal, and possibly glial, lineage specification. Initial observations suggest that differentiation of BP patient-derived neurons to dorsal telencephalic derivatives may be impaired, possibly due to alterations in WNT, Hedgehog or Nodal pathway signaling. These investigations strongly support a developmental contribution to BP and identify novel pathways, mechanisms and opportunities for improved treatments.


Assuntos
Transtorno Bipolar/patologia , Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Transtorno Bipolar/genética , Transtorno Bipolar/metabolismo , Sinalização do Cálcio , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Neurogênese , Transcriptoma
8.
Stem Cell Reports ; 5(6): 954-962, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26626176

RESUMO

We demonstrate that dissociated human pluripotent stem cells (PSCs) are intrinsically programmed to form lumens. PSCs form two-cell cysts with a shared apical domain within 20 hr of plating; these cysts collapse to form monolayers after 5 days. Expression of pluripotency markers is maintained throughout this time. In two-cell cysts, an apical domain, marked by EZRIN and atypical PKCζ, is surrounded by apically targeted organelles (early endosomes and Golgi). Molecularly, actin polymerization, regulated by ARP2/3 and mammalian diaphanous-related formin 1 (MDIA), promotes lumen formation, whereas actin contraction, mediated by MYOSIN-II, inhibits this process. Finally, we show that lumenal shape can be manipulated in bioengineered micro-wells. Since lumen formation is an indispensable step in early mammalian development, this system can provide a powerful model for investigation of this process in a controlled environment. Overall, our data establish that lumenogenesis is a fundamental cell biological property of human PSCs.


Assuntos
Células-Tronco Pluripotentes/citologia , Actinas/metabolismo , Actinas/ultraestrutura , Animais , Técnicas de Cultura de Células , Linhagem Celular , Separação Celular , Forma Celular , Cães , Humanos , Camundongos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/ultraestrutura
9.
Stem Cell Res ; 14(1): 79-94, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25535864

RESUMO

Multipotent, self-renewing stem cells are present throughout the developing nervous system remaining in discrete regions of the adult brain. In the subventricular zone (SVZ) signaling molecules, including the bone morphogenetic proteins and their secreted inhibitor, noggin appear to play a critical role in controlling neural stem cell (NSC) behavior. To examine the function of this signaling pathway in the intact nervous system, we developed a transgenic mouse model in which noggin expression can be induced specifically in NSC via a nestin-driven reverse tetracycline-controlled transactivator (rtTA). In adult animals, the induction of noggin expression promotes the proliferation of neural progenitors in the SVZ, and shifts the differentiation of B cells (NSC) from mature astrocytes to transit amplifying C cells and oligodendrocyte precursor cells without depleting the NSC population. Noggin expression significantly increases neuronal and oligodendrocyte differentiation both in vivo and in vitro when NSCs are grown as neurospheres. These results demonstrate that noggin/BMP interactions tightly control cell fate in the SVZ.


Assuntos
Proteínas de Transporte/metabolismo , Ventrículos Laterais/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Astrócitos , Proteínas Morfogenéticas Ósseas/química , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Nestina/metabolismo , Células-Tronco Neurais/citologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Transdução de Sinais
11.
Stem Cells Dev ; 23(21): 2613-25, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25014361

RESUMO

The establishment of a reliable prenatal source of autologous, transgene-free progenitor cells has enormous potential in the development of regenerative-medicine-based therapies for infants born with devastating birth defects. Here, we show that a largely CD117-negative population of human amniotic fluid mesenchymal stromal cells (AF-MSCs) obtained from fetuses with or without prenatally diagnosed anomalies are readily abundant and have limited baseline differentiation potential when compared with bone-marrow-derived MSCs and other somatic cell types. Nonetheless, the AF-MSCs could be easily reprogrammed into induced pluripotent stem cells (iPSCs) using nonintegrating Sendai viral vectors encoding for OCT4, SOX2, KLF4, and cMYC. The iPSCs were virtually indistinguishable from human embryonic stem cells in multiple assays and could be used to generate a relatively homogeneous population of neural progenitors, expressing PAX6, SOX2, SOX3, Musashi-1, and PSA-NCAM, for potential use in neurologic diseases. Further, these neural progenitors showed engraftment potential in vivo and were capable of differentiating into mature neurons and astrocytes in vitro. This study demonstrates the usefulness of AF-MSCs as an excellent source for the generation of human transgene-free iPSCs ideally suited for autologous perinatal regenerative medicine applications.


Assuntos
Líquido Amniótico/citologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Diferenciação Celular/genética , Células Cultivadas , Reprogramação Celular/genética , Proteínas do Olho/genética , Feminino , Citometria de Fluxo , Proteínas de Homeodomínio/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Células-Tronco Mesenquimais/metabolismo , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genética , Molécula L1 de Adesão de Célula Nervosa/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Gravidez , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Ácidos Siálicos/genética , Transgenes/genética , Transplante Autólogo
12.
J Biol Chem ; 289(30): 20858-70, 2014 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-24876386

RESUMO

COPII-coated vesicles mediate the transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi. SEC24 is the COPII component primarily responsible for recruitment of protein cargoes into nascent vesicles. There are four Sec24 paralogs in mammals, with mice deficient in SEC24A, -B, and -D exhibiting a wide range of phenotypes. We now report the characterization of mice with deficiency in the fourth Sec24 paralog, SEC24C. Although mice haploinsufficient for Sec24c exhibit no apparent abnormalities, homozygous deficiency results in embryonic lethality at approximately embryonic day 7. Tissue-specific deletion of Sec24c in hepatocytes, pancreatic cells, smooth muscle cells, and intestinal epithelial cells results in phenotypically normal mice. Thus, SEC24C is required in early mammalian development but is dispensable in a number of tissues, likely as a result of compensation by other Sec24 paralogs. The embryonic lethality resulting from loss of SEC24C occurs considerably later than the lethality previously observed in SEC24D deficiency; it is clearly distinct from the restricted neural tube phenotype of Sec24b null embryos and the mild hypocholesterolemic phenotype of adult Sec24a null mice. Taken together, these results demonstrate that the four Sec24 paralogs have developed unique functions over the course of vertebrate evolution.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Embrião de Mamíferos/citologia , Fígado/citologia , Fígado/embriologia , Camundongos , Camundongos Mutantes , Especificidade de Órgãos/fisiologia , Pâncreas/citologia , Pâncreas/embriologia , Proteínas de Transporte Vesicular/genética
13.
Tissue Eng Part C Methods ; 20(9): 731-40, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24447109

RESUMO

The reliable derivation of induced pluripotent stem cells (iPSCs) from a noninvasive autologous source at birth would facilitate the study of patient-specific in vitro modeling of congenital diseases and would enhance ongoing efforts aimed at developing novel cell-based treatments for a wide array of fetal and pediatric disorders. Accordingly, we have successfully generated iPSCs from human fetal chorionic somatic cells extracted from term pregnancies by ectopic expression of OCT4, SOX2, KLF4, and cMYC. The isolated parental somatic cells exhibited an immunophenotypic profile consistent with that of chorionic mesenchymal stromal cells (CMSCs). CMSC-iPSCs maintained pluripotency in feeder-free systems for more than 15 passages based on morphology, immunocytochemistry, and gene expression studies and were capable of embryoid body formation with spontaneous trilineage differentiation. CMSC-iPSCs could be selectively differentiated in vitro into various germ layer derivatives, including neural stem cells, beating cardiomyocytes, and definitive endoderm. This study demonstrates the feasibility of term placental chorion as a novel noninvasive alternative to dermal fibroblasts and cord blood for human perinatal iPSC derivation and may provide additional insights regarding the reprogramming capabilities of extra-embryonic tissues as they relate to developmental ontogeny and perinatal tissue engineering applications.


Assuntos
Córion/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Placenta/citologia , Engenharia Tecidual/métodos , Diferenciação Celular , Linhagem da Célula , Corpos Embrioides/citologia , Endoderma/citologia , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Miócitos Cardíacos/citologia , Gravidez
14.
PLoS One ; 8(3): e58813, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23505563

RESUMO

Despite the crucial role of innate immunity in preventing or controlling pathogen-induced damage in most, if not all, cell types, very little is known about the activity of this essential defense system in central nervous system neurons, especially in humans. In this report we use both an established neuronal cell line model and an embryonic stem cell-based system to examine human neuronal innate immunity and responses to neurotropic alphavirus infection in cultured cells. We demonstrate that neuronal differentiation is associated with increased expression of crucial type I interferon signaling pathway components, including interferon regulatory factor-9 and an interferon receptor heterodimer subunit, which results in enhanced interferon stimulation and subsequent heightened antiviral activity and cytoprotective responses against neurotropic alphaviruses such as western equine encephalitis virus. These results identify important differentiation-dependent changes in innate immune system function that control cell-autonomous neuronal responses. Furthermore, this work demonstrates the utility of human embryonic stem cell-derived cultures as a platform to study the interactions between innate immunity, virus infection, and pathogenesis in central nervous system neurons.


Assuntos
Diferenciação Celular , Interferon Tipo I/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Transdução de Sinais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Expressão Gênica , Ordem dos Genes , Humanos , Imunidade Inata , Interferon Tipo I/imunologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/imunologia , RNA Mensageiro/genética , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/metabolismo
15.
Bipolar Disord ; 15(2): 177-87, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23360497

RESUMO

OBJECTIVES: Bipolar disorder (BD) is a mental illness of unknown neuropathology and has several genetic associations. Antipsychotics are effective for the treatment of acute mania, psychosis, or mixed states in individuals with BD. We aimed to identify gene transcripts differentially expressed in postmortem brains from antipsychotics-exposed individuals with BD (hereafter the 'exposed' group), non-exposed individuals with BD (hereafter the 'non-exposed' group), and controls. METHODS: We quantified the abundance of gene transcripts in postmortem brains from seven exposed individuals, seven non-exposed individuals, and 12 controls with the Affymetrix U133P2 GeneChip microarrays and technologies. We applied a q-value of ≤0.005 to identify statistically significant transcripts with mean abundance differences between the exposed, non-exposed and control groups. RESULTS: We identified 2191 unique genes with significantly altered expression levels in non-exposed brains compared to those in the control and exposed groups. The expression levels of these genes were not significantly different between exposed brains and controls, suggesting a normalization effect of antipsychotics on the expression of these genes. Gene ontology (GO) enrichment analysis showed significant (Bonferroni p ≤ 0.05) clustering of subgroups of the 2191 genes under many GO terms; notably, the protein products of genes enriched are critical to the function of synapses, affecting, for example, intracellular trafficking and synaptic vesicle biogenesis, transport, release and recycling, as well as organization and stabilization of the node of Ranvier. CONCLUSIONS: These results support a hypothesis of synaptic and intercellular communication impairment in BD. The apparent normalization of expression patterns with exposure to antipsychotic medication may represent a physiological process that relates both to etiology and improvement patterns of the disorder.


Assuntos
Transtorno Bipolar/patologia , Encéfalo/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Adulto , Antipsicóticos/uso terapêutico , Transtorno Bipolar/tratamento farmacológico , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Mudanças Depois da Morte
16.
Dev Dyn ; 242(3): 230-53, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23288605

RESUMO

BACKGROUND: Delineating the cascades of growth and transcription factor expression that shape the developing nervous system will improve our understanding of its molecular histogenesis and suggest strategies for cell replacement therapies. In the current investigation, we examined the ability of the proneural gene, Neurogenin1 (Neurog1; also Ngn1, Neurod3), to drive differentiation of pluripotent embryonic stem cells (ESC). RESULTS: Transient expression of Neurog1 in ESC was sufficient to initiate neuronal differentiation, and produced neuronal subtypes reflecting its expression pattern in vivo. To begin to address the molecular mechanisms involved, we used microarray analysis to identify potential down-stream targets of Neurog1 expressed at sequential stages of neuronal differentiation. CONCLUSIONS: ESC expressing Neurogenin1 begin to withdraw from cycle and form precursors that differentiate exclusively into neurons. This work identifies unique patterns of gene expression following expression of Neurog1, including genes and signaling pathways involved in process outgrowth and cell migration, regional differentiation of the nervous system, and cell cycle.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Células-Tronco Neurais/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , Movimento Celular/fisiologia , Células-Tronco Embrionárias/citologia , Perfilação da Expressão Gênica , Camundongos , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Neurônios/citologia , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/fisiologia
17.
Stem Cells Dev ; 22(8): 1177-89, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23249188

RESUMO

Geminin is a nuclear protein that performs the related functions of modulating cell cycle progression by binding Cdt1, and controlling differentiation by binding transcription factors. Since embryonic stem cells (ESC) and the epiblast share a similar gene expression profile and an attenuated cell cycle, ESC form an accessible and tractable model system to study lineage choice at gastrulation. We derived several ESC lines in which Geminin can be inducibly expressed, and employed short hairpin RNAs targeting Geminin. As in the embryo, a lack of Geminin protein resulted in DNA damage and cell death. In monolayer culture, in defined medium, Geminin supported neural differentiation; however, in three-dimensional culture, overexpression of Geminin promoted mesendodermal differentiation and epithelial-to-mesenchymal transition. In vitro, ESC overexpressing Geminin rapidly recolonized a wound, downregulated E-cadherin expression, and activated Wnt signaling. We suggest that Geminin may promote differentiation via binding Groucho/TLE proteins and upregulating canonical Wnt signaling.


Assuntos
Proteínas de Ciclo Celular/genética , Células-Tronco Embrionárias/metabolismo , Transição Epitelial-Mesenquimal/genética , Gastrulação/genética , Proteínas Nucleares/genética , Animais , Caderinas/genética , Caderinas/metabolismo , Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular , Movimento Celular/genética , Doxiciclina/farmacologia , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Geminina , Expressão Gênica/efeitos dos fármacos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Microscopia de Fluorescência , Neurônios/citologia , Neurônios/metabolismo , Proteínas/genética , Proteínas/metabolismo , Interferência de RNA , RNA não Traduzido , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Teratoma/genética , Teratoma/metabolismo , Teratoma/patologia , Via de Sinalização Wnt/genética
18.
Stem Cells Dev ; 21(13): 2395-409, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22335560

RESUMO

Geminin is a multifunctional protein previously suggested to both maintain the bone morphogenetic protein inhibition required for neural induction and to control cell-cycle progression and cell fate in the early embryo. Since Geminin is required in the blastocyst on E3.5, we employed shRNA to examine its role during postimplantation development. Geminin knockdown inhibited the epithelial to mesenchymal transition (EMT) required at gastrulation and neural crest delamination, resulting in anterior-posterior axis and patterning defects, while overexpression promoted EMT at both locations. Geminin was negatively correlated with expression of E-cadherin, which is critically involved in controlling epithelial architecture. In addition, Geminin expression level was correlated with Wnt signaling and expression of the Wnt target gene Axin2 and with Msx2, and negatively correlated with the expression of Bmp4 and Neurog1 in quantitative reverse transcriptase-polymerase chain reaction analysis of RNAs from individual embryos. These results suggest that in addition to patterning the early embryo, Geminin plays a previously unrecognized role in EMT via its ability to affect Wnt signaling and E-cadherin expression.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Transição Epitelial-Mesenquimal , Gastrulação , Proteínas Nucleares/metabolismo , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Padronização Corporal , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Caderinas/metabolismo , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Movimento Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Geminina , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Crista Neural/citologia , Crista Neural/embriologia , Crista Neural/metabolismo , Neurogênese , Proteínas Nucleares/genética , Gravidez , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Via de Sinalização Wnt
19.
Mol Cell Neurosci ; 49(2): 104-9, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22122823

RESUMO

The auditory sensory epithelium in non-mammalian vertebrates can replace lost hair cells by transdifferentiation of supporting cells, but this regenerative ability is lost in the mammalian cochlea. Future cell-based treatment of hearing loss may depend on stem cell transplantation or on transdifferentiation of endogenous cells in the cochlea. For both approaches, identification of cells with stem cell features within the mature cochlea may be useful. Here we use a Nestin-ß-gal mouse to examine the presence of Nestin positive cells in the mature auditory epithelium, and determine how overstimulation of the ear impacts these cells. Nestin positive cells were found in the apical turn of the cochlea lateral to the outer hair cell area. This pattern of expression persisted into mature age. The area of Nestin positive cells was increased after the noise lesion. This increase in area coincided with an increase in expression of the Nestin mRNA. The data suggest that cells with potential stem cell features remain in the mature mammalian cochlea, restricted to the apical turn, and that an additional set of signals is necessary to trigger their contribution to cell replacement therapy in the ear. As such, this population of cells could serve to generate cochlear stem cells for research and potential therapy, and may be a target for treatments based on induced transdifferentiation of endogenous cochlear cells.


Assuntos
Diferenciação Celular , Transdiferenciação Celular/fisiologia , Cóclea/citologia , Proteínas de Filamentos Intermediários/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Órgão Espiral/metabolismo , Células-Tronco/metabolismo , Animais , Proliferação de Células , Cóclea/metabolismo , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/metabolismo , Camundongos , Nestina , Ruído , Órgão Espiral/citologia , Ratos
20.
Nat Protoc ; 6(7): 1037-43, 2011 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-21720316

RESUMO

The culture of human embryonic stem (hES) cells in defined and xenogeneic-free conditions will contribute substantially to future biotechnological and medical applications. To achieve this goal, we developed the first fully defined synthetic polymer coating poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH) that sustains long-term growth of hES cells in different culture media. Here we describe a detailed protocol for the reproducible fabrication of PMEDSAH coating on tissue culture polystyrene dishes, and for the feeder-free culture of hES cells on PMEDSAH coating in defined culture medium. This culture system represents a key step toward the fully defined and xenogeneic-free culture of hES cells.


Assuntos
Técnicas de Cultura de Células , Células-Tronco Embrionárias/citologia , Metacrilatos/síntese química , Polímeros/síntese química , Compostos de Amônio Quaternário/síntese química , Técnicas de Cultura de Células/instrumentação , Colágeno , Meios de Cultura , Combinação de Medicamentos , Humanos , Laminina , Proteoglicanas
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