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1.
Int J Mol Sci ; 24(8)2023 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-37108604

RESUMO

Autism spectrum disorder (ASD) is an umbrella term that encompasses several disabling neurodevelopmental conditions. These conditions are characterized by impaired manifestation in social and communication skills with repetitive and restrictive behaviors or interests. Thus far, there are no approved biomarkers for ASD screening and diagnosis; also, the current diagnosis depends heavily on a physician's assessment and family's awareness of ASD symptoms. Identifying blood proteomic biomarkers and performing deep blood proteome profiling could highlight common underlying dysfunctions between cases of ASD, given its heterogeneous nature, thus laying the foundation for large-scale blood-based biomarker discovery studies. This study measured the expression of 1196 serum proteins using proximity extension assay (PEA) technology. The screened serum samples included ASD cases (n = 91) and healthy controls (n = 30) between 6 and 15 years of age. Our findings revealed 251 differentially expressed proteins between ASD and healthy controls, of which 237 proteins were significantly upregulated and 14 proteins were significantly downregulated. Machine learning analysis identified 15 proteins that could be biomarkers for ASD with an area under the curve (AUC) = 0.876 using support vector machine (SVM). Gene Ontology (GO) analysis of the top differentially expressed proteins (TopDE) and weighted gene co-expression analysis (WGCNA) revealed dysregulation of SNARE vesicular transport and ErbB pathways in ASD cases. Furthermore, correlation analysis showed that proteins from those pathways correlate with ASD severity. Further validation and verification of the identified biomarkers and pathways are warranted.


Assuntos
Transtorno do Espectro Autista , Humanos , Transtorno do Espectro Autista/genética , Projetos Piloto , Proteômica , Biomarcadores/metabolismo , Proteoma/metabolismo
2.
Stem Cells ; 38(9): 1124-1136, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32510174

RESUMO

Although the application of human mesenchymal stem cells (hMSCs) to repair damaged or diseased tissues has proven relatively effective, both the donor-to-donor variability in ex vivo expansion rates and the maintenance of stemness remain a bottleneck to widespread translation. Previous work from this laboratory stratified donors into those yielding hMSCs with high- or low-growth capacity; global transcriptomic analysis revealed that high-growth-capacity hMSCs were characterized by a loss of the gene encoding glutathione S-transferase theta 1 (GSTT1). These GSTT1-null hMSCs demonstrated increased proliferative rates, clonogenic potential, and longer telomeres compared with low-growth capacity hMSCs that were GSTT1-positive. Thus, this study identifies GSTT1 as a novel genomic DNA biomarker for hMSC scalability.


Assuntos
Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Genoma Humano , Células-Tronco Mesenquimais/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Proliferação de Células/genética , Células Clonais , Genótipo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Homozigoto , Humanos , Células-Tronco Mesenquimais/metabolismo , Transcriptoma/genética
3.
Mol Cell ; 51(3): 349-59, 2013 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-23932716

RESUMO

Long noncoding RNAs (lncRNAs) are abundant in the mammalian transcriptome, and many are specifically expressed in the brain. We have identified a group of lncRNAs, including rhabdomyosarcoma 2-associated transcript (RMST), which are indispensable for neurogenesis. Here, we provide mechanistic insight into the role of human RMST in modulating neurogenesis. RMST expression is specific to the brain, regulated by the transcriptional repressor REST, and increases during neuronal differentiation, indicating a role in neurogenesis. RMST physically interacts with SOX2, a transcription factor known to regulate neural fate. RMST and SOX2 coregulate a large pool of downstream genes implicated in neurogenesis. Through RNA interference and genome-wide SOX2 binding studies, we found that RMST is required for the binding of SOX2 to promoter regions of neurogenic transcription factors. These results establish the role of RMST as a transcriptional coregulator of SOX2 and a key player in the regulation of neural stem cell fate.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Processamento Alternativo , Sítios de Ligação , Diferenciação Celular , Linhagem Celular , Proteínas Correpressoras , Proteínas de Ligação a DNA , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Neurais , Neurogênese , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , RNA Longo não Codificante/genética , RNA Interferente Pequeno
4.
Genome Res ; 27(11): 1783-1794, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29030469

RESUMO

The stochastic dynamics and regulatory mechanisms that govern differentiation of individual human neural precursor cells (NPC) into mature neurons are currently not fully understood. Here, we used single-cell RNA-sequencing (scRNA-seq) of developing neurons to dissect/identify NPC subtypes and critical developmental stages of alternative lineage specifications. This study comprises an unsupervised, high-resolution strategy for identifying cell developmental bifurcations, tracking the stochastic transcript kinetics of the subpopulations, elucidating regulatory networks, and finding key regulators. Our data revealed the bifurcation and developmental tracks of the two NPC subpopulations, and we captured an early (24 h) transition phase that leads to alternative neuronal specifications. The consequent up-regulation and down-regulation of stage- and subpopulation-specific gene groups during the course of maturation revealed biological insights with regard to key regulatory transcription factors and lincRNAs that control cellular programs in the identified neuronal subpopulations.


Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Células-Tronco Neurais/citologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Neurogênese , RNA Longo não Codificante/genética , Fatores de Transcrição/genética
5.
Neurobiol Dis ; 124: 1-13, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30391288

RESUMO

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of polyglutamine region in the androgen receptor. To gain insights into mechanisms of SBMA, four wild-type and five SBMA iPSC lines were differentiated to spinal motor neurons (sMNs) with high efficiency. SBMA sMNs showed neurite defects, reduced sMN survival and decreased protein synthesis levels. Microarray analysis revealed a dysregulation in various neuronal-related signalling pathways in SBMA sMNs. Strikingly, FAM135B a novel gene of unknown function, was found drastically downregulated in SBMA sMNs. Knockdown of FAM135B in wild-type sMNs reduced their survival and contributed to neurite defects, similar to SBMA sMNs, suggesting a functional role of FAM135B in SBMA. The degenerative phenotypes and dysregulated genes revealed could be potential therapeutic targets for SBMA.


Assuntos
Atrofia Bulboespinal Ligada ao X/metabolismo , Atrofia Bulboespinal Ligada ao X/patologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Neuritos/metabolismo , Neuritos/patologia , Atrofia Bulboespinal Ligada ao X/genética , Diferenciação Celular , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fenótipo , Transdução de Sinais
6.
Hum Mol Genet ; 26(2): 367-382, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-28365779

RESUMO

The cellular and molecular mechanisms underlying neurodevelopmental conditions such as autism spectrum disorders have been studied intensively for decades. The ability to generate patient-specific induced pluripotent stem cells (iPSCs) now offers a novel strategy for modelling human diseases. Recent studies have reported the derivation of iPSCs from patients with neurological disorders. The key challenge remains the demonstration of disease-related phenotypes and the ability to model the disease. Here we report a case study with signs of neurodevelopmental disorders (NDDs) harbouring chromosomal rearrangements that were sequenced using long-insert DNA paired-end tag (DNA-PET) sequencing approach. We identified the disruption of a specific gene, GTDC1. By deriving iPSCs from this patient and differentiating them into neural progenitor cells (NPCs) and neurons we dissected the disease process at the cellular level and observed defects in both NPCs and neuronal cells. We also showed that disruption of GTDC1 expression in wild type human NPCs and neurons showed a similar phenotype as patient's iPSCs. Finally, we utilized a zebrafish model to demonstrate a role for GTDC1 in the development of the central nervous system. Our findings highlight the importance of combining sequencing technologies with the iPSC technology for NDDs modelling that could be applied for personalized medicine.


Assuntos
Transtorno do Espectro Autista/genética , Glicosiltransferases/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/patologia , Diferenciação Celular/genética , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Genoma Humano , Glicosiltransferases/biossíntese , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Neurais/patologia , Neurônios/metabolismo , Neurônios/patologia , Medicina de Precisão , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
7.
EMBO J ; 33(11): 1271-83, 2014 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-24802670

RESUMO

Several transcription factors (TFs) have been implicated in neuroectoderm (NE) development, and recently, the TF PAX6 was shown to be critical for human NE specification. However, microRNA networks regulating human NE development have been poorly documented. We hypothesized that microRNAs activated by PAX6 should promote NE development. Using a genomics approach, we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells (hESC). PAX6 binding to active enhancers was found in the proximity of several microRNAs, including hsa-miR-135b. MiR-135b was activated during NE development, and ectopic expression of miR-135b in hESC promoted differentiation toward NE. MiR-135b promotes neural conversion by targeting components of the TGF-ß and BMP signaling pathways, thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-miRNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , MicroRNAs/genética , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Sítios de Ligação , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas do Olho/genética , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , MicroRNAs/metabolismo , Modelos Moleculares , Mutação , Placa Neural , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/genética , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética
8.
Mol Ther ; 25(2): 342-355, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28153087

RESUMO

Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 enables us to generate targeted sequence changes in the genomes of cells and organisms. However, off-target effects have been a persistent problem hampering the development of therapeutics based on CRISPR/Cas9 and potentially confounding research results. Efforts to improve Cas9 specificity, like the development of RNA-guided FokI-nucleases (RFNs), usually come at the cost of editing efficiency and/or genome targetability. To overcome these limitations, we engineered improved chimeras of RFNs that enable higher cleavage efficiency and provide broader genome targetability, while retaining high fidelity for genome editing in human cells. Furthermore, we developed a new RFN ortholog derived from Staphylococcus aureus Cas9 and characterize its utility for efficient genome engineering. Finally, we demonstrate the feasibility of RFN orthologs to functionally hetero-dimerize to modify endogenous genes, unveiling a new dimension of RFN target design opportunities.


Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Edição de Genes , Engenharia de Proteínas , RNA Guia de Cinetoplastídeos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Endonucleases/metabolismo , Variação Genética , Humanos , Modelos Biológicos , Mutação , Células-Tronco Pluripotentes/metabolismo , Ligação Proteica , Multimerização Proteica , Fatores de Transcrição de Fator Regulador X/química , Fatores de Transcrição de Fator Regulador X/genética
9.
BMC Genomics ; 18(1): 383, 2017 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-28514956

RESUMO

BACKGROUND: Development of primary open-angle glaucoma (POAG) is associated with the malfunctioning trabecular meshwork (TM). Cell therapy offers great potential for the treatment of POAG, but requires the generation of functional TM cells in vitro to replace the lost/dysfunctional cells. TM differentiation in vitro from various stem cell types must be monitored by the expression of specific markers. However, no single definitive marker of the TM has been identified. RESULTS: To identify robust markers of TM differentiation, we performed global transcriptome profiling using high-density oligonucleotide microarray on ex vivo TM tissue and cultured TM progenitors. Corneal and scleral tissues were also used in the analysis. After removal of genes expressed in the cornea and sclera, 18 genes were identified that were differentially expressed in the TM relative to the other samples. CDH23, F5, KCNAB1, FGF9, SPP1, and HEY1 were selected among the genes highly expressed in the TM, together with BDNF which was repressed, compared to progenitors for further investigation. Expression analysis by qPCR verified the differential expression and immunofluorescence of the anterior segment confirmed strong expression in the TM. CONCLUSIONS: Three independent cohort of expression studies have identified novel markers, fitting in identifying TM cells and in evaluating directed TM differentiation in vitro.


Assuntos
Diferenciação Celular/genética , Perfilação da Expressão Gênica , Malha Trabecular/citologia , Malha Trabecular/metabolismo , Idoso , Biomarcadores/metabolismo , Córnea/metabolismo , Humanos , Pessoa de Meia-Idade , Esclera/metabolismo , Células-Tronco/citologia
10.
EMBO J ; 32(7): 938-53, 2013 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-23474895

RESUMO

How regulatory information is encoded in the genome is poorly understood and poses a challenge when studying biological processes. We demonstrate here that genomic redistribution of Oct4 by alternative partnering with Sox2 and Sox17 is a fundamental regulatory event of endodermal specification. We show that Sox17 partners with Oct4 and binds to a unique 'compressed' Sox/Oct motif that earmarks endodermal genes. This is in contrast to the pluripotent state where Oct4 selectively partners with Sox2 at 'canonical' binding sites. The distinct selection of binding sites by alternative Sox/Oct partnering is underscored by our demonstration that rationally point-mutated Sox17 partners with Oct4 on pluripotency genes earmarked by the canonical Sox/Oct motif. In an endodermal differentiation assay, we demonstrate that the compressed motif is required for proper expression of endodermal genes. Evidently, Oct4 drives alternative developmental programs by switching Sox partners that affects enhancer selection, leading to either an endodermal or pluripotent cell fate. This work provides insights in understanding cell fate transcriptional regulation by highlighting the direct link between the DNA sequence of an enhancer and a developmental outcome.


Assuntos
Embrião de Mamíferos/embriologia , Endoderma/embriologia , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas HMGB/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXF/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Diferenciação Celular/fisiologia , Linhagem Celular , Embrião de Mamíferos/citologia , Endoderma/citologia , Proteínas HMGB/genética , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXF/genética , Transcrição Gênica/fisiologia
11.
Stem Cells ; 34(1): 124-34, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26507573

RESUMO

The transcription factor REST is a key suppressor of neuronal genes in non-neuronal tissues. REST has been shown to suppress proneuronal microRNAs in neural progenitors indicating that REST-mediated neurogenic suppression may act in part via microRNAs. We used neural differentiation of Rest-null mouse ESC to identify dozens of microRNAs regulated by REST during neural development. One of the identified microRNAs, miR-375, was upregulated during human spinal motor neuron development. We found that miR-375 facilitates spinal motor neurogenesis by targeting the cyclin kinase CCND2 and the transcription factor PAX6. Additionally, miR-375 inhibits the tumor suppressor p53 and protects neurons from apoptosis in response to DNA damage. Interestingly, motor neurons derived from a spinal muscular atrophy patient displayed depressed miR-375 expression and elevated p53 protein levels. Importantly, SMA motor neurons were significantly more susceptible to DNA damage induced apoptosis suggesting that miR-375 may play a protective role in motor neurons.


Assuntos
MicroRNAs/genética , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Degeneração Neural/patologia , Animais , Apoptose/genética , Sequência de Bases , Humanos , Camundongos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Atrofia Muscular Espinal/genética , Degeneração Neural/genética , Neurogênese/genética , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/metabolismo
12.
Stem Cells ; 34(4): 860-72, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26864965

RESUMO

During development, lineage specification is controlled by several signaling pathways involving various transcription factors (TFs). Here, we studied the RE-1-silencing transcription factor (REST) and identified an important role of this TF in cardiac differentiation. Using mouse embryonic stem cells (ESC) to model development, we found that REST knockout cells lost the ability to differentiate into the cardiac lineage. Detailed analysis of specific lineage markers expression showed selective downregulation of endoderm markers in REST-null cells, thus contributing to a loss of cardiogenic signals. REST regulates cardiac differentiation of ESCs by negatively regulating the Wnt/ß-catenin signaling pathway and positively regulating the cardiogenic TF Gata4. We propose here a new role for REST in cell fate specification besides its well-known repressive role of neuronal differentiation.


Assuntos
Diferenciação Celular/genética , Fator de Transcrição GATA4/biossíntese , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Repressoras/genética , Animais , Linhagem da Célula/genética , Fator de Transcrição GATA4/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Via de Sinalização Wnt
13.
Trends Genet ; 29(8): 461-8, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23562612

RESUMO

The central nervous system (CNS) is a complex biological system composed of numerous cell types working in concert. The intricate development and functioning of this highly ordered structure depends upon exquisite spatial and temporal control of gene expression in the cells comprising the CNS. Thus, gene regulatory networks that control cell fates and functions play critical roles in the CNS. Failure to develop and maintain intricate regulatory networks properly leads to impaired development or neural dysfunction, which might manifest as neurological disorders. Long noncoding RNAs (lncRNAs) are emerging as important components of gene regulatory networks, working in concert with transcription factors and epigenetic regulators of gene expression. Interestingly, many lncRNAs are highly expressed in the adult and developing brain, often showing precise temporal and spatial patterns of expression. This specificity of expression and growing awareness of the importance of lncRNAs suggest that they play key roles in CNS development and function. In this review, we highlight the growing evidence for the importance of lncRNAs in the CNS and the indications that their dysregulation underlies some neurological disorders.


Assuntos
Sistema Nervoso Central/fisiopatologia , RNA Longo não Codificante/genética , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiopatologia , Sistema Nervoso Central/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Humanos , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/fisiopatologia , Transcrição Gênica
14.
EMBO J ; 31(3): 522-33, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22193719

RESUMO

Long non-coding RNAs (lncRNAs) are a numerous class of newly discovered genes in the human genome, which have been proposed to be key regulators of biological processes, including stem cell pluripotency and neurogenesis. However, at present very little functional characterization of lncRNAs in human differentiation has been carried out. In the present study, we address this using human embryonic stem cells (hESCs) as a paradigm for pluripotency and neuronal differentiation. With a newly developed method, hESCs were robustly and efficiently differentiated into neurons, and we profiled the expression of thousands of lncRNAs using a custom-designed microarray. Some hESC-specific lncRNAs involved in pluripotency maintenance were identified, and shown to physically interact with SOX2, and PRC2 complex component, SUZ12. Using a similar approach, we identified lncRNAs required for neurogenesis. Knockdown studies indicated that loss of any of these lncRNAs blocked neurogenesis, and immunoprecipitation studies revealed physical association with REST and SUZ12. This study indicates that lncRNAs are important regulators of pluripotency and neurogenesis, and represents important evidence for an indispensable role of lncRNAs in human brain development.


Assuntos
Diferenciação Celular/genética , Cromatina/metabolismo , Neurônios/citologia , RNA não Traduzido/fisiologia , Fatores de Transcrição/metabolismo , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , RNA não Traduzido/genética , RNA não Traduzido/metabolismo
15.
Stem Cells ; 33(7): 2126-34, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25809870

RESUMO

The variant histone protein H2A.Z plays a critical role in early development. Likewise, Nanog, a master regulator of embryonic stem cells (ESCs), is essential for proper development in early embryogenesis. In this study, we establish that these two factors work together to maintain pluripotency. It is shown that H2A.Z influences the protein level of Nanog through the ubiquitin-proteasome pathway. Knockdown of H2A.Z causes differentiation of mouse ESCs and disrupts the reprogramming of somatic cells, which can be partially rescued by overexpression of Nanog. We conclude that the H2A.Z-Nanog partnership is involved in ESC pluripotency and reprogramming of somatic cells. Stem Cells 2015;33:2126-2134.


Assuntos
Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Animais , Diferenciação Celular , Humanos , Camundongos , Proteína Homeobox Nanog , Células-Tronco Pluripotentes/metabolismo
16.
Stem Cells ; 33(6): 1878-91, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25752682

RESUMO

This study sought to identify critical determinants of mesenchymal stem cell (MSC) potency using in vitro and in vivo attributes of cells isolated from the bone marrow of age- and sex-matched donors. Adherence to plastic was not indicative of potency, yet capacity for long-term expansion in vitro varied considerably between donors, allowing the grouping of MSCs from the donors into either those with high-growth capacity or low-growth capacity. Using this grouping strategy, high-growth capacity MSCs were smaller in size, had greater colony-forming efficiency, and had longer telomeres. Cell-surface biomarker analysis revealed that the International Society for Cellular Therapy (ISCT) criteria did not distinguish between high-growth capacity and low-growth capacity MSCs, whereas STRO-1 and platelet-derived growth factor receptor alpha were preferentially expressed on high-growth capacity MSCs. These cells also had the highest mean expression of the mRNA transcripts TWIST-1 and DERMO-1. Irrespective of these differences, both groups of donor MSCs produced similar levels of key growth factors and cytokines involved in tissue regeneration and were capable of multilineage differentiation. However, high-growth capacity MSCs produced approximately double the volume of mineralized tissue compared to low-growth capacity MSCs when assessed for ectopic bone-forming ability. The additional phenotypic criteria presented in this study when combined with the existing ISCT minimum criteria and working proposal will permit an improved assessment of MSC potency and provide a basis for establishing the quality of MSCs prior to their therapeutic application.


Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células-Tronco Mesenquimais/citologia , Animais , Células Cultivadas , Citocinas/metabolismo , Humanos , Camundongos , Cicatrização/fisiologia
18.
PLoS Genet ; 9(10): e1003852, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24204288

RESUMO

Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.


Assuntos
Padronização Corporal/genética , Proteínas de Homeodomínio/genética , Elementos Reguladores de Transcrição/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Animais , Sítios de Ligação , Regulação da Expressão Gênica no Desenvolvimento , Genômica , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismo
19.
J Neurosci Res ; 93(8): 1203-14, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25691247

RESUMO

Adult neural stem cell (aNSC) activity is tuned by external stimuli through the recruitment of transcription factors. This study examines the RE1 silencing transcription factor (REST) in neural stem/progenitor cells isolated from the subventricular zone of adult mouse brain and provides the first extensive characterization of REST-mediated control of the cellular and molecular properties. This study shows that REST knockdown affects the capacity of progenitor cells to generate neurospheres, reduces cell proliferation, and triggers cell differentiation despite the presence of growth factors. Genome- and transcriptome-wide analyses show that REST binding sites are significantly enriched in genes associated with synaptic transmission and nervous system development and function. Seeking candidate regulators of aNSC function, this study identifies a member of the bone morphogenetic protein (BMP) family, BMP6, the mRNA and protein of which increased after REST knockdown. The results of this study extend previous findings, demonstrating a reciprocal control of REST expression by BMPs. Administration of exogenous BMP6 inhibits aNSC proliferation and induces the expression of the astrocytic marker glial fibrillary acidic protein, highlighting its antimitogenic and prodifferentiative effects. This study suggests that BMP6 produced in a REST-regulated manner together with other signals can contribute to regulation of NSC maintenance and fate.


Assuntos
Células-Tronco Adultas/fisiologia , Inativação Gênica/fisiologia , Ventrículos Laterais/citologia , Ventrículos Laterais/fisiologia , Células-Tronco Neurais/fisiologia , Proteínas Repressoras/fisiologia , Animais , Proteína Morfogenética Óssea 6/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Masculino , Camundongos , Fatores de Transcrição/fisiologia
20.
Stem Cells ; 32(3): 609-22, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24549637

RESUMO

An important goal in stem cell biology is to develop methods for efficient generation of clinically interesting cell types from relevant stem cell populations. This is particularly challenging for different types of neurons of the central nervous system where hundreds of distinct neuronal cell types are generated during embryonic development. We previously used a strategy based on forced transcription factor expression in embryonic stem cell-derived neural progenitors to generate specific types of neurons, including dopamine and serotonin neurons. Here, we extend these studies and show that noradrenergic neurons can also be generated from pluripotent embryonic stem cells by forced expression of the homeobox transcription factor Phox2b under the signaling influence of fibroblast growth factor 8 (FGF8) and bone morphogenetic proteins. In neural progenitors exposed to FGF8 and sonic hedgehog both Phox2b and the related Phox2a instead promoted the generation of neurons with the characteristics of mid- and hindbrain motor neurons. The efficient generation of these neuron types enabled a comprehensive genome-wide gene expression analysis that provided further validation of the identity of generated cells. Moreover, we also demonstrate that the generated cell types are amenable to drug testing in vitro and we show that variants of the differentiation protocols can be applied to cultures of human pluripotent stem cells for the generation of human noradrenergic and visceral motor neurons. Thus, these studies provide a basis for characterization of yet an additional highly clinically relevant neuronal cell type.


Assuntos
Neurônios Adrenérgicos/citologia , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Neurônios Motores/citologia , Fatores de Transcrição/metabolismo , Neurônios Adrenérgicos/metabolismo , Animais , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Engenharia Genética , Genoma/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Neurônios Motores/metabolismo , Transdução de Sinais
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