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1.
Stem Cells Int ; 2019: 1375807, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30863449

RESUMO

The eukaryotic nucleus is a highly complex structure that carries out multiple functions primarily needed for gene expression, and among them, transcription seems to be the most fundamental. Diverse approaches have demonstrated that transcription takes place at discrete sites known as transcription factories, wherein RNA polymerase II (RNAP II) is attached to the factory and immobilized while transcribing DNA. It has been proposed that transcription factories promote chromatin loop formation, creating long-range interactions in which relatively distant genes can be transcribed simultaneously. In this study, we examined long-range interactions between the POU5F1 gene and genes previously identified as being POU5F1 enhancer-interacting, namely, CDYL, TLE2, RARG, and MSX1 (all involved in transcriptional regulation), in human pluripotent stem cells (hPSCs) and their early differentiated counterparts. As a control gene, RUNX1 was used, which is expressed during hematopoietic differentiation and not associated with pluripotency. To reveal how these long-range interactions between POU5F1 and the selected genes change with the onset of differentiation and upon RNAP II inhibition, we performed three-dimensional fluorescence in situ hybridization (3D-FISH) followed by computational simulation analysis. Our analysis showed that the numbers of long-range interactions between specific genes decrease during differentiation, suggesting that the transcription of monitored genes is associated with pluripotency. In addition, we showed that upon inhibition of RNAP II, long-range associations do not disintegrate and remain constant. We also analyzed the distance distributions of these genes in the context of their positions in the nucleus and revealed that they tend to have similar patterns resembling normal distribution. Furthermore, we compared data created in vitro and in silico to assess the biological relevance of our results.

2.
IEEE Trans Med Imaging ; 37(12): 2630-2641, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29994200

RESUMO

The existence of diverse image datasets accompanied by reference annotations is a crucial prerequisite for an objective benchmarking of bioimage analysis methods. Nevertheless, such a prerequisite is hard to satisfy for time lapse, multidimensional fluorescence microscopy image data, manual annotations of which are laborious and often impracticable. In this paper, we present a simulation system capable of generating 3-D time-lapse sequences of single motile cells with filopodial protrusions of user-controlled structural and temporal attributes, such as the number, thickness, length, level of branching, and lifetime of filopodia, accompanied by inherently generated reference annotations. The proposed simulation system involves three globally synchronized modules, each being responsible for a separate task: the evolution of filopodia on a molecular level, linear elastic deformation of the entire cell with filopodia, and the synthesis of realistic, time-coherent cell texture. Its flexibility is demonstrated by generating multiple synthetic 3-D time-lapse sequences of single lung cancer cells of two different phenotypes, qualitatively and quantitatively resembling their real counterparts acquired using a confocal fluorescence microscope.


Assuntos
Imageamento Tridimensional/métodos , Pseudópodes/fisiologia , Análise de Célula Única/métodos , Imagem com Lapso de Tempo/métodos , Células A549 , Humanos , Microscopia de Fluorescência/métodos
3.
Biotechnol Bioeng ; 115(4): 850-862, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29278409

RESUMO

Fibroblast growth factors (FGFs) serve numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability. Here we tackle this problem using a generalizable computer-assisted protein engineering strategy to create a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function. The data from the characterization of stabilized FGF2 showed a remarkable prediction potential of in silico methods and provided insight into the unfolding mechanism of the protein. The molecule holds a considerable promise for stem cell research and medical or pharmaceutical applications.


Assuntos
Desenho Assistido por Computador , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Engenharia de Proteínas , Estabilidade Proteica , Sequência de Aminoácidos , Animais , Simulação por Computador , Evolução Molecular Direcionada , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fator 2 de Crescimento de Fibroblastos/química , Humanos , Mutação Puntual , Dobramento de Proteína
4.
Stem Cells Transl Med ; 2(4): 246-54, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23486835

RESUMO

The generation of human pluripotent stem cells (hPSCs) of sufficient quantity and quality remains a major challenge for biomedical application. Here we present an efficient feeder-free, high-density monolayer system in which hPSCs become SSEA-3-high and gradually more viable than their feeder-dependent counterparts without changes attributed to culture adaptation. As a consequence, monolayer hPSCs possess advantages over their counterparts in embryoid body development, teratoma formation, freezing as a single-cell suspension, and colony-forming efficiency. Importantly, this monolayer culture system is reversible, preserving the competence of hPSCs to gradually reacquire features of colony growth, if necessary. Therefore, the monolayer culture system is highly suitable for long-term, large-scale propagation of hPSCs, which is necessary in drug development and pluripotent stem cell-based therapies.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes/citologia , Animais , Biomarcadores/metabolismo , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Ensaio de Unidades Formadoras de Colônias , Células Alimentadoras/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Células-Tronco Pluripotentes/metabolismo , Teratoma/patologia
5.
Stem Cells ; 31(4): 693-702, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23315699

RESUMO

The inevitable accumulation of chromosomal abnormalities in human embryonic stem cells (hESCs) during in vitro expansion represents a considerable obstacle for cell replacement therapies. To determine the source of chromosomal abnormalities, we examined hESCs maintained in culture for over 55 months for defects in telomere maintenance and DNA repair. Although prolonged culture affected neither telomerase activity nor nonhomologous end joining, the efficiency of base excision repair (BER) was significantly decreased and correlated with reduced expression of apurinic/apyrimidinic endonuclease 1 (APE1), the major nuclease required for BER. Interestingly, the expression of other BER enzymes was unchanged. Addition of human recombinant APE1 protein to nuclear extracts from late passage hESCs increased BER efficiency to the level typical of early passage hESCs. The link between BER and double-strand breaks (DSB) was demonstrated by decreased DSB release after downregulation of APE1 in early passage hESCs via siRNA. Correspondingly lower APE1 level in late passage hESC resulted in slower and less intensive but long lasting DSB release upon ionizing radiation (IR). Downregulation of APE1 in early passage hESCs also led to approximately 30% decrease in γ-H2AX signaling following IR, similar to that in late passage hESCs. We suggest that downregulation of APE1 significantly contributes to the failure of BER during long-term culture of hESCs, and further that BER failure is one of the factors affecting the genomic instability of hESCs by altering BER-dependent DSB release and cell cycle/checkpoint signaling.


Assuntos
Reparo do DNA/fisiologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Células-Tronco Embrionárias/enzimologia , Células-Tronco Embrionárias/metabolismo , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Humanos , Imuno-Histoquímica , Cariotipagem
6.
Reprod Biomed Online ; 21(5): 676-86, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20884295

RESUMO

For human embryonic stem cells (ESC) to be used in cell replacement therapies, they must be grown under good manufacturing conditions in a chemically defined medium that lacks animal proteins. This study examined the ability of a newly designed medium containing the plant-derived serum replacement VegetaCell and other reagents of human origin to support undifferentiated growth and pluripotency of human ESC. This medium was tested in several culture systems, using human fibroblasts as a feeder layer or Matrigel in a feeder-free culture. Even under the most stringent feeder-free conditions without conditioned medium, human ESC exhibited an undifferentiated morphology, expressed markers of undifferentiated cells, demonstrated high alkaline phosphatase activity and multilineage differentiation and retained a normal karyotype. Compared with human ESC grown in standard culture conditions, human ESC maintained in humanized VegetaCell medium show longer cell cycles and decreased cell death. The availability of an animal protein-free medium supplemented with the low-cost VegetaCell reagent expands the repertoire of media for culturing human ESC as well as induced pluripotent stem cells for drug testing and cell replacement therapy.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura Livres de Soro/farmacologia , Células-Tronco Pluripotentes/citologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno , Meios de Cultura , Meios de Cultivo Condicionados/farmacologia , Combinação de Medicamentos , Células-Tronco Embrionárias/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas , Laminina , Proteoglicanas
7.
Stem Cells ; 27(8): 1847-57, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19544431

RESUMO

The transcription program that is responsible for the pluripotency of human ESCs (hESCs) is believed to be comaintained by exogenous fibroblast growth factor-2 (FGF-2), which activates FGF receptors (FGFRs) and stimulates the mitogen-activated protein kinase (MAPK) pathway. However, the same pathway is stimulated by insulin receptors, insulin-like growth factor 1 receptors, and epidermal growth factor receptors. This mechanism is further complicated by intracrine FGF signals. Thus, the molecular mechanisms by which FGF-2 promotes the undifferentiated growth of hESCs are unclear. Here we show that, in undifferentiated hESCs, exogenous FGF-2 stimulated the expression of stem cell genes while suppressing cell death and apoptosis genes. Inhibition of autocrine FGF signaling caused upregulation of differentiation-related genes and downregulation of stem cell genes. Thus, exogenous FGF-2 reinforced the pluripotency maintenance program of intracrine FGF-2 signaling. Consistent with this hypothesis, expression of endogenous FGF-2 decreased during hESC differentiation and FGF-2 knockdown-induced hESC differentiation. In addition, FGF-2 signaling via FGFR2 activated MAPK kinase/extracellular signal-regulated kinase and AKT kinases, protected hESC from stress-induced cell death, and increased hESC adhesion and cloning efficiency. This stimulation of self-renewal, cell survival, and adhesion by exogenous and endogenous FGF-2 may synergize to maintain the undifferentiated growth of hESCs.


Assuntos
Células-Tronco Embrionárias/citologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Regulação para Baixo , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Ativação Enzimática , Fator 2 de Crescimento de Fibroblastos/genética , Expressão Gênica , Humanos , Immunoblotting , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Oncogênica v-akt/metabolismo , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
8.
Int J Dev Biol ; 52(4): 353-63, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18415935

RESUMO

Various types of feeder cells have been adopted for the culture of human embryonic stem cells (hESCs) to improve their attachment and provide them with stemness-supporting factors. However, feeder cells differ in their capacity to support the growth of undifferentiated hESCs. Here, we compared the expression and secretion of four well-established regulators of hESC pluripotency and/or differentiation among five lines of human foreskin fibroblasts and primary mouse embryonic fibroblasts throughout a standard hESC culture procedure. We found that human and mouse feeder cells secreted comparable levels of TGF beta 1. However, mouse feeder cells secreted larger quantities of activin A than human feeder cells. Conversely, FGF-2, which was produced by human feeder cells, could not be detected in culture media from mouse feeder cells. The quantity of BMP-4 was at about the level of detectability in media from all feeder cell types, although BMP-4 dimers were present in all feeder cells. Production of TGF beta 1, activin A, and FGF-2 varied considerably among the human-derived feeder cell lines. Low- and high-producing human feeder cells as well as mouse feeder cells were evaluated for their ability to support the undifferentiated growth of hESCs. We found that a significantly lower proportion of hESCs maintained on human feeder cell types expressed SSEA3, an undifferentiated cell marker. Moreover, SSEA3 expression and thus the pluripotent hESC compartment could be partially rescued by addition of activin A. Cumulatively, these results suggest that the ability of a feeder layer to promote the undifferentiated growth of hESCs is attributable to its characteristic growth factor production.


Assuntos
Técnicas de Cocultura/métodos , Células-Tronco Embrionárias/citologia , Ativinas/biossíntese , Ativinas/genética , Animais , Antígenos Glicosídicos Associados a Tumores/genética , Sequência de Bases , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/análise , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular , Proliferação de Células , Meios de Cultivo Condicionados/análise , Primers do DNA/genética , Células-Tronco Embrionárias/metabolismo , Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator 2 de Crescimento de Fibroblastos/genética , Expressão Gênica , Glicoesfingolipídeos/genética , Humanos , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade da Espécie , Antígenos Embrionários Estágio-Específicos , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/genética
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