RESUMO
Intercellular exchange of protein and RNA-containing microparticles is an increasingly important mode of cell-cell communication. Here we investigate if mesenchymal stem cells (MSCs) known for secreting therapeutic paracrine factors also secrete RNA-containing microparticles. We observed that human embryonic stem cell (hESC)-derived MSC conditioned medium contained small RNAs (less than 300 nt) encapsulated in cholesterol-rich phospholipid vesicles as evidenced by their RNase sensitivity only in the presence of a sodium dodecyl sulfate-based cell lysis buffer, phospholipase A2 and a chelator of cholesterol, cyclodextrin and the restoration of their lower than expected density by detergent or phospholipase A2 treatment. MicroRNAs (miRNAs) such as hsa-let-7b and hsa-let-7g were present in a high precursor (pre)- to mature miRNA ratio by microarray analysis and quantitative reverse transcription-polymerase chain reaction. The pre-miRNAs were cleaved to mature miRNA by RNase III in vitro. High performance liquid chromatography-purified RNA-containing vesicles have a hydrodynamic radius of 55-65 nm and were readily taken up by H9C2 cardiomyocytes. This study suggests that MSCs could facilitate miRNA-mediated intercellular communication by secreting microparticles enriched for pre-miRNA.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Precursores de RNA/metabolismo , Linhagem Celular , Centrifugação com Gradiente de Concentração , Cromatografia Líquida de Alta Pressão , Humanos , MicroRNAs/química , Miócitos Cardíacos/metabolismo , Fosfolipídeos/metabolismo , Precursores de RNA/química , Ribonuclease PancreáticoRESUMO
The therapeutic effects of mesenchymal stem cells (MSCs) transplantation are increasingly thought to be mediated by MSC secretion. We have previously demonstrated that human ESC-derived MSCs (hESC-MSCs) produce cardioprotective microparticles in pig model of myocardial ischemia/reperfusion (MI/R) injury. As the safety and availability of clinical grade human ESCs remain a concern, MSCs from fetal tissue sources were evaluated as alternatives. Here we derived five MSC cultures from limb, kidney and liver tissues of three first trimester aborted fetuses and like our previously described hESC-derived MSCs; they were highly expandable and had similar telomerase activities. Each line has the potential to generate at least 10(16-19) cells or 10(7-10) doses of cardioprotective secretion for a pig model of MI/R injury. Unlike previously described fetal MSCs, they did not express pluripotency-associated markers such as Oct4, Nanog or Tra1-60. They displayed a typical MSC surface antigen profile and differentiated into adipocytes, osteocytes and chondrocytes in vitro. Global gene expression analysis by microarray and qRT-PCR revealed a typical MSC gene expression profile that was highly correlated among the five fetal MSC cultures and with that of hESC-MSCs (r(2)>0.90). Like hESC-MSCs, they produced secretion that was cardioprotective in a mouse model of MI/R injury. HPLC analysis of the secretion revealed the presence of a population of microparticles with a hydrodynamic radius of 50-65 nm. This purified population of microparticles was cardioprotective at approximately 1/10 dosage of the crude secretion.
Assuntos
Técnicas de Cultura de Células , Células-Tronco Embrionárias/citologia , Células-Tronco Mesenquimais/citologia , Animais , Antígenos/química , Diferenciação Celular , Cromatografia Líquida de Alta Pressão/métodos , Perfilação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Microesferas , Análise de Sequência com Séries de Oligonucleotídeos , Tamanho da Partícula , Traumatismo por Reperfusão/patologia , Suínos , Distribuição TecidualRESUMO
A large number of CHO glycosylation mutants were isolated by Ricinus communis agglutinin-I (RCA-I). Complementation tests revealed that all these mutant lines possessed a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. Sequencing analyses on the GnT I cDNAs isolated from 16 mutant lines led to the identification of nine different single base pair mutations. Some mutations result in a premature stop codon whereas others cause a single amino acid substitution in the GnT I protein. Interestingly, expression of the normal GnT I cDNA in mutant cells resulted in enhanced sialylation of N-glycans. The sialylation of recombinant erythropoietin (EPO) produced in mutant cells that were co-transfected with GnT I was enhanced compared to that of EPO produced in wild type CHO cells. The enhanced sialylation of EPO produced by JW152 cells in the presence of GnT I over CHO-K1 cells is a result of increased sialylated glycan structures with higher antennary branching. These findings represent a new strategy that may be utilized by the biotechnology industry to produce highly sialylated therapeutic glycoproteins.
Assuntos
Eritropoetina/metabolismo , N-Acetilglucosaminiltransferases/genética , Ácido N-Acetilneuramínico/metabolismo , Lectinas de Plantas/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Glicosilação , Mutação Puntual , Proteínas RecombinantesRESUMO
An intact-cell mass spectrometry (ICM) method using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) was evaluated for the screening of stable recombinant Chinese hamster ovary (CHO) cell lines, an important mammalian cell line in bioprocessing. With rapid and simple cell pretreatments, viabilities of cells could be rapidly distinguished on the different fingerprints of mass spectra. Detectable m/z values on cell surfaces and their relative intensities were processed by two biostatistical methods, principle components analysis (PCA) and partial least squares (PLS), with promising results. Discrimination among cell lines with different expressed recombinant proteins or different productivities could be achieved. The ICM method has the advantage of providing multiple parameters simultaneously and possesses the potential to become a powerful method for routine monitoring of bioprocesses.
Assuntos
Células CHO/química , Células CHO/citologia , Biologia Computacional/métodos , Engenharia de Proteínas/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Células CHO/metabolismo , Sobrevivência Celular , Análise por Conglomerados , Cricetinae , Cricetulus , Humanos , Interferon gama/biossíntese , Análise dos Mínimos Quadrados , Análise de Componente Principal , Proteínas RecombinantesRESUMO
Genome-scale flux analysis of Escherichia coli DH5alpha growth in a complex medium was performed to investigate the relationship between the uptake of various nutrients and their metabolic outcomes. During the exponential growth phase, we observed a sequential consumption order of serine, aspartate and glutamate in the complex medium as well as the complete consumption of key carbohydrate nutrients, glucose and trehalose. Based on the consumption and production rates of the measured metabolites, constraints-based flux analysis of a genome-scale E. coli model was then conducted to elucidate their utilization in the metabolism. The in silico analysis revealed that the cell exploited biosynthetic precursors taken up directly from the complex medium, through growth-related anabolic pathways. This suggests that the cell could be functioning in an energetically more efficient manner by reducing the energy needed to produce amino acids. The in silico simulation also allowed us to explain the observed rapid consumption of serine: excessively consumed external serine from the complex medium was mainly converted into pyruvate and glycine, which in turn, led to the acetate accumulation. The present work demonstrates the application of an in silico modeling approach to characterizing microbial metabolism under complex medium condition. This work further illustrates the use of in silico genome-scale analysis for developing better strategies related to improving microbial growth and enhancing the productivity of desirable metabolites.
Assuntos
Biologia Computacional/métodos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Acetatos/metabolismo , Aminoácidos/metabolismo , Reatores Biológicos , Simulação por Computador , Meios de Cultura , Escherichia coli/genética , Genoma Bacteriano , Glucose/metabolismo , Redes e Vias Metabólicas/fisiologia , Modelos Biológicos , Serina/metabolismoRESUMO
Advances in high-throughput techniques have led to the creation of increasing amounts of glycome data. The storage and analysis of this data would benefit greatly from a compact notation for describing glycan structures that can be easily stored and interpreted by computers. Towards this end, we propose a fixed-length alpha-numeric code for representing N-linked glycan structures commonly found in secreted glycoproteins from mammalian cell cultures. This code, GlycoDigit, employs a pre-assigned alpha-numeric index to represent the monosaccharides attached in different branches to the core glycan structure. The present branch-centric representation allows us to visualize the structure while the numerical nature of the code makes it machine readable. In addition, a difference operator can be defined to quantitatively differentiate between glycan structures for further analysis. The usefulness and applicability of GlycoDigit were demonstrated by constructing and visualizing an N-linked glycosylation network.
Assuntos
Biotecnologia/métodos , Biologia Computacional/métodos , Glicoproteínas/química , Polissacarídeos/química , Acetilglucosamina/química , Algoritmos , Animais , Células CHO , Cricetinae , Cricetulus , Galactose/química , Glicoproteínas/metabolismo , Glicosilação , Manose/química , Ácido N-Acetilneuramínico/química , SoftwareRESUMO
A CHO mutant line, MAR-11, was isolated using a cytotoxic lectin, Maackia amurensis agglutinin (MAA). This mutant has decreased levels of cell surface sialic acid relative to both wild-type CHO-K1 and Lec2 mutant CHO cells. The CMP-sialic acid transporter (CMP-SAT) gene in the MAR-11 mutant cell has a C-T mutation that results in a premature stop codon. As a result, MAR-11 cells express a truncated version of CMP-SAT which contains only 100 amino acids rather than the normal CMP-SAT which contains 336 amino acids. Biochemical analyses indicate that recombinant interferon-gamma (IFN-gamma) produced by the mutant cells lack sialic acid. Using MAR-11 as host cells, an EPO/IEF assay for the structure-function study of CMP-SAT was developed. This assay seems more sensitive than previous assays that were used to analyze sialylation in Lec2 cells. Cotransfection of constructs that express CMP-SAT into MAR-11 cells completely converted the recombinant EPO to a sialylation pattern that is similar to the EPO produced by the wild-type CHO cells. Using this assay, we showed that CMP-SAT lacking C-terminal 18 amino acids from the cytosolic tail was able to allow high levels of EPO sialylation. Substitution of the Gly residues with Ile in three different transmembrane domains of CMP-SAT resulted in dramatic decreases in transporter's activity. The CMP-SAT only lost partial activity if the same Gly residues were substituted with Ala, suggesting that the lack of side chain in Gly residues in the transmembrane domains is essential for transport activity.
Assuntos
Ácido N-Acetilneuramínico do Monofosfato de Citidina/metabolismo , Complexo de Golgi/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Simportadores/metabolismo , Animais , Sequência de Bases , Células CHO , Cricetinae , Cricetulus , DNA Complementar/genética , DNA Complementar/metabolismo , Eritropoetina/genética , Eritropoetina/metabolismo , Galectinas/metabolismo , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Dados de Sequência Molecular , Transportadores de Ânions Orgânicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Simportadores/genética , TransfecçãoRESUMO
Twelve nucleotides and seven nucleotide sugars in Chinese Hamster ovary (CHO) cells were determined by capillary electrophoresis (CE). The CE operating conditions of buffer pH value, ion strength, capillary temperature, polymer additive and cell extraction method were investigated. Optimum separation was achieved with 40 mM sodium tetraborate buffer (pH 9.5) containing 1% (w/v) polyethylene glycol (PEG) at a capillary temperature of 22 degrees C. Acetonitrile and chloroform were used for intracellular extraction. This method can be used to monitor intracellular carbohydrate metabolism.
Assuntos
Eletroforese Capilar/métodos , Açúcares de Nucleosídeo Difosfato/análise , Nucleotídeos/análise , Animais , Células CHO , Extratos Celulares/análise , Cricetinae , CricetulusRESUMO
Culturing recombinant CHO (Chinese-hamster ovary) cells at low temperatures (30-33 degrees C) increases specific recombinant protein productivity by 2-5-fold. However, even though the specific productivity is increased, cell growth is decreased in low-temperature culture such that the final recombinant protein titre remains unchanged or is even diminished, owing to the lower cell density. Exposing mammalian cells to low temperatures results in a change in the expression of many 'cold-stress' genes. CIRP (cold-inducible RNA-binding protein) is a cold-stress protein that is highly expressed at 32 degrees C, but not at 37 degrees C. In the present study we demonstrated that overexpression of CIRP at 37 degrees C can increase the recombinant-protein titre in CHO cells. Stable overexpression of CIRP at 37 degrees C improved the final titre of CHO IFN-gamma, a recombinant CHO cell line producing human IFN-gamma (interferon-gamma), by 25% in adherent culture and up to 40% in suspension culture. Real-time PCR analysis showed that the increase in the recombinant IFN-gamma titre could be attributed to increased recombinant IFN-gamma mRNA levels, while growth data showed that CIRP overexpression did not result in growth arrest in CHO IFN-gamma cells. Glycan analysis showed that the increase in IFN-gamma titre as a result of CIRP overexpression did not affect the site occupancy, glycan structures or sialic acid content of IFN-gamma. Using this strategy, the final IFN-gamma titre was increased by 40% compared with current temperature-based strategies. Furthermore, there is no decrease in cell growth or recombinant-protein glycosylation quality, as previously observed in low-temperature culture.
Assuntos
Melhoramento Genético/métodos , Interferon gama/metabolismo , Engenharia de Proteínas/métodos , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/biossíntese , Animais , Células CHO , Temperatura Baixa , Cricetinae , Cricetulus , Humanos , Interferon gama/genética , Regulação para CimaRESUMO
Human ESC-derived mesenchymal stem cell (MSC)-conditioned medium (CM) was previously shown to mediate cardioprotection during myocardial ischemia/reperfusion injury through large complexes of 50-100 nm. Here we show that these MSCs secreted 50- to 100-nm particles. These particles could be visualized by electron microscopy and were shown to be phospholipid vesicles consisting of cholesterol, sphingomyelin, and phosphatidylcholine. They contained coimmunoprecipitating exosome-associated proteins, e.g., CD81, CD9, and Alix. These particles were purified as a homogeneous population of particles with a hydrodynamic radius of 55-65 nm by size-exclusion fractionation on a HPLC. Together these observations indicated that these particles are exosomes. These purified exosomes reduced infarct size in a mouse model of myocardial ischemia/reperfusion injury. Therefore, MSC mediated its cardioprotective paracrine effect by secreting exosomes. This novel role of exosomes highlights a new perspective into intercellular mediation of tissue injury and repair, and engenders novel approaches to the development of biologics for tissue repair.
Assuntos
Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Isquemia Miocárdica/terapia , Traumatismo por Reperfusão/terapia , Animais , Antígenos CD/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cardiotônicos/uso terapêutico , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Exossomos/fisiologia , Humanos , Glicoproteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Microscopia Eletrônica , Tetraspanina 28 , Tetraspanina 29RESUMO
Glycosylation has profound effects on the quality of recombinant proteins produced in mammalian cells. The biosynthetic pathways of N-linked glycans on glycoproteins involves a relatively small number of enzymes and nucleotide sugars. Many of these glycoconjugate enzymes can utilize multiple N-glycans as substrates, thus generating a large number of glycan intermediates, and making the biosynthetic pathway resemble a network with diverging and converging paths. The N-glycans on secreted glycoprotein molecules include not only terminal glycans, but also pathway intermediates. To better assess the glycan distribution and the potential route of their synthesis, we created GlycoVis, a visualization program that displays the distribution and the potential reaction paths leading to each N-glycan on the reaction network. The substrate specificities of the enzymes involved were organized into a relationship matrix. With the input of glycan distribution data, the program outputs a reaction pathway map which labels the relative abundance levels of different glycans with different colors. The program also traces all possible reaction paths leading to each glycan and identifies each pathway on the map. Glycoform distribution of Chinese Hamster Ovary cell-derived tissue plasminogen activator (TPA), and human and mouse IgG were used as illustrations for the application of GlycoVis. In addition, the intracellular and secreted IgG from an NS0 producer cell line were isolated, and their glycoform profiles were displayed using GlycoVis for comparison. This visualization tool facilitates the analysis of potential reaction paths utilized under different physiological or culture conditions, and may provide insight on the potential targets for metabolic engineering.