Phenotypic and global gene expression profile changes between low passage and high passage MIN-6 cells

The long-term potential to routinely use replacement β cells/islets as cell therapy for type 1 diabetes relies on our ability to culture such cells/islets, in vitro, while maintaining their functional status. Previous β cell studies, by ourselves and other researchers, have indicated that the glucose-stimulated insulin secretion (GSIS) phenotype is relatively unstable, in long-term culture. This study aimed to investigate phenotypic and gene expression changes associated with this loss of GSIS, using the MIN-6 cell line as model. Phenotypic differences between MIN-6(L, low passage) and MIN-6(H, high passage) were determined by ELISA (assessing GSIS and cellular (pro)insulin content), proliferation assays, phase contrast light microscopy and analysis of alkaline phosphatase expression. Differential mRNA expression was investigated using microarray, bioinformatics and real-time PCR technologies. Long-term culture was found to be associated with many phenotypic changes, including changes in growth rate and cellular morphology, as well as loss of GSIS. Microarray analyses indicate expression of many mRNAs, including many involved in regulated secretion, adhesion and proliferation, to be significantly affected by passaging/ long-term culture. Loss/reduced levels, in high passage cells, of certain transcripts associated with the mature β cell, together with increased levels of neuron/glia-associated mRNAs, suggest that, with time in culture, MIN-6 cells may revert to an early (possibly multi-potential), poorly differentiated, ‘precursor-like’ cell type. This observation is supported by increased expression of the stem cell marker, alkaline phosphatase.

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Long-term passage of tachyzoites in tissue culture can attenuate virulence of Neospora caninum in vivo

Parasitology ◽

10.1017/s0031182006000539 ◽

2006 ◽

Vol 133 (4) ◽

pp. 421-432 ◽

Cited By ~ 47

Author(s):

P. M. BARTLEY ◽

S. WRIGHT ◽

J. SALES ◽

F. CHIANINI ◽

D. BUXTON ◽

...

Keyword(s):

Tissue Culture ◽

Body Weight ◽

Growth Rates ◽

Clinical Symptoms ◽

Neospora Caninum ◽

High Passage ◽

Low Passage

To determine whether prolonged in vitro passage would result in attenuation of virulence in vivo, Neospora caninum tachyzoites were passaged for different lengths of time in vitro and compared for their ability to cause disease in mice. Groups of Balb/c mice were inoculated intraperitoneally with 5×106 or 1×107 of low-passage or high-passage N. caninum tachyzoites. The mice were monitored for changes in their demeanour and body weight, and were culled when severe clinical symptoms of murine neosporosis were observed. Mice inoculated with the high-passage parasites survived longer (P<0·05), and showed fewer clinical symptoms of murine neosporosis, compared to the mice receiving the low-passage parasites. The parasite was detected in the brains of inoculated mice using immunohistochemistry and ITS1 PCR. Tissue cysts containing parasites were seen in mice inoculated with both low-passage and high-passage parasites. When the in vitro growth rates of the parasites were compared, the high-passage parasites initially multiplied more rapidly (P<0·001) than the low-passage parasites, suggesting that the high-passage parasites had become more adapted to tissue culture. These results would suggest that it is possible to attenuate the virulence of N. caninum tachyzoites in mice through prolonged in vitro passage.

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qPCR analysis of mesenchymal stem cell marker expression during the long-term culture of canine adipocyte derived stem cells

Medical Journal of Cell Biology ◽

10.2478/acb-2020-0017 ◽

2020 ◽

Vol 8 (4) ◽

pp. 139-145

Author(s):

Rut Bryl ◽

Claudia Dompe ◽

Maurycy Jankowski ◽

Katarzyna Stefańska ◽

Afsaneh Golkar Narenji ◽

...

Keyword(s):

Stem Cells ◽

Molecular Mechanisms ◽

Cultured Cells ◽

Stem Cell Marker ◽

Pcr Analysis ◽

Marker Genes ◽

Long Term Culture ◽

Marker Expression

AbstractDue to its availability and accessibility, adipose tissue has been the subject of various studies in many different medical fields and is believed to be a useful source of stem cells. The ability of ASCs to differentiate towards different cell lineages, with possibility of directing this differentiation, increases their possible clinical applications, and they have been widely employed in multiple therapies and treatment of different pathologies. However, a deeper understanding of the molecular mechanisms underlying the ASCs osteoblastic and chondrocyte differentiation may lead to novel applications treating a multitude of different bone-related diseases through techniques more likely meeting worldwide consensus. In this study, the RT-qPCR method was used to determine the changes in expression of ASC specific markers (CD105, CD73, CD14, CD34, CD90 and CD45) before and after long-term (14-day) in vitro cultures. To confirm the identity of the investigated cells, flow cytometry was used to evaluate the presence of positive (CD44, CD90) and negative (CD45, CD34) ASC markers. Overall, the results of the PCR analysis showed a significant change in expression of most of the marker genes, indicating significant changes in the cultured cells caused by their long-term culture, potentially altering their original stem-like characteristics.Running title: ASC marker expression during long-term in vitro culture

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Differential Gene Expression Profiling of Enriched Human Spermatogonia after Short- and Long-Term Culture

BioMed Research International ◽

10.1155/2014/138350 ◽

2014 ◽

Vol 2014 ◽

pp. 1-17 ◽

Cited By ~ 15

Author(s):

Sabine Conrad ◽

Hossein Azizi ◽

Maryam Hatami ◽

Mikael Kubista ◽

Michael Bonin ◽

...

Keyword(s):

Gene Expression ◽

Germ Cell ◽

Cultured Cells ◽

Embryonic Stem ◽

Molecular Signature ◽

Molecular Profile ◽

Short Term ◽

Long Term Culture

This study aimed to provide a molecular signature for enriched adult human stem/progenitor spermatogonia during short-term (<2 weeks) and long-term culture (up to more than 14 months) in comparison to human testicular fibroblasts and human embryonic stem cells. Human spermatogonia were isolated by CD49f magnetic activated cell sorting and collagen−/laminin+matrix binding from primary testis cultures obtained from ten adult men. For transcriptomic analysis, single spermatogonia-like cells were collected based on their morphology and dimensions using a micromanipulation system from the enriched germ cell cultures. Immunocytochemical, RT-PCR and microarray analyses revealed that the analyzed populations of cells were distinct at the molecular level. The germ- and pluripotency-associated genes and genes of differentiation/spermatogenesis pathway were highly expressed in enriched short-term cultured spermatogonia. After long-term culture, a proportion of cells retained and aggravated the “spermatogonial” gene expression profile with the expression of germ and pluripotency-associated genes, while in the majority of long-term cultured cells this molecular profile, typical for the differentiation pathway, was reduced and more genes related to the extracellular matrix production and attachment were expressed. The approach we provide here to study the molecular status ofin vitrocultured spermatogonia may be important to optimize the culture conditions and to evaluate the germ cell plasticity in the future.

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The influence of titanium surfaces treated by alkalis on macrophage and osteoblast-like cell adhesion and gene expression in vitro

RSC Advances ◽

10.1039/c5ra10701f ◽

2015 ◽

Vol 5 (99) ◽

pp. 81378-81387 ◽

Cited By ~ 6

Author(s):

Ting Ma ◽

Xi-Yuan Ge ◽

Sheng-Nan Jia ◽

Xi Jiang ◽

Yu Zhang ◽

...

Keyword(s):

Gene Expression ◽

Alkaline Phosphatase ◽

Cell Adhesion ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Related Gene ◽

Titanium Surfaces

The effect of alkali-treated titanium surfaces on inflammation-related gene expression of macrophages and alkaline phosphatase activity of osteoblast-like cells.

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Slush nitrogen vitrification of human ovarian tissue does not alter gene expression and improves follicle health and progression in long-term in vitro culture

Fertility and Sterility ◽

10.1016/j.fertnstert.2018.08.020 ◽

2018 ◽

Vol 110 (7) ◽

pp. 1356-1366 ◽

Cited By ~ 3

Author(s):

Vincenza Barbato ◽

Roberto Gualtieri ◽

Teresa Capriglione ◽

Maria Michela Pallotta ◽

Sabrina Braun ◽

...

Keyword(s):

Gene Expression ◽

In Vitro Culture ◽

Ovarian Tissue ◽

Human Ovarian Tissue

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Simulated microgravity using the Random Positioning Machine inhibits differentiation and alters gene expression profiles of 2T3 preosteoblasts

AJP Cell Physiology ◽

10.1152/ajpcell.00222.2004 ◽

2005 ◽

Vol 288 (6) ◽

pp. C1211-C1221 ◽

Cited By ~ 94

Author(s):

Steven J. Pardo ◽

Mamta J. Patel ◽

Michelle C. Sykes ◽

Manu O. Platt ◽

Nolan L. Boyd ◽

...

Keyword(s):

Gene Expression ◽

Alkaline Phosphatase ◽

Bone Loss ◽

Simulated Microgravity ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Underlying Mechanism ◽

Bone Biology ◽

Random Positioning Machine

Exposure to microgravity causes bone loss in humans, and the underlying mechanism is thought to be at least partially due to a decrease in bone formation by osteoblasts. In the present study, we examined the hypothesis that microgravity changes osteoblast gene expression profiles, resulting in bone loss. For this study, we developed an in vitro system that simulates microgravity using the Random Positioning Machine (RPM) to study the effects of microgravity on 2T3 preosteoblast cells grown in gas-permeable culture disks. Exposure of 2T3 cells to simulated microgravity using the RPM for up to 9 days significantly inhibited alkaline phosphatase activity, recapitulating a bone loss response that occurs in real microgravity conditions without altering cell proliferation and shape. Next, we performed DNA microarray analysis to determine the gene expression profile of 2T3 cells exposed to 3 days of simulated microgravity. Among 10,000 genes examined using the microarray, 88 were downregulated and 52 were upregulated significantly more than twofold using simulated microgravity compared with the static 1-g condition. We then verified the microarray data for some of the genes relevant in bone biology using real-time PCR assays and immunoblotting. We confirmed that microgravity downregulated levels of alkaline phosphatase, runt-related transcription factor 2, osteomodulin, and parathyroid hormone receptor 1 mRNA; upregulated cathepsin K mRNA; and did not significantly affect bone morphogenic protein 4 and cystatin C protein levels. The identification of gravisensitive genes provides useful insight that may lead to further hypotheses regarding their roles in not only microgravity-induced bone loss but also the general patient population with similar pathological conditions, such as osteoporosis.

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Eaf1 Links the NuA4 Histone Acetyltransferase Complex to Htz1 Incorporation and Regulation of Purine Biosynthesis

Eukaryotic Cell ◽

10.1128/ec.00004-15 ◽

2015 ◽

Vol 14 (6) ◽

pp. 535-544 ◽

Cited By ~ 9

Author(s):

Xue Cheng ◽

Andréanne Auger ◽

Mohammed Altaf ◽

Simon Drouin ◽

Eric Paquet ◽

...

Keyword(s):

Gene Expression ◽

Cross Talk ◽

Global Gene Expression ◽

Histone Variant ◽

Purine Biosynthesis ◽

Expression Array ◽

Link Type ◽

Molecular Events ◽

Nucleosome Disassembly

ABSTRACT Proper modulation of promoter chromatin architecture is crucial for gene regulation in order to precisely and efficiently orchestrate various cellular activities. Previous studies have identified the stimulatory effect of the histone-modifying complex NuA4 on the incorporation of the histone variant H2A.Z (Htz1) at the PHO5 promoter (A. Auger, L. Galarneau, M. Altaf, A. Nourani, Y. Doyon, R. T. Utley, D. Cronier, S. Allard, and J. Côté, Mol Cell Biol 28:2257–2270, 2008, http://dx.doi.org/10.1128/MCB.01755-07 ). In vitro studies with a reconstituted system also indicated an intriguing cross talk between NuA4 and the H2A.Z-loading complex, SWR-C (M. Altaf, A. Auger, J. Monnet-Saksouk, J. Brodeur, S. Piquet, M. Cramet, N. Bouchard, N. Lacoste, R. T. Utley, L. Gaudreau, J. Côté, J Biol Chem 285:15966–15977, 2010, http://dx.doi.org/10.1074/jbc.M110.117069 ). In this work, we investigated the role of the NuA4 scaffold subunit Eaf1 in global gene expression and genome-wide incorporation of Htz1. We found that loss of Eaf1 affects Htz1 levels mostly at the promoters that are normally highly enriched in the histone variant. Analysis of eaf1 mutant cells by expression array unveiled a relationship between NuA4 and the gene network implicated in the purine biosynthesis pathway, as EAF1 deletion cripples induction of several ADE genes. NuA4 directly interacts with Bas1 activation domain, a key transcription factor of adenine genes. Chromatin immunoprecipitation (ChIP) experiments demonstrate that nucleosomes on the inactive ADE17 promoter are acetylated already by NuA4 and enriched in Htz1. Upon derepression, these poised nucleosomes respond rapidly to activate ADE gene expression in a mechanism likely reminiscent of the PHO5 promoter, leading to nucleosome disassembly. These detailed molecular events depict a specific case of cross talk between NuA4-dependent acetylation and incorporation of histone variant Htz1, presetting the chromatin structure over ADE promoters for subsequent chromatin remodeling and activated transcription.

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Growth, Drug Susceptibility, and Gene Expression Profiling of Plasmodium falciparum Cultured in Medium Supplemented with Human Serum

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057107310638 ◽

2007 ◽

Vol 12 (8) ◽

pp. 1109-1114 ◽

Cited By ~ 14

Author(s):

Kshipra Singh ◽

Ameeta Agarwal ◽

Shabana I. Khan ◽

Larry A. Walker ◽

Babu L. Tekwani

Keyword(s):

Gene Expression ◽

Plasmodium Falciparum ◽

Human Serum ◽

Global Gene Expression ◽

Drug Susceptibility ◽

Growth Cycle ◽

In Vitro Cultivation ◽

Human Malaria Parasite ◽

In Vitro Drug Susceptibility

In vitro cultivation of Plasmodium falciparum has been extremely useful in understanding the biology of the human malaria parasite as well as research on the discovery of new antimalarial drugs and vaccines. A chemically defined serum-free medium supplemented with lipid-rich bovine serum albumin (AlbuMAX I) offers the following advantages over human serum-supplemented media for the in vitro culture of P. falciparum: 1) improved growth profile, with more than a 2-fold higher yield of the parasites at any stage of the growth cycle; 2) suitability for in vitro antimalarial screening, as the parasites grown in AlbuMAX and human serum-supplemented media show similar sensitivity to standard and novel antimalarials as well as natural product extracts in the in vitro drug susceptibility assays; and 3) DNA microarray analysis comparing the global gene expression profile of sorbitol-synchronized P. falciparum trophozoites grown in the 2 different media, indicating minimal differences. ( Journal of Biomolecular Screening 2007:1109-1114)

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The neutrophil-mobilizing cytokine interleukin-26 in the airways of long-term tobacco smokers

Clinical Science ◽

10.1042/cs20180057 ◽

2018 ◽

Vol 132 (9) ◽

pp. 959-983 ◽

Cited By ~ 7

Author(s):

Karlhans Fru Che ◽

Ellen Tufvesson ◽

Sara Tengvall ◽

Elisa Lappi-Blanco ◽

Riitta Kaarteenaho ◽

...

Keyword(s):

Gene Expression ◽

Chronic Bronchitis ◽

Pathogenic Bacteria ◽

Cell Model ◽

Water Soluble ◽

Chronic Obstructive ◽

Neutrophil Accumulation

Long-term tobacco smokers with chronic obstructive pulmonary disease (COPD) or chronic bronchitis display an excessive accumulation of neutrophils in the airways; an inflammation that responds poorly to established therapy. Thus, there is a need to identify new molecular targets for the development of effective therapy. Here, we hypothesized that the neutrophil-mobilizing cytokine interleukin (IL)-26 (IL-26) is involved in airway inflammation amongst long-term tobacco smokers with or without COPD, chronic bronchitis or colonization by pathogenic bacteria. By analyzing bronchoalveolar lavage (BAL), bronchail wash (BW) and induced sputum (IS) samples, we found increased extracellular IL-26 protein in the airways of long-term smokers in vivo without further increase amongst those with clinically stable COPD. In human alveolar macrophages (AM) in vitro, the exposure to water-soluble tobacco smoke components (WTC) enhanced IL-26 gene and protein. In this cell model, the same exposure increased gene expression of the IL-26 receptor complex (IL10R2 and IL20R1) and nuclear factor κ B (NF-κB); a proven regulator of IL-26 production. In the same cell model, recombinant human IL-26 in vitro caused a concentration-dependent increase in the gene expression of NF-κB and several pro-inflammatory cytokines. In the long-term smokers, we also observed that extracellular IL-26 protein in BAL samples correlates with measures of lung function, tobacco load, and several markers of neutrophil accumulation. Extracellular IL-26 was further increased in long-term smokers with exacerbations of COPD (IS samples), with chronic bronchitis (BAL samples ) or with colonization by pathogenic bacteria (IS and BW samples). Thus, IL-26 in the airways emerges as a promising target for improving the understanding of the pathogenic mechanisms behind several pulmonary morbidities in long-term tobacco smokers.

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Construction and optimization of herpes simplex virus vectors for central nervous system gene delivery based on CRISPR/Cas9-mediated genome editing

Current Gene Therapy ◽

10.2174/1566523219666210618154326 ◽

2021 ◽

Vol 21 ◽

Author(s):

Xinwei Huang ◽

Xiuqing Li ◽

Lijuan Yang ◽

Pengfei Wang ◽

Jingyuan Yan ◽

...

Keyword(s):

Gene Expression ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Transgene Expression ◽

Mouse Hippocampus ◽

Simplex Virus ◽

Hsv 1

Aims: We aim to define parameters affecting the safety and long-term transgene expression of attenuated HSV-1 vectors and optimize the expression cassettes to achieve robust and sustained expression in CNS. Background: Engineered, attenuated Herpes simplex virus (HSV) vectors are promising vehicles for gene delivery to the peripheral and central nervous systems. The virus latent promoter (LAP) is commonly used to drive exogenous gene expression; however, parameters affecting the safety and long-term transgene expression of attenuated HSV-1 vectors have not been fully understood. Objective: This study aimed to construct attenuated HSV-1 vectors using the CRISPR-Cas9 system and examine the influence of transgene cassette construction and insertion site on transgene expression and vector safety. Method: In this study, we used a CRISPR-Cas9 system to accurately and efficiently edit attenuated HSV-1 strain 1716, and constructed two series of recombinant virus LMR and LMRx with different sets of gene cassettes insertion in Exon1(LAP2) and 2.0 kb intron downstream of LAP, respectively. The transgene expression and viral gene transcriptional kinetics were compared in in-vitro cell lines. The reporter gene expression and safety profiles of each vector were further evaluated in the mouse hippocampus gene transduction model. Result: The in-vitro cell line analysis indicated that the insertion of a gene expression cassette would disrupt virus gene transcription. Mouse hippocampus transducing analysis suggested that complete expression cassette insertion at 2.0 kb intron could achieve robust and longtime gene expression than the other constructs. Recombinants with gene expression cassettes lacked Poly (A), which induced significant neuronal inflammation due to persistent viral antigen expression and microglia activation. Conclusion: Our results indicated that the integrity of LAT transcripts was not necessary for the establishment of long-term latent expression. Exogenous strong promoters (like cBh promoter) could remain active during latency when placed in Exon1 or 2.0 Kb Intron of LAT locus, although their transcriptional activity declined with time. Consistent with previous research, the foreign gene expression would last much longer when the gene cassette was located downstream of Exon1, which suggested a role of LAP2 in maintaining promoter activity during latency. Besides, over-transcription of the downstream part of LAT may induce continuous activation of the attenuated vectors, suggesting an important role of LAT in maintaining viral reactivation potential.

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