Evolutionary insights into primate skeletal gene regulation using a comparative cell culture model

Mapping Intimacies ◽

10.1101/2021.09.30.462680 ◽

2021 ◽

Author(s):

Genevieve Housman ◽

Emilie Briscoe ◽

Yoav Gilad

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Bone Cells ◽

Cell Types ◽

Differentially Expressed ◽

Functional Genomic ◽

Cell Culture Model ◽

Culture Model ◽

Study Gene Regulation

AbstractThe evolution of complex skeletal traits in primates was likely influenced by both genetic and environmental factors. Because skeletal tissues are notoriously challenging to study using functional genomic approaches, they remain poorly characterized even in humans, let alone across multiple species. The challenges involved in obtaining functional genomic data from the skeleton, combined with the difficulty of obtaining such tissues from nonhuman apes, motivated us to consider an alternative in vitro system with which to comparatively study gene regulation in skeletal cell types. Specifically, we differentiated six human and six chimpanzee induced pluripotent stem cell lines (iPSCs) into mesenchymal stem cells (MSCs) and subsequently into osteogenic cells (bone cells). We validated differentiation using standard methods and collected single-cell RNA sequencing data from over 100,000 cells across multiple samples and replicates at each stage of differentiation. While most genes that we examined display conserved patterns of expression across species, hundreds of genes are differentially expressed (DE) between humans and chimpanzees within and across stages of osteogenic differentiation. Some of these interspecific DE genes show functional enrichments relevant in skeletal tissue trait development. Moreover, topic modeling indicates that interspecific gene programs become more pronounced as cells mature. Overall, we propose that this in vitro model can be used to identify interspecific regulatory differences that may have contributed to skeletal trait differences between species.Author SummaryPrimates display a range of skeletal morphologies and susceptibilities to skeletal diseases, but the molecular basis of these phenotypic differences is unclear. Studies of gene expression variation in primate skeletal tissues are extremely restricted due to the ethical and practical challenges associated with collecting samples. Nevertheless, the ability to study gene regulation in primate skeletal tissues is crucial for understanding how the primate skeleton has evolved. We therefore developed a comparative primate skeletal cell culture model that allows us to access a spectrum of human and chimpanzee cell types as they differentiate from stem cells into bone cells. While most gene expression patterns are conserved across species, we also identified hundreds of differentially expressed genes between humans and chimpanzees within and across stages of differentiation. We also classified cells by osteogenic stage and identified additional interspecific differentially expressed genes which may contribute to skeletal trait differences. We anticipate that this model will be extremely useful for exploring questions related to gene regulation variation in primate bone biology and development.

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Impact on gene expression in response to silver-decorated titania nanomatrix using an in vitro satellite cell culture model

Polymer Bulletin ◽

10.1007/s00289-015-1581-3 ◽

2015 ◽

Vol 73 (7) ◽

pp. 1855-1874

Author(s):

Touseef Amna ◽

M. Shamshi Hassan ◽

Salem S. Al-Deyab ◽

Myung-Seob Khil ◽

Inho Hwang

Keyword(s):

Gene Expression ◽

Cell Culture ◽

Satellite Cell ◽

Cell Culture Model ◽

Culture Model

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1120: Citrate and Vitamin E Blunt the SWL-Induced Free Radical Surge in an In-Vitro MDCK Cell Culture Model

The Journal of Urology ◽

10.1016/s0022-5347(18)38357-5 ◽

2004 ◽

Vol 171 (4S) ◽

pp. 295-295

Author(s):

Fernando C. Delvecchio ◽

Ricardo M. Brizuela ◽

Karen J. Byer ◽

W. Patrick Springhart ◽

Saeed R. Khan ◽

...

Keyword(s):

Cell Culture ◽

Free Radical ◽

Vitamin E ◽

Mdck Cell ◽

Cell Culture Model ◽

Culture Model

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CircFAM13B promotes the proliferation of hepatocellular carcinoma by sponging miR-212, upregulating E2F5 expression and activating the P53 pathway

Cancer Cell International ◽

10.1186/s12935-021-02120-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Ying Xie ◽

Xiaofeng Hang ◽

Wensheng Xu ◽

Jing Gu ◽

Yuanjing Zhang ◽

...

Keyword(s):

Gene Expression ◽

Hepatocellular Carcinoma ◽

Gene Expression Omnibus ◽

Differentially Expressed ◽

In Vivo Studies ◽

Circular Rnas ◽

Novel Target ◽

Underlying Mechanisms

Abstract Background Most of the biological functions of circular RNAs (circRNAs) and the potential underlying mechanisms in hepatocellular carcinoma (HCC) have not yet been discovered. Methods In this study, using circRNA expression data from HCC tumor tissues and adjacent tissues from the Gene Expression Omnibus database, we identified out differentially expressed circRNAs and verified them by qRT-PCT. Functional experiments were performed to evaluate the effects of circFAM13B in HCC in vitro and in vivo. Results We found that circFAM13B was the most significantly differentially expressed circRNA in HCC tissue. Subsequently, in vitro and in vivo studies also demonstrated that circFAM13B promoted the proliferation of HCC. Further studies revealed that circFAM13B, a sponge of miR-212, is involved in the regulation of E2F5 gene expression by competitively binding to miR-212, inhibits the activation of the P53 signalling pathway, and promotes the proliferation of HCC cells. Conclusions Our findings revealed the mechanism underlying the regulatory role played by circFAM13B, miR-212 and E2F5 in HCC. This study provides a new theoretical basis and novel target for the clinical prevention and treatment of HCC.

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An In Vitro Cell Culture Model for Pyoverdine-Mediated Virulence

Pathogens ◽

10.3390/pathogens10010009 ◽

2020 ◽

Vol 10 (1) ◽

pp. 9

Author(s):

Donghoon Kang ◽

Natalia V. Kirienko

Keyword(s):

Cell Culture ◽

Virulence Factors ◽

Model Organisms ◽

Cell Culture Model ◽

Chronic Infections ◽

In Vitro Cell Culture ◽

Mitochondrial Homeostasis ◽

Culture Model ◽

Wide Range

Pseudomonas aeruginosa is a multidrug-resistant, opportunistic pathogen that utilizes a wide-range of virulence factors to cause acute, life-threatening infections in immunocompromised patients, especially those in intensive care units. It also causes debilitating chronic infections that shorten lives and worsen the quality of life for cystic fibrosis patients. One of the key virulence factors in P. aeruginosa is the siderophore pyoverdine, which provides the pathogen with iron during infection, regulates the production of secreted toxins, and disrupts host iron and mitochondrial homeostasis. These roles have been characterized in model organisms such as Caenorhabditis elegans and mice. However, an intermediary system, using cell culture to investigate the activity of this siderophore has been absent. In this report, we describe such a system, using murine macrophages treated with pyoverdine. We demonstrate that pyoverdine-rich filtrates from P. aeruginosa exhibit substantial cytotoxicity, and that the inhibition of pyoverdine production (genetic or chemical) is sufficient to mitigate virulence. Furthermore, consistent with previous observations made in C. elegans, pyoverdine translocates into cells and disrupts host mitochondrial homeostasis. Most importantly, we observe a strong correlation between pyoverdine production and virulence in P. aeruginosa clinical isolates, confirming pyoverdine’s value as a promising target for therapeutic intervention. This in vitro cell culture model will allow rapid validation of pyoverdine antivirulents in a simple but physiologically relevant manner.

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Development of an in vitro cell culture model to study milk to plasma ratios of therapeutic drugs

Indian Journal of Pharmacology ◽

10.4103/0253-7613.114994 ◽

2013 ◽

Vol 45 (4) ◽

pp. 325 ◽

Cited By ~ 2

Author(s):

Anurupa Maitra ◽

Shahnaz Patel ◽

VijayR Bhate ◽

VilliS Toddywalla ◽

MaithiliA Athavale

Keyword(s):

Cell Culture ◽

Cell Culture Model ◽

In Vitro Cell Culture ◽

Therapeutic Drugs ◽

Culture Model

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Effects of Ethanol and Acetaldehyde on Tight Junction Integrity: In Vitro Study in a Three Dimensional Intestinal Epithelial Cell Culture Model

PLoS ONE ◽

10.1371/journal.pone.0035008 ◽

2012 ◽

Vol 7 (4) ◽

pp. e35008 ◽

Cited By ~ 60

Author(s):

Elhaseen Elamin ◽

Daisy Jonkers ◽

Kati Juuti-Uusitalo ◽

Sven van IJzendoorn ◽

Freddy Troost ◽

...

Keyword(s):

Cell Culture ◽

Epithelial Cell ◽

Intestinal Epithelial Cell ◽

Three Dimensional ◽

In Vitro Study ◽

Intestinal Epithelial ◽

Cell Culture Model ◽

Epithelial Cell Culture ◽

Culture Model

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Transcriptome analysis ofSchistosoma mansonilarval development using serial analysis of gene expression (SAGE)

Parasitology ◽

10.1017/s0031182009005733 ◽

2009 ◽

Vol 136 (5) ◽

pp. 469-485 ◽

Cited By ~ 22

Author(s):

A. S. TAFT ◽

J. J. VERMEIRE ◽

J. BERNIER ◽

S. R. BIRKELAND ◽

M. J. CIPRIANO ◽

...

Keyword(s):

Gene Expression ◽

Sequence Data ◽

Subsequent Development ◽

Differentially Expressed ◽

Cdna Libraries ◽

Genome Wide ◽

A Genome ◽

Genome Wide Expression ◽

Cell Conditioned Medium

SUMMARYInfection of the snail,Biomphalaria glabrata, by the free-swimming miracidial stage of the human blood fluke,Schistosoma mansoni, and its subsequent development to the parasitic sporocyst stage is critical to establishment of viable infections and continued human transmission. We performed a genome-wide expression analysis of theS. mansonimiracidia and developing sporocyst using Long Serial Analysis of Gene Expression (LongSAGE). Five cDNA libraries were constructed from miracidia andin vitrocultured 6- and 20-day-old sporocysts maintained in sporocyst medium (SM) or in SM conditioned by previous cultivation with cells of theB. glabrataembryonic (Bge) cell line. We generated 21 440 SAGE tags and mapped 13 381 to theS. mansonigene predictions (v4.0e) either by estimating theoretical 3′ UTR lengths or using existing 3′ EST sequence data. Overall, 432 transcripts were found to be differentially expressed amongst all 5 libraries. In total, 172 tags were differentially expressed between miracidia and 6-day conditioned sporocysts and 152 were differentially expressed between miracidia and 6-day unconditioned sporocysts. In addition, 53 and 45 tags, respectively, were differentially expressed in 6-day and 20-day cultured sporocysts, due to the effects of exposure to Bge cell-conditioned medium.

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Establishment of a Human Induced Pluripotent Stem Cell-Derived Neuromuscular Co-Culture Under Optogenetic Control

10.1101/2020.04.10.036400 ◽

2020 ◽

Cited By ~ 1

Author(s):

Elliot W. Swartz ◽

Greg Shintani ◽

Jijun Wan ◽

Joseph S. Maffei ◽

Sarah H. Wang ◽

...

Keyword(s):

Motor Neurons ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Calcium Flux ◽

Electrode Arrays ◽

Spinal Motor Neurons ◽

Culture Model ◽

Skeletal Myotubes ◽

Induced Pluripotent

SummaryThe failure of the neuromuscular junction (NMJ) is a key component of degenerative neuromuscular disease, yet how NMJs degenerate in disease is unclear. Human induced pluripotent stem cells (hiPSCs) offer the ability to model disease via differentiation toward affected cell types, however, the re-creation of an in vitro neuromuscular system has proven challenging. Here we present a scalable, all-hiPSC-derived co-culture system composed of independently derived spinal motor neurons (MNs) and skeletal myotubes (sKM). In a model of C9orf72-associated disease, co-cultures form functional NMJs that can be manipulated through optical stimulation, eliciting muscle contraction and measurable calcium flux in innervated sKM. Furthermore, co-cultures grown on multi-electrode arrays (MEAs) permit the pharmacological interrogation of neuromuscular physiology. Utilization of this co-culture model as a tunable, patient-derived system may offer significant insights into NMJ formation, maturation, repair, or pathogenic mechanisms that underlie NMJ dysfunction in disease.

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Enhancing droplet-based single-nucleus RNA-seq resolution using the semi-supervised machine learning classifier DIEM

10.1101/786285 ◽

2019 ◽

Cited By ~ 4

Author(s):

Marcus Alvarez ◽

Elior Rahmani ◽

Brandon Jew ◽

Kristina M. Garske ◽

Zong Miao ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cell Types ◽

Supervised Machine Learning ◽

Data Sets ◽

Rna Seq ◽

Novel Approach ◽

Single Nucleus ◽

Downstream Analysis

AbstractSingle-nucleus RNA sequencing (snRNA-seq) measures gene expression in individual nuclei instead of cells, allowing for unbiased cell type characterization in solid tissues. Contrary to single-cell RNA seq (scRNA-seq), we observe that snRNA-seq is commonly subject to contamination by high amounts of extranuclear background RNA, which can lead to identification of spurious cell types in downstream clustering analyses if overlooked. We present a novel approach to remove debris-contaminated droplets in snRNA-seq experiments, called Debris Identification using Expectation Maximization (DIEM). Our likelihood-based approach models the gene expression distribution of debris and cell types, which are estimated using EM. We evaluated DIEM using three snRNA-seq data sets: 1) human differentiating preadipocytes in vitro, 2) fresh mouse brain tissue, and 3) human frozen adipose tissue (AT) from six individuals. All three data sets showed various degrees of extranuclear RNA contamination. We observed that existing methods fail to account for contaminated droplets and led to spurious cell types. When compared to filtering using these state of the art methods, DIEM better removed droplets containing high levels of extranuclear RNA and led to higher quality clusters. Although DIEM was designed for snRNA-seq data, we also successfully applied DIEM to single-cell data. To conclude, our novel method DIEM removes debris-contaminated droplets from single-cell-based data fast and effectively, leading to cleaner downstream analysis. Our code is freely available for use at https://github.com/marcalva/diem.

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