Serum-Based Culture Conditions Provoke Gene Expression Variability in Mouse Embryonic Stem Cells as Revealed by Single-Cell Analysis

Guo, Guoji; Pinello, Luca; Han, Xiaoping; Lai, Shih‐Wei; Shen, Li; Lin, Ta-Wei; Zou, Keyong; Yuan, Guo‐Cheng; Orkin, Stuart H.

doi:10.1016/j.celrep.2015.12.089

Cited by 75 publications

(74 citation statements)

References 39 publications

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“…), and it is well known that high concentrations of serum in media can differentially affect the culture of fibroblasts (Guo et al. ). Accordingly, reproducibility between different laboratories becomes almost impossible (Faller & Bracher, ; Ng & Ikeda, ).…”

Section: Discussionmentioning

confidence: 99%

“…Secondly, there are multiple media recipes in which skin cells have been isolated and cultured, leading to a lack of consistency of practice. Historically, epidermal cell isolation has been performed using many media additives (Rheinwald & Green, 1975;Bertolero et al 1984), and it is well known that high concentrations of serum in media can differentially affect the culture of fibroblasts (Guo et al 2016). Accordingly, reproducibility between different laboratories becomes almost impossible (Faller & Bracher, 2002;Ng & Ikeda, 2011).…”

Section: Discussionmentioning

confidence: 99%

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Bioengineering the microanatomy of human skin

et al. 2019

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Recreating the structure of human tissues in the laboratory is valuable for fundamental research, testing interventions, and reducing the use of animals. Critical to the use of such technology is the ability to produce tissue models that accurately reproduce the microanatomy of the native tissue. Current artificial cell‐based skin systems lack thorough characterisation, are not representative of human skin, and can show variation. In this study, we have developed a novel full thickness model of human skin comprised of epidermal and dermal compartments. Using an inert porous scaffold, we created a dermal construct using human fibroblasts that secrete their own extracellular matrix proteins, which avoids the use of animal‐derived materials. The dermal construct acts as a foundation upon which epidermal keratinocytes were seeded and differentiated into a stratified keratinised epithelium. In‐depth morphological analyses of the model demonstrated very close similarities with native human skin. Extensive immunostaining and electron microscopy analysis revealed ultrastructural details such as keratohyalin granules and lamellar bodies within the stratum granulosum , specialised junctional complexes, and the presence of a basal lamina. These features reflect the functional characteristics and barrier properties of the skin equivalent. Robustness and reproducibility of in vitro models are important attributes in experimental practice, and we demonstrate the consistency of the skin construct between different users. In summary, a new model of full thickness human skin has been developed that possesses microanatomical features reminiscent of native tissue. This skin model platform will be of significant interest to scientists researching the structure and function of human skin.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Bioengineering the microanatomy of human skin

et al. 2019

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“…(Importantly, Padovan-Merhar et al followed up by experiments to explicitly demonstrate that this results from a global volume-dependent transcriptional control mechanism to maintain transcript concentration rather than just count.) Additional factors influencing variability in expression include cell cycle (Buettner et al, 2015; Zopf et al, 2013), mitochondrial variability (Guantes et al, 2015; Johnston et al, 2012), heterogeneity in cell culture medium (Guo et al, 2016), temperature (Arnaud et al, 2015) and a plethora of other factors (Battich et al, 2015). …”

Section: Why Are Cells Different From Each Other?mentioning

confidence: 99%

What’s Luck Got to Do with It: Single Cells, Multiple Fates, and Biological Nondeterminism

2016

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The field of single cell biology has morphed from a philosophical digression at its inception to a playground for quantitative biologists, to a major area of biomedical research. The last several years have witnessed an explosion of new technologies, allowing us to apply ever more of the modern molecular biology toolkit to single cells. Conceptual progress, however, has been comparatively slow. Here, we provide a framework for classifying both the origins of the differences between individual cells and the consequences of those differences. We discuss how the concept of “random” differences is context dependent, and propose that rigorous definitions of inputs and outputs may bring clarity to the discussion. We also categorize ways in which probabilistic behavior may influence cellular function, highlighting studies that point to exciting future directions in the field.

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“…Several groups have reported the detection of transcriptome-wide cell-to-cell heterogeneity in gene expression by scRNA-seq in inbred (34,35) and hybrid mESC lines (8,36). However, most of these studies only considered the overall heterogeneity in gene expression but not the TBi noise, resulting in incomplete characterization of the general mechanism underlying heterogeneous gene expression.…”

Section: Discussionmentioning

confidence: 99%

Genome-wide analysis of transcriptional bursting-induced noise in mammalian cells

Ochiai

Hayashi

Umeda

et al. 2019

Preprint

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Transcriptional bursting is stochastic activation and inactivation of promoters, leading to discontinuous production of mRNA, and is considered to be a contributing factor to cell-to-cell heterogeneity in gene expression. However, it remains elusive how the kinetic properties of transcriptional bursting (e.g., burst size, burst frequency, and noise induced by transcriptional bursting) are regulated in mammalian cells. In this study, we performed a genome-wide analysis of transcriptional bursting in mouse embryonic stem cells (mESCs) using single-cell RNA-sequencing.We found that the kinetics of transcriptional bursting was determined by a combination of promoter and gene body binding proteins, including polycomb repressive complex 2 and transcription elongation-related factors. Furthermore, large-scale CRISPR-Cas9-based screening and functional analysis revealed that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncover key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.

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Serum-Based Culture Conditions Provoke Gene Expression Variability in Mouse Embryonic Stem Cells as Revealed by Single-Cell Analysis

Cited by 75 publications

References 39 publications

Bioengineering the microanatomy of human skin

Bioengineering the microanatomy of human skin

What’s Luck Got to Do with It: Single Cells, Multiple Fates, and Biological Nondeterminism

Genome-wide analysis of transcriptional bursting-induced noise in mammalian cells

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