The Maintenance of Telomere Length in CD28+ T Cells During T Lymphocyte Stimulation

Huang, Ejun; Tedone, Enzo; O’Hara, Ryan; Cornelius, Crystal; Lai, Tsung‐Po; Ludlow, Andrew T.; Wright, Woodring E.

doi:10.1038/s41598-017-05174-7

Cited by 36 publications

(32 citation statements)

References 39 publications

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“…After trypsinization, PBS was added to adjust the cell density to ∼1 × 10 6 cells/mL; 1 × 10 5 cell pellets were collected to be simultaneously run alongside single-cell samples as pooled control samples. Single-cell isolation was performed as described previously (Huang et al 2017). In brief, cells were diluted in 1× PBS + 0.1% FBS to 2000 cells/mL, and 1-µL aliquots were placed on a glass slide.…”

Section: Single-cell Mtdna Quantificationmentioning

confidence: 99%

“…Additionally, the use of a reference gene subjects qPCR results to compounding errors, further reducing the sensitivity of qPCR measurement. Recent work has strengthened the utility and flexibility of droplet digital PCR (ddPCR) technology (Ludlow et al 2014;Huang et al 2017). The ddPCR technology uses oil emulsion to partition samples into thousands of droplets, each representing an independent PCR system.…”

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confidence: 99%

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Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution

et al. 2019

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Mitochondria are involved in a number of diverse cellular functions, including energy production, metabolic regulation, apoptosis, calcium homeostasis, cell proliferation, and motility, as well as free radical generation. Mitochondrial DNA (mtDNA) is present at hundreds to thousands of copies per cell in a tissue-specific manner. mtDNA copy number also varies during aging and disease progression and therefore might be considered as a biomarker that mirrors alterations within the human body. Here, we present a new quantitative, highly sensitive droplet digital PCR (ddPCR) method, droplet digital mitochondrial DNA measurement (ddMDM), to measure mtDNA copy number not only from cell populations but also from single cells. Our developed assay can generate data in as little as 3 h, is optimized for 96-well plates, and also allows the direct use of cell lysates without the need for DNA purification or nuclear reference genes. We show that ddMDM is able to detect differences between samples whose mtDNA copy number was close enough as to be indistinguishable by other commonly used mtDNA quantitation methods. By utilizing ddMDM, we show quantitative changes in mtDNA content per cell across a wide variety of physiological contexts including cancer progression, cell cycle progression, human T cell activation, and human aging.

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Section: Single-cell Mtdna Quantificationmentioning

confidence: 99%

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confidence: 99%

Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution

et al. 2019

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“…Additionally, the use of a reference gene subjects qPCR results to compounding error, further reducing the sensitivity of qPCR measurement. Recent work has strengthened the utility and flexibility of droplet digital PCR (ddPCR) technology (Ludlow et al 2014;Huang et al 2017).…”

Section: Introductionmentioning

confidence: 99%

Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single cell resolution: a focus on aging and cancer

O’Hara

Tedone

Ludlow

et al. 2019

Preprint

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Mitochondria are involved in a number of diverse cellular functions, including energy production, metabolic regulation, apoptosis, calcium homeostasis, cell proliferation and motility as well as free radical generation. Mitochondrial DNA (mtDNA) is present at hundreds to thousands of copies per cell in a tissue-specific manner. Importantly, mtDNA copy number also varies during aging and disease progression and therefore might be considered as a biomarker that mirrors alterations within the human body. Here we present a new quantitative, highly sensitive droplet digital PCR (ddPCR) method (ddMDM; droplet digital mitochondrial DNA measurement) to measure mtDNA copy number not only from cell populations but also from single cells. Our developed assay can generate data in as little as 3 hours, is optimized for 96-well plates and also allows the direct use of cell lysates without the need for DNA purification or nuclear reference genes. Importantly, we show that ddMDM is able to detect differences between samples whose mtDNA copy number was close enough as to be indistinguishable by other commonly used mtDNA quantitation methods. By utilizing ddMDM, we show quantitative changes in mtDNA content per cell across a wide variety of physiological contexts including cancer progression, cell cycle progression, human T cell activation, and human aging.

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“…Initially, telomerase activity was only associated with advanced human tumors and cancer cell lines while most somatic tissues tested were telomerase negative (13). Further investigations detected telomerase activity in a subset of fast proliferating normal human cells, including hematopoietic tissues (14)(15)(16)(17), skin (18,19), hair follicles (20), and intestinal mucosa (21). Epithelial cell turnover in the lung occurs at a slower pace in the absence of damage.…”

Section: Introductionmentioning

confidence: 99%

Human Lung Epithelial Cells Divide >200 Population Doublings without Engaging a Telomere Maintenance Mechanism

Peters-Hall

Min

Tedone

et al. 2018

Preprint

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The “Hayflick limit” is a “mitotic clock” and primary cells have a finite lifespan that correlates with telomere length. However, introduction of the telomerase catalytic protein component (TERT) is insufficient to immortalize most, but not all, human cell types under typical cell culture conditions. Originally, telomerase activity was only detected in cancer cells but is now recognized as being detectable in transit amplifying cells in tissues undergoing regeneration or in extreme conditions of wound repair. Here we report thatin vitrolow stress culture conditions allow normal human lung basal epithelial cells to grow for over 200 population doublings without engaging any telomere maintenance mechanism. This suggests that most reported instances of telomere-based replicative senescence are due to cell culture stress-induced premature senescence.One Sentence SummaryHuman lung cells growing in reduced stress conditions can divide well beyond the Hayflick limit.

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The Maintenance of Telomere Length in CD28+ T Cells During T Lymphocyte Stimulation

Cited by 36 publications

References 39 publications

Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution

Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution

Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single cell resolution: a focus on aging and cancer

Human Lung Epithelial Cells Divide >200 Population Doublings without Engaging a Telomere Maintenance Mechanism

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