Live cell imaging and single cell tracking of mesenchymal stromal cells<i>in vitro</i>

Cornwell, James; Gutierrez, Maria Z.; Harvey, Richard P.; Nordon, Robert E.

doi:10.1002/9781118907474.ch24

Cited by 3 publications

(9 citation statements)

References 93 publications

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“…Now, we address these limitations by single-cell tracking of SAOS-2 cells co-cultured with HDFs. This method records the behaviour and fate of individual cells and their progeny across multiple cell divisions and generations [ 38 , 39 , 40 ]. By identifying the fate of individual cells, single-cell tracking overcomes the limitations of pooled cell assays that average the outcomes for thousands of cells.…”

Section: Introductionmentioning

confidence: 99%

“…By identifying the fate of individual cells, single-cell tracking overcomes the limitations of pooled cell assays that average the outcomes for thousands of cells. An interesting finding of single-cell tracking studies is that sister cells are more similar to each other than they are to their mother cell or their own progeny [ 38 , 39 , 40 ].…”

Section: Introductionmentioning

confidence: 99%

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Cell-Projection Pumping of Fibroblast Contents into Osteosarcoma SAOS-2 Cells Correlates with Increased SAOS-2 Proliferation and Migration, as well as Altered Morphology

et al. 2021

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We earlier reported that cell-projection pumping transfers fibroblast contents to cancer cells and this alters the cancer cell phenotype. Here, we report on single-cell tracking of time lapse recordings from co-cultured fluorescent fibroblasts and SAOS-2 osteosarcoma cells, tracking 5201 cells across 7 experiments. The fluorescent lipophilic marker DiD was used to label fibroblast organelles and to trace the transfer of fibroblast cytoplasm into SAOS-2 cells. We related SAOS-2 phenotypic change to levels of fluorescence transfer from fibroblasts to SAOS-2 cells, as well as what we term ‘compensated fluorescence’, that numerically projects mother cell fluorescence post-mitosis into daughter cells. The comparison of absolute with compensated fluorescence allowed us to deduct if the phenotypic effects in mother SAOS-2 cells were inherited by their daughters. SAOS-2 receipt of fibroblast fluorescence correlated by Kendall’s tau with cell-profile area and without evidence of persistence in daughter cells (median tau = 0.51, p < 0.016); negatively and weakly with cell circularity and with evidence of persistence (median tau = −0.19, p < 0.05); and very weakly with cell migration velocity and without evidence of persistence (median tau = 0.01, p < 0.016). In addition, mitotic SAOS-2 cells had higher rates of prior fluorescence uptake (median = 64.9 units/day) than non-dividing cells (median = 35.6 units/day, p < 0.016) and there was no evidence of persistence post-mitosis. We conclude that there was an appreciable impact of cell-projection pumping on cancer cell phenotype relevant to cancer histopathological diagnosis, clinical spread and growth, with most effects being ‘reset’ by cancer cell mitosis.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cell-Projection Pumping of Fibroblast Contents into Osteosarcoma SAOS-2 Cells Correlates with Increased SAOS-2 Proliferation and Migration, as well as Altered Morphology

et al. 2021

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“…Single-cell tracking software was adapted from that previously developed in MATLAB (TrackPad: https://github.com/Jamcor/TrackPad), and was used to follow the fate of individual cells and their progeny in phase contrast time-lapse recordings [38,39]. Cells present at the beginning of experiments, were defined as the 'starting population', while sequential cell divisions generated 'first', 'second', 'third' and very occasionally 'fourth' generations of progeny.…”

Section: Single-cell Tracking and Analysismentioning

confidence: 99%

“…Moving cell location was determined during single-cell tracking in Trackpad, while further analysis was in RStudio, similar to earlier reports [38,39]. In brief, mean cell migration velocity was calculated for all tracked cells, by firstly identifying cell centroids in phase contrast images at relevant time points at 2 h intervals, performed in Trackpad.…”

Section: Determination Of Cell Migration Velocitymentioning

confidence: 99%

“…We now address these limitations by single-cell tracking of SAOS-2 co-cultured with HDF. This method records the behavior and fate of individual cells and their progeny across multiple cell divisions and generations [38][39][40]. By identifying the fate of individual cells, single-cell tracking overcomes the limitations of pooled cell assays that average the outcomes for thousands of cells.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Cell-Projection Pumping of Fibroblast Contents into Osteosarcoma SAOS-2 Cells Correlates with Increased SAOS-2 Proliferation and Migration, and also with Altered Morphology

Mahadevan

Cornwell

Chami

et al. 2021

Preprint

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We earlier reported that cell-projection pumping transfers fibroblast contents to cancer cells, and this alters cancer cell phenotype. We now report on single-cell tracking of time lapse recordings from co-cultured fluorescent fibroblasts and SAOS-2 osteosarcoma cells, tracking 5,201 cells across 7 experiments. The fluorescent lipophilic marker DiD was used to label fibroblast organelles, and to trace transfer of fibroblast cytoplasm into SAOS-2. We related SAOS-2 phenotypic change to levels of fluorescence transfer from fibroblasts to SAOS-2, and also to what we term ‘compensated fluorescence’, that numerically projects mother cell fluorescence post-mitosis, into daughter cells. Comparison of absolute with compensated fluorescence, allowed deduction if phenotypic effects in mother SAOS-2, were inherited by their daughters. SAOS-2 receipt of fibroblast fluorescence correlated by Kendall’s tau: with cell-profile area, and without evidence for persistence in daughter cells (median tau = 0.51, p < 0. 016); negatively and weakly with cell circularity, and with evidence for persistence (median tau = −0.19, p < 0.05); and very weakly with cell migration velocity, and without evidence for persistence (median tau = 0.01, p < 0.016). Also, mitotic SAOS-2 had higher rates of prior fluorescence uptake (median = 64.9 units/day), compared with non dividing cells (median = 35.6 units/day, p < 0.016), and there was no evidence for persistence post-mitosis. We conclude there is appreciable impact of cell-projection pumping on cancer cell phenotype, relevant to cancer histopathological diagnosis, clinical spread, and growth, with most effects ‘reset’ by cancer cell mitosis.

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A Novel Cartesian Plot Analysis for Fixed Monolayers That Relates Cell Phenotype to Transfer of Contents between Fibroblasts and Cancer Cells by Cell-Projection Pumping

Mahadevan

Kwong

et al. 2022

IJMS

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We recently described cell-projection pumping as a mechanism transferring cytoplasm between cells. The uptake of fibroblast cytoplasm by co-cultured SAOS-2 osteosarcoma cells changes SAOS-2 morphology and increases cell migration and proliferation, as seen by single-cell tracking and in FACS separated SAOS-2 from co-cultures. Morphological changes in SAOS-2 seen by single cell tracking are consistent with previous observations in fixed monolayers of SAOS-2 co-cultures. Notably, earlier studies with fixed co-cultures were limited by the absence of a quantitative method for identifying sub-populations of co-cultured cells, or for quantitating transfer relative to control populations of SAOS-2 or fibroblasts cultured alone. We now overcome that limitation by a novel Cartesian plot analysis that identifies individual co-cultured cells as belonging to one of five distinct cell populations, and also gives numerical measure of similarity to control cell populations. We verified the utility of the method by first confirming the previously established relationship between SAOS-2 morphology and uptake of fibroblast contents, and also demonstrated similar effects in other cancer cell lines including from melanomas, and cancers of the ovary and colon. The method was extended to examine global DNA methylation, and while there was no clear effect on SAOS-2 DNA methylation, co-cultured fibroblasts had greatly reduced DNA methylation, similar to cancer associated fibroblasts.

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Live cell imaging and single cell tracking of mesenchymal stromal cellsin vitro

Cited by 3 publications

References 93 publications

Cell-Projection Pumping of Fibroblast Contents into Osteosarcoma SAOS-2 Cells Correlates with Increased SAOS-2 Proliferation and Migration, as well as Altered Morphology

Cell-Projection Pumping of Fibroblast Contents into Osteosarcoma SAOS-2 Cells Correlates with Increased SAOS-2 Proliferation and Migration, as well as Altered Morphology

Cell-Projection Pumping of Fibroblast Contents into Osteosarcoma SAOS-2 Cells Correlates with Increased SAOS-2 Proliferation and Migration, and also with Altered Morphology

A Novel Cartesian Plot Analysis for Fixed Monolayers That Relates Cell Phenotype to Transfer of Contents between Fibroblasts and Cancer Cells by Cell-Projection Pumping

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