Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods

Abstract Human mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably mo...

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Main Authors:	Weichao Zhai, Jerome Tan, Tobias Russell, Sixun Chen, Dennis McGonagle, May Win Naing, Derrick Yong, Elena Jones
Format:	article
Language:	EN
Published:	Nature Portfolio 2021
Subjects:	Medicine R Science Q
Online Access:	https://doaj.org/article/b7f21e41506c4da7b9df7d2a24745571
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spelling	oai:doaj.org-article:b7f21e41506c4da7b9df7d2a247455712021-12-02T14:12:08ZMulti-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods10.1038/s41598-020-79831-92045-2322https://doaj.org/article/b7f21e41506c4da7b9df7d2a247455712021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-79831-9https://doaj.org/toc/2045-2322Abstract Human mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difficult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture’s viability, differentiation potential and ultimately leads to reduced therapeutic efficacies. Histochemical $$\upbeta $$ β -galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label-free. In this study, we have investigated alternatives in quantification of hMSCs senescence, which included flow cytometry methods that are based on a combination of cell size measurements and fluorescence detection of SA- $$\upbeta $$ β -galactosidase activity using the fluorogenic substrate, C $${_{12}}$$ 12 FDG; and autofluorescence methods that measure fluorescence output from endogenous fluorophores including lipopigments. For identification of senescent cells in the hMSC batches produced, the non-destructive and label-free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the final output of the therapeutic-grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced flow cytometry and autofluorescence methods, which were benchmarked with the current standard in $$\upbeta $$ β -galactosidase staining. Both the advanced methods demonstrated statistically significant values, (r = 0.76, p $$\le $$ ≤ 0.001 for the fluorogenic C $${_{12}}$$ 12 FDG method, and r = 0.72, p $$\le $$ ≤ 0.05 for the forward scatter method), and good fold difference ranges (1.120–4.436 for total autofluorescence mean and 1.082–6.362 for lipopigment autofluorescence mean) between early and senescent passage hMSCs. Our autofluroescence imaging and spectra decomposition platform offers additional benefit in label-free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers.Weichao ZhaiJerome TanTobias RussellSixun ChenDennis McGonagleMay Win NaingDerrick YongElena JonesNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Weichao Zhai Jerome Tan Tobias Russell Sixun Chen Dennis McGonagle May Win Naing Derrick Yong Elena Jones Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods
description	Abstract Human mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difficult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture’s viability, differentiation potential and ultimately leads to reduced therapeutic efficacies. Histochemical $$\upbeta $$ β -galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label-free. In this study, we have investigated alternatives in quantification of hMSCs senescence, which included flow cytometry methods that are based on a combination of cell size measurements and fluorescence detection of SA- $$\upbeta $$ β -galactosidase activity using the fluorogenic substrate, C $${_{12}}$$ 12 FDG; and autofluorescence methods that measure fluorescence output from endogenous fluorophores including lipopigments. For identification of senescent cells in the hMSC batches produced, the non-destructive and label-free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the final output of the therapeutic-grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced flow cytometry and autofluorescence methods, which were benchmarked with the current standard in $$\upbeta $$ β -galactosidase staining. Both the advanced methods demonstrated statistically significant values, (r = 0.76, p $$\le $$ ≤ 0.001 for the fluorogenic C $${_{12}}$$ 12 FDG method, and r = 0.72, p $$\le $$ ≤ 0.05 for the forward scatter method), and good fold difference ranges (1.120–4.436 for total autofluorescence mean and 1.082–6.362 for lipopigment autofluorescence mean) between early and senescent passage hMSCs. Our autofluroescence imaging and spectra decomposition platform offers additional benefit in label-free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers.
format	article
author	Weichao Zhai Jerome Tan Tobias Russell Sixun Chen Dennis McGonagle May Win Naing Derrick Yong Elena Jones
author_facet	Weichao Zhai Jerome Tan Tobias Russell Sixun Chen Dennis McGonagle May Win Naing Derrick Yong Elena Jones
author_sort	Weichao Zhai
title	Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods
title_short	Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods
title_full	Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods
title_fullStr	Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods
title_full_unstemmed	Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods
title_sort	multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/b7f21e41506c4da7b9df7d2a24745571
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_version_	1718391806075338752

Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods

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