Abstract
Mesenchymal stromal cells (MSC) are used in cell therapies, however cellular senescence increases heterogeneity of cell populations and leads to uncertainty in therapies’ outcomes. The determination of cellular senescence is time consuming and logistically intensive. Here, we propose the use of endogenous autofluorescence as real-time quantification of cellular senescence in human MSC, based on label-free flow cytometry analysis. We correlated cell autofluorescence to senescence using senescence-associated beta-galactosidase assay (SA-β-Gal) with chromogenic (X-GAL) and fluorescent (C12FDG) substrates, gene expression of senescence markers (such as p16INK4A, p18INK4C, CCND2 and CDCA7) and telomere length. Autofluorescence was further correlated to MSC differentiation assays (adipogenesis, chondrogenesis and osteogenesis), MSC stemness markers (CD90/CD106) and cytokine secretion (IL-6 and MCP-1). Increased cell autofluorescence significantly correlated with increased SA-β-Gal signal (both X-GAL and C12FDG substrates), cell volume and cell granularity, IL-6/MCP-1 secretion and with increased p16INK4A and CCND2 gene expression. Increased cell autofluorescence was negatively associated with the expression of the CD90/CD106 markers, osteogenic and chondrogenic differentiation potentials and p18INK4C and CDCA7 gene expression. Cell autofluorescence correlated neither with telomere length nor with adipogenic differentiation potential. We conclude that autofluorescence can be used as fast and non-invasive senescence assay for comparing MSC populations under controlled culture conditions.
Highlights
Human mesenchymal stromal cells (MSC) are multipotent cells with the ability to replicate[1,2] and differentiate into several mesodermal cell lineages, such as adipocytes, chondrocytes, myocytes and osteoblasts[3]
Analysis of cellular autofluorescence can be quickly and extensively used as it relies on common flow cytometry instruments which detect excitation of endogenous fluorophores produced by cell constituents, such as oxidative chain respiratory proteins (nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and cytochrome C), structural proteins, vitamins and undegredable waste material[21]
In order to characterize cellular senescence, bone marrow isolated MSC were initially categorized by their senescence-associated beta-galactosidase (SA-β-Gal) activity, evaluated with chromogenic (X-GAL, Fig. 1a) and fluorescent substrates (C12FDG, Fig. 1b)
Summary
Human mesenchymal stromal cells (MSC) are multipotent cells with the ability to replicate[1,2] and differentiate into several mesodermal cell lineages, such as adipocytes, chondrocytes, myocytes and osteoblasts[3]. Senescent cells express ligands and adhesion molecules that signal to natural killer and other immune cells to attack them[15] This normally stimulates surrounding progenitor cells to regenerate the compromised tissue[13]. Cells is associated to decreased tissue regeneration capacity and life expectancy, and their elimination in a mouse model resulted in increased lifespan[16]. This identifies cellular senescence as an ideal target for the development of new anti-ageing therapies. Riboflavin has already been recognized as a biomarker associated selectively with epithelial cancer stem cells[25], while fluorescence has been proposed as a non-invasive indicator of embryonic stem cell differentiation state, to distinguishing pluripotent from more mature cells[26]
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