Poster M12 - Examining the Effects of Quercetin on Mesenchymal Stem Cell Potency

Introduction: Mesenchymal Stem Cells (MSCs) are a type of adult stem cell capable of differentiation and self-renewal with relevance to tissue repair and regeneration. Researchers are actively searching for ways to make these cells viable for cellular therapy treatments to aid in the treatment of pathologies such as arthritis, osteoporosis, and more. However, for these cells to be effective, scientists must be able to generate sufficient numbers of cells with high differentiation and reparative properties. A large obstacle to this goal is in vitro cellular aging. During extensive subculturing to achieve clinically relevant number of cells, MSCs undergo replicative senescence that results in suboptimal functional capacities in late passage cells compared to early passage cells. Cellular aging, or senescence, occurs with repeated culturing and is marked by cells losing their ability to replicate, a decreased differentiation ability, and detrimental effects on neighboring cells, resulting in in vitro senescence.
In creating MSCs for cellular therapies, senescence is a phenomenon that must be avoided, as it makes the cells less suitable for intended applications. Senolytics are a class of drugs defined by their function of targeting and removing senescent cells while preserving young and healthy ones. In this study, we examine short-term senolytic treatment effects on MSC growth, differentiation capabilities, and gene expression utilizing a senolytic quercetin, through in vitro functionality assessments. It was hypothesized that in late passage cultures, quercetin would clear senescent cell populations and leave healthy populations intact, resulting in increased potency of late passage cultures.

Materials and

Methods: Quercetin Treatment: The treatment groups were treated with quercetin diluted in DMSO to 10 uM. The control groups received equivalent volumes of DMSO. The groups were treated with adipogenic or osteogenic media and quercetin or DMSO for the first week of differentiation. After 7 days, cells were treated with only adipogenic or osteogenic media.
Differentiation: Young and senescent cultures were seeded into 6-well plates at 5,000 cells/cm2. Osteogenic groups were cultured in Dulbecco's Modified Eagle Media (DMEM) with L-glutamine and penicillin-streptomycin (complete DMEM) media along with beta-glycerol phosphate, ascorbic acid, and dexamethasone. Adipogenic groups were cultured using high glucose complete DMEM media, insulin, dexamethasone, 3-isobutyl-1-methylxanthine, and indomethacin.
Staining: Around 21 days post-differentiation, cultures were fixed with 4% formaldehyde. The adipogenic cultures were stained with 0.36% Oil-red-O. The osteogenic cultures were stained with Alizarin Red, followed by microscopic examination.
Gene expression studies: At day 14 post-induction of differentiation, cells were lysed with RLT buffer. Lysates were collected and frozen at -80C. RNA was isolated using the QIAGEN RNeasy Mini Kit. Real time-qPCR was performed for the genes ALP, COL1A1, and SP7.
IDO Assay: Early passage cells were seeded at 5,000 cells/cm2 and treated with 2 uM quercetin or 100 ng/mL of IFNγ or a combination of both. After 72 hours, supernatants and lysates were collected, followed by ELISA to detect IDO secretion.

Results, Conclusions, and Discussions: In both young and senescent cells across various donors, quercetin treatment did not consistently and significantly affect osteogenic or adipogenic differentiation, with some donor MSCs demonstrating better differentiation when treated with quercetin. This is evident from the post-differentiation qualitative staining performed on adipogenic and osteogenic cultures.
Results from real-time PCR showed similar findings. In the analysis of the osteogenic markers ALP, COL1A1, and SP7, only one donor-derived MSC group treated with quercetin showed greater gene expression than the control group that was statistically significant. However, it must be noted that no group treated with quercetin exhibited less gene expression than a control group that was statistically significant. It should also be noted that quercetin treatment either kept gene expression levels relatively close to the control or increased the expression. However, the increase tended to be within the margin of error.
Finally, IDO analysis also yielded similar results. MSCs were analyzed for secretion of IDO (indoleamine 2,3 dioxygenase) after short-term treatment with IFNγ and quercetin using ELISA. It was noted that IDO activity was highest for (IFN-/quercetin-) in both passages assessed. It was also noted that presence of IFNγ reduces IDO activity significantly more than presence of quercetin when analyzing the lysate. However, the effects of quercetin when compared to groups without quercetin appear to be inconsistent.
Thus, it was concluded that quercetin has relatively insignificant effects on MSC function when comparing the phenotypes of cells treated with quercetin to control groups using various methods such as differentiation, gene analysis, and secretion of IDO. The use of senolytics, however, still appears promising; more research is needed to investigate different senolytics, different dosages, and different combinations of drugs in the interest of delaying cellular aging.

Acknowledgements (Optional): We acknowledge funding support to PI Dr. Samsonraj from MTF Biologics Foundation Investigator Grant, Women’s Giving Circle University of Arkansas College of Engineering, National Institutes of Health, and the Arkansas Biosciences Institute. We also thank the University of Arkansas Honors College for supporting Thomas Needy’s undergraduate research. We also thank all members of the Cellular Therapy and Biomanufacturing Laboratory for all help and support given in this project.