Three-Dimensional Compaction Switches Stress Response Programs and Enhances Therapeutic Efficacy of Endometrial Mesenchymal Stem/Stromal Cells

Abstract

Mesenchymal stem cells are currently tested as a promising tool for the treatment of a wide range of human diseases. Enhanced therapeutic potential of spheroids formed from these cells has been proved in numerous studies, however, the fundamental basics of this effect are still being discussed. In this work, we showed that endometrial mesenchymal stem/stromal cells (eMSCs) assembled in spheroids possess a higher therapeutic efficacy compared to cells grown in monolayer in the treatment of the defects that are non-specific for eMSC tissue origin -skin wounds. With the purpose to elucidate the possible causes of superior spheroid potency, we compared the tolerance of eMSC cultivated in spheres and monolayer to the stress insults. Using genetically encoded hydrogen peroxide biosensor HyPer, we showed that three-dimensional configuration (3D) helped to shield the inner cell layers of spheroid from the external H 2 O 2 -induced oxidative stress. However, the viability of oxidatively damaged eMSCs in spheroids appeared to be much lower than that of monolayer cells. An extensive analysis, which included administration of heat shock and irradiation stress, revealed that cells in spheroids damaged by stress factors activate the apoptosis program, while in monolayer cells stress-induced premature senescence is developed. We found that basal down-regulation of anti-apoptotic and autophagy-related genes provides the possible molecular basis of the high commitment of eMSCs cultured in 3D to apoptosis. We conclude that predisposition to apoptosis provides the programmed elimination of damaged cells and contributes to the transplant safety of spheroids. In addition, to investigate the role of paracrine secretion in the wound healing potency of spheroids, we exploited the in vitro wound model (scratch assay) and found that culture medium conditioned by eMSC spheroids accelerates the migration of adherent cells. We showed that 3D eMSCs upregulate transcriptional activator, hypoxia-inducible factor (HIF)-1, and secret ten-fold more HIF-1-inducible pro-angiogenic factor VEGF

Impact of 3D cell culture on bone regeneration potential of mesenchymal stromal cells

“…In contrast, it has been reported that cultivation of human periodontal ligament fibroblasts, a cell type which is phenotypically indistinguishable from MSCs [ 95 ], in 3D methylcellulose hydrogels results in suppression of TNF-α-induced cellular senescence and an increase in proliferation and migration [ 96 ]. In an attempt to study the stress response in human endometrial MSCs cultivated as 3D spheroids in comparison to conventional 2D culture, Domnina and colleagues exposed cells cultivated under both conditions to heat shock and treatment with H 2 O 2 [ 97 ]. This study demonstrated that 2D monolayer MSCs developed premature senescence, while MSCs in 3D exhibited decreased viability without cell cycle arrest and other signs of senescence.…”

Cell-Fiber-Based 3D Tissue Array for Drug Response Assay

“…4), EC50 of CF fragments = 0.8 mM, HeLa ; EC50 of 2D culture = 75 µM 39 , EC50 of CF fragment = 85 µM). This is because H 2 O 2 acts on all cells at once in 2D culture, while in 3D tissue, only cells on the tissue surface are affected by H 2 O 2 40 , suggesting that the EC50 is higher in 3D tissue. In 2D cultures, β-estradiol has been reported to have proliferative and trophic effects at low concentrations, but these effects are suppressed at high concentrations 34,35 .…”

Monovalent Ions and Stress-Induced Senescence in Human Mesenchymal Endometrial Stem Cells

“…As reported recently and also shown in our study, cardiac glycosides do not induce apoptosis in both hMESCs and their senescent partners 105 . Obviously, the high resistance of hMESCs to cardiac glycosides is provided by a highly expressed anti-apoptotic program in these cells 32,64,65,106,107 . Taken together, the above data suggest that the depletion of cellular K + is necessary event in cardiac glycoside-induced apoptosis; however, a separate apoptotic signal is required to kill a cell, which acts together with low cellular K + .…”