Enhanced Autophagy of Adipose-Derived Stem Cells Grown on Chitosan Substrates

Abstract

Autophagy is an important protein quality control mechanism for cells under stress conditions to promote cell survival. Modulation of autophagy on biomaterial substrates is rarely reported. In this study, the autophagy of adipose-derived stem cells (ADSCs) cultured on chitosan (CS) substrates was examined. Compared to the traditional monolayer culture, ADSCs cultured on CS substrates showed spheroid formation as well as a prolonged upregulation of autophagosomal marker-microtubule-associated protein 1 light chain 3 (LC3) II protein expression. In addition, the green fluorescent protein tagged-LC3 (GFP-LC3) expressing ADSCs also revealed more GFP-LC3 puncta on CS substrates. The enhanced autophagy on CS substrates was associated with Ca2+, while ethylene glycol tetraacetic acid (EGTA), a Ca2+ chelator, repressed the autophagy in a dose-dependent manner. Moreover, ADSC spheroids on CS substrates demonstrated a higher survival rate and autophagy response upon H2O2 treatment. The upstream components of autophagy signal pathway-UNC51-like kinase 1 (Ulk1), autophagy-related protein 13 (Atg13), and autophagy/beclin-1 regulator 1 (Ambra1) genes were more highly expressed in ADSC spheroids before and after adding H2O2 than those in the conventional culture. EGTA also decreased the cell viability and autophagy-associated gene expression for ADSC spheroids on CS substrates after H2O2 treatment. Therefore, we suggest that three-dimensional (3D) cell culture on CS may confer ADSCs the ability to increase the autophagic flux in response to stimulations in a Ca2+-dependent manner.

3D-cultured adipose tissue-derived stem cells inhibit liver cancer cell migration and invasion through suppressing epithelial-mesenchymal transition

“…It is generally accepted that molecules secreted from ADSCs may influence the effects of ADSCs on tumor growth. Therefore, the culture conditions of ADSCs may have an important role in determining the association between ADSCs and tumor cells, since various culture conditions could affect the secretion of molecules from ADSCs ( 32 , 33 ). Notably, Tian et al reported that human MSCs may inhibit proliferation of cancer cells in vitro and enhance tumor growth in vivo ( 34 ), thus suggesting that MSCs exert a dual effect on the same tumor under various growing conditions.…”

“…It has been reported that sphere or 3D culture methods may promote the secretion of cytokines and chemo kines from ADSCs ( 32 , 33 ); consequently, sphere or 3D culture conditions may theoretically enhance the effects of ADSCs on tumor cells. As predicted, the present study demonstrated that sphere or 3D culture methods could enhance the inhibitory effects of ADSCs on liver cancer cell migration, and could accelerate the effects of ADSCs on liver cancer cell apoptosis.…”

“…For example, Bhang et al demonstrated that ADSCs cultured under three-dimensional (3D) culture conditions could produce higher concentrations of angiogenic and/or anti-apoptotic factors, including vascular endothelial growth factor, fibroblast growth factor-2, hepatocyte growth factor and C-X-C motif chemokine ligand 12 compared with cells cultured under two-dimensional (2D) conditions ( 32 ). In addition, Yang et al demonstrated that the 3D culture method could enhance the activity of ADSCs and increase the autophagic response upon hydrogen peroxide (H 2 O 2 ) treatment compared with the 2D culture method ( 33 ). Tian et al revealed that human MSCs inhibited proliferation of cancer cells in vitro , whereas they enhanced tumor formation and growth in vivo ( 34 ), thus indicating the dual effects of MSCs on the same tumor.…”

Modulating autophagy in mesenchymal stem cells effectively protects against hypoxia- or ischemia-induced injury

“…Furthermore, MSCs cultured on chitosan substrates show spheroid formation and upregulated expression of the autophagosomal marker LC3 II in a Ca 2+ -dependent manner, and these changes are accompanied by a higher survival rate in response to H 2 O 2 than in cells grown as a monolayer culture. In addition, chitosan substrates also increase the expression levels of the upper autophagy signal pathway-related proteins, including Ulk1, Atg-13, and autophagy/Beclin-1 regulator 1, both before and after the addition of H 2 O 2 compared with general culture conditions [58]. Jun et al argued that melatonin increases the levels of manganese superoxide dismutase (MnSOD) and catalase while suppressing H 2 O 2 -induced ER stress and autophagy to reduce the apoptosis rate of MSCs via upregulation of prion protein (PrP C ) [59].…”

Cannabinoid-Induced Autophagy and Heme Oxygenase-1 Determine the Fate of Adipose Tissue-Derived Mesenchymal Stem Cells under Stressful Conditions

“…Autophagy, a process of cellular self-digestion, is considered to be a cytoprotective response that may occur after the withdrawal of growth factors or under stress conditions (for review see [ 24 ]). In this context, autophagy has also been identified as an essential mechanism for protecting MSCs and regulating bioenergetic requirements for stem cell activation [ 25 , 26 , 27 , 28 ]. On the other hand, initial data from experiments with ADMSCs have also shown that the downregulation of nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1 promotes autophagy-dependent osteoblastic differentiation, which indicates a negative regulation of autophagy by HO-1 in this cell type [ 29 ].…”