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.
Highlights
Autophagy is a conserved catabolic mechanism for degradation of nonessential or dysfunctional cellular organelles and proteins.[1,2] Upon induction, the Ulk[1] protein kinase complex (Ulk[1], Atg[13], FIP200, and Atg101) initiates autophagosome formation[3] and the autophagy/beclin-1 regulator 1 (Ambra1) enhances Ulk[1] kinase activity.[4]
Induction of autophagy in adipose-derived stem cells (ADSCs) on CS substrates To examine whether cell culture on CS substrates increased autophagy, the expression of endogenous light chain 3 (LC3)-II, an autophagosomal marker, was detected by Western blots
Formation of green fluorescent protein tagged-LC3 (GFP-LC3) puncta in ADSCs on CS substrates was clearly observed and the number of GFP-LC3 puncta was more than that on TCPS (Fig. 1B,C). These results indicated that autophagy was more pronounced and prolonged in ADSCs when cultured on CS substrates
Summary
Autophagy is a conserved catabolic mechanism for degradation of nonessential or dysfunctional cellular organelles and proteins.[1,2] Upon induction, the Ulk[1] protein kinase complex (Ulk[1], Atg[13], FIP200, and Atg101) initiates autophagosome formation[3] and the autophagy/beclin-1 regulator 1 (Ambra1) enhances Ulk[1] kinase activity.[4] Microtubule- associated protein. The poor cell viability may further lead to the limited therapeutic efficacy.[9] Recent works have shown that autophagy protects the bone marrowderived MSCs from apoptosis under hypoxia and serum deprivation.[10,11,12]
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