1Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
2Department of Anatomical Sciences, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran.
Mesenchymal stem cells (MSCs) are valuable for cell-based therapy. However, their application is limited owing to their low survival rate when exposed to stressful conditions. Autophagy, the process by which cells recycle the cytoplasm and dispose of defective organelles, is activated by stress stimuli to adapt, tolerate adverse conditions, or trigger the apoptotic machinery. This study aimed to determine whether regulation of autophagy would affect the survival of MSCs under stress conditions.
Autophagy was induced in bone marrow-derived MSCs (BM-MSCs) by rapamycin, and was inhibited via shRNA-mediated knockdown of the autophagy specific gene,
Of 4 specific
Autophagy modulation in MSCs can be proposed as a new strategy to improve their survival rate in stressful microenvironments.
Mesenchymal stem cells (MSCs) are valuable cells in cell-based therapy and have been extensively investigated as a new modality for treatment of several diseases . Different tissue-derived MSCs were characterized and expanded
Practical strategies should therefore be employed to improve the underlying mechanisms of MSC resistance to harsh stress conditions, thus leading to higher survival rates and consequently higher engraftment rates after transplantation .
Autophagy (self-digestion), also described as the garbage disposal system of the cell, involves lysosomal-mediated catabolism of long-lived proteins and damaged organelles, which are sequestered by autophagosomes or specific autophagy vacuoles . Basal autophagy regulates a wide range of physiological and pathophysiological processes, from starvation adaptation and homeostasis to anti-aging and cell death . Autophagy was initially described as a survival mechanism that recycles degraded intracellular components to serve as new building blocks for the synthesis of macromolecules and metabolites, which were then used as the cellular energy source for cell survival . However, autophagy seems to play dual roles depending on the cell type and context. In the context of cellular death, the persistent and extensive occurrence of autophagy leads to the catabolism of cytoplasmic materials, to an extent that causes metabolic and bio-energetic cell collapse. Recent studies have shown that autophagy might act as a double-edged sword, leading to either cell death or survival by inactivation of key autophagy genes, particularly
Interestingly, few studies have investigated autophagy in stem cells , including MSCs, and its role in this cell type is still controversial. This study therefore aimed to address whether autophagy protects or exacerbates MSCs following exposure to various types of stress conditions.
Human MSCs were isolated from the bone marrow of healthy volunteers in the bone marrow transplantation (BMT) center of Shariati hospital under the institutional protocol and consent procedure. These human bone marrow-derived-mesenchymal stem cells (hBM-MSCs) were successfully isolated using their adhesive characteristics as previously described . hBM-MSC surface markers were detected by flow cytometry and their differentiation potentiality for osteogenesis, chondrogenesis, and adipogenesis was analyzed as previously described .
To suppress autophagy in hBM-MSCs, the RNAi (RNA interference) technique was performed, since it is the most specific strategy for knockdown studies. SureSilencing short hairpin RNA (shRNA) plasmids targeting human
Following plasmid extraction, hBM-MSCs were transfected with the negative control and each of the 4 shRNA vectors using the transfection reagent FuGENE HD (Roche, Germany) according to the manufacturer's protocol. The transfected cells were named MSC-shRNA 1, MSC-shRNA 2, MSC-shRNA 3, MSC-shRNA 4 according to the number of the shRNA vector, and MSC-shRNA Cont for the negative control shRNA transfected MSCs. A plasmid containing EGFP conjugated ubiquitous microtubule-associated protein 1A/1B-light chain 3 (pEGFP-LC3m) was obtained from Dr. Y. Kuwahara, Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Miyagi, Japan as a gift. hBM-MSCs were also transfected with pEGFP-LC3m, and were named MSC-LC3. After transfection for 48 hours, MSC-LC3 cells were observed under a fluorescence microscope. RT-PCR analysis was performed to select proper shRNA vectors, which could effectively knock down
Rapamycin (Rapa), a well-known inducer of autophagy, was used to activate the autophagy pathway. To optimize rapamycin (Invitrogen, USA) concentration, hBM-MSCs were cultured in 96-well plates (Orange Scientific, Belgium), and different rapamycin doses (10, 200, and 500 nM) were added and incubated for 24 hours in a standard cell-culturing incubator. The rapamycin-treated hBM-MSCs were named MSC-10 nM Rapa, MSC-200 nM Rapa, and MSC-500 nM Rapa according to the rapamycin dose applied. MSC-LC3, MSC-shRNA Cont, and MSC-shRNA 3 treated with the optimized rapamycin dose were named MSC-LC3-Rapa, MSC-shRNA Cont-Rapa and MSC-shRNA 3-Rapa, respectively. These rapamycin-treated hBM-MSCs were then subjected to western blot analysis.
Ubiquitous microtubule-associated protein 1A/1B-light chain 3 (LC3) is a soluble protein in mammalian cells. During the autophagy process, the cytosolic form of LC3 (LC3-I) is converted to a lipid bound form (LC3-II) . Western blot analysis was performed to evaluate these autophagy markers (LC3-I and LC3-II) at the translational level. Whole cell extract was isolated from different rapamycin-treated cells as well as the untreated control. Samples were run on 16% SDS-polyacrylamide gel, and then, transferred to a PDVF membrane. Samples were then incubated with diluted rabbit anti-LC3-I and LC3-II (1:1,000) (Cell Signaling Technology, USA) primary antibodies for 3 hrs. After primary antibody incubation, the membrane was washed 3 times, and then incubated with an HRP-conjugated goat anti-rabbit secondary antibody for 1.5 hrs. Finally, enhanced chemiluminescence (ECL) (Abcam, UK) substrate solutions were used to visualize the proteins on the PVDF membrane using a chemiluminescence imaging system. β-actin served as an internal loading control protein.
Total RNA was extracted from transfected hBM-MSCs using the Tripure reagent (Roche, Germany) according to the manufacturer's instructions. The first-strand cDNA was generated from 1 µg of the isolated total RNA using the cDNA synthesis kit (Bioneer, USA) according to the manufacturer's instructions. Appropriate primer sets for
PCR reactions were performed using Taq DNA polymerase (Roche, Germany).
For further confirmation of the RT-PCR results, cDNA from MSC-shRNA 3 and MSC-shRNA Cont was subjected to qPCR analysis to quantify the suppression level of
To investigate the effects of autophagy on cellular survival rate, the different groups of hBM-MSCs including autophagy-suppressed hBM-MSCs (MSC-shRNA 3), Rapamycin-treated hBM-MSCs (MSC-Rapa), MSC-shRNA Cont, and normal h-BM-MSCs without any treatment (MSC-Cont) were cultured under hypoxic, serum-deprived, and oxidative stress conditions. Cells were seeded at a density of 2×104 cells/well in a 96-well plate. To induce oxidative stress, cells were exposed to different doses of 1, 2, 3, 4, and 5 mM hydrogen peroxide (H2O2) (Sigma, Germany) for 1 hour. For induction of serum deprivation, cells were washed three times with serum-free medium and maintained in this condition for various lengths of time. The hypoxic condition was created by transferring plates to a Galaxy 48 R hypoxic incubator (New Brunswick, Germany). These cells were incubated at 37℃ in the presence of 5% CO2, 1% O2, and N2 for different lengths of time. Each test was done in triplicate and the cytotoxic effects of H2O2, hypoxia, or serum deprivation on cells were determined by a WST-1 cytotoxicity assay. The viability of the four MSC groups: MSC-shRNA3, MSC-Rapa, MSC-shRNA Cont and MSC-Cont, were also studied under normal conditions
A WST-1 assay was performed to evaluate the viability of MSCs. The WST-1 reagent (Sigma, Germany) was added to cells at a concentration of 10 µL/well (Sigma, Germany) in 100 µL culture medium and the samples were incubated at 37℃ for 4 hours. The optical density (OD) of each sample was measured using a microplate reader at 450 nM.
The quantitative results were analyzed using the SPSS statistical software (version 19) (SPSS Inc., Chicago, IL, USA). Statistically significant differences (
Next, qPCR was performed to evaluate the level of
Rapamycin was then added to cells at different concentrations in order to induce autophagy. Following activation of the autophagy pathway, the conversion of LC3I to LC3II leads to a higher intensity of the LC3II band and an elevated relative ratio of LC3 II to LC3 I. The induction of autophagy was detected after 24-hour-treatment with 500 nM rapamycin (Fig. 2A).
Autophagy was also investigated in hBM-MSCs transfected with GFP-LC3 plasmids (MSC-LC3). MSC-LC3 were exposed to 500 nM rapamycin (the optimized autophagy inducing dose) for 24 hours (MSC-LC3-Rapa), and then observed under a fluorescence microscope. Rapamycin induced stimulation of autophagy resulted in the formation of shiny green dots in MSC-LC3-Rapa (Fig. 2B.I), which were not observed in untreated MSC-LC3 (Fig. 2B.II). MSC-shRNA 3 and MSC-shRNA Cont were also treated with 500 nM rapamycin (MSC-shRNA 3-Rapa and MSC-shRNA Cont-Rapa, respectively). Induction of autophagy was observed in MSC-shRNA Cont-Rapa as determined by the presence of shiny green dots (Fig. 2B.III). However this was not observed in MSC-shRNA 3-Rapa, indicating that rapamycin treatment could not induce autophagy under
Western blot analysis of MSC-shRNA Cont-Rapa and MSC-shRNA 3-Rapa also indicated that the down-regulation of
To determine the effects of autophagy induction or inhibition on the survival rate of MSCs, cells were cultivated under normal conditions for five days, and cell viability was evaluated using the WST-1 assay. Survival of MSCs increased with the induction of autophagy and decreased with autophagy inhibition, however the differences were not significant (
Next, all hBM-MSCs groups including autophagy-suppressed MSCs (MSC-shRNA 3), autophagy-induced MSCs (MSC-Rapa) and their relevant controls, MSC-shRNA Cont, and normal MSCs without any treatment (MSC-Cont) were exposed to hypoxic, serum-deprived, and oxidative stress conditions. As shown in Fig. 3B, the cells with
Following 24-hour exposure to hypoxic conditions, the viability of MSC-shRNA 3 was higher than that of MSC-Rapa and the control group. This indicates that
The advancement of MSC-based cell therapy is hindered by the low viability of infused MSCs. Harsh microenvironments such as those induced by hypoxia, serum deprivation, and oxidative stress are well-known causes of MSC death
Autophagy was one of the most ambiguous concepts, due to its dual role in cellular life and death depending on the cell source and the type of stress the cell is exposed to .
Our findings indicate that inhibition of
More recently, Song et al. suggested that autophagy induction is a survival response to oxidative stress in murine bone marrow-derived mesenchymal stromal cells . In their study, autophagy was inhibited via non-specific genes (
Here, we provide evidence that the induction of autophagy induces cell death while its inhibition protects MSCs in response to severe stressful microenvironments. Our findings highlight the importance of autophagy regulation in the fate of MSCs, and suggest a possible new strategy to prevent the death of engrafted cells in MSC-based cell therapy. It also emphasizes that management of cellular responses to stress can be harnessed in practical applications.
In conclusion, autophagy modulation can be proposed as a new strategy for improving the efficacy of MSC-based cell therapies as a result of enhanced survival rates. However, further ongoing studies including
Confirmation of autophagy suppression via
Confirmation of autophagy induction with rapamycin.
WST-1 assay to detect the effects of autophagy on hBM-MSC survival.