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AICAR and nicotinamide treatment synergistically augment the proliferation and attenuate senescence-associated changes in mesenchymal stromal cells.

ABSTRACT: BACKGROUND:Mesenchymal stromal cell (MSC) stemness capacity diminishes over prolonged in vitro culture, which negatively affects their application in regenerative medicine. To slow down the senescence of MSCs, here, we have evaluated the in vitro effects of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, and nicotinamide (NAM), an activator of sirtuin1 (SIRT1). METHODS:Human adipose-derived MSCs were cultured to passage (P) 5. Subsequently, the cells were grown in either normal medium alone (control group), the medium supplemented with AICAR (1?mM) and NAM (5?mM), or in the presence of both for 5 weeks to P10. Cell proliferation, differentiation capacity, level of apoptosis and autophagy, morphological changes, total cellular reactive oxygen species (ROS), and activity of mTORC1 and AMPK were compared among different treatment groups. RESULTS:MSCs treated with AICAR, NAM, or both displayed an increase in proliferation and osteogenic differentiation, which was augmented in the group receiving both. Treatment with AICAR or NAM led to decreased expression of ?-galactosidase, reduced accumulation of dysfunctional lysosomes, and characteristic morphologic features of young MSCs. Furthermore, while NAM administration could significantly reduce the total cellular ROS in aged MSCs, AICAR treatment did not. Moreover, AICAR-treated cells possess a high proliferation capacity; however, they also show the highest level of cellular apoptosis. The observed effects of AICAR and NAM were in light of the attenuated mTORC1 activity and increased AMPK activity and autophagy. CONCLUSIONS:Selective inhibition of mTORC1 by AICAR and NAM boosts autophagy, retains MSCs' self-renewal and multi-lineage differentiation capacity, and postpones senescence-associated changes after prolonged in vitro culture. Additionally, co-administration of AICAR and NAM shows an additive or probably a synergistic effect on cellular senescence.

SUBMITTER: Khorraminejad-Shirazi M

PROVIDER: S-EPMC6998366 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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AICAR and nicotinamide treatment synergistically augment the proliferation and attenuate senescence-associated changes in mesenchymal stromal cells.

Khorraminejad-Shirazi Mohammadhossein M Sani Mahsa M Talaei-Khozani Tahereh T Dorvash Mohammadreza M Mirzaei Malihe M Faghihi Mohammad Ali MA Monabati Ahmad A Attar Armin A

Stem cell research & therapy 20200203 1

<h4>Background</h4>Mesenchymal stromal cell (MSC) stemness capacity diminishes over prolonged in vitro culture, which negatively affects their application in regenerative medicine. To slow down the senescence of MSCs, here, we have evaluated the in vitro effects of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, and nicotinamide (NAM), an activator of sirtuin1 (SIRT1).<h4>Methods</h4>Human adipose-derived MSCs were cultured to passage (P) 5. Subsequently, the cells were ...[more]

PMID: 32014016

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Project description:Mesenchymal stromal cells (MSCs) are potent regulators of immune responses largely through paracrine signaling. MSC secreted extracellular vesicles (MSC-EVs) are increasingly recognized as the key paracrine factors responsible for the biological and therapeutic function of MSCs. We report the first comprehensive study demonstrating the immunomodulatory effect of MSC-EVs on dendritic cell (DC) maturation and function. MSC-EVs were isolated from MSC conditioned media using differential ultracentrifugation. Human monocyte-derived DCs were generated in the absence or presence of MSC-EVs (20 ug/ml) then subjected to phenotypic and functional analysis in vitro. MSC-EV treatment impaired antigen uptake by immature DCs and halted DC maturation resulting in reduced expression of the maturation and activation markers CD83, CD38, and CD80, decreased secretion of pro-inflammatory cytokines IL-6 and IL-12p70 and increased production of anti-inflammatory cytokine TGF-β. MSC-EV treated DCs also demonstrated a diminished CCR 7 expression after LPS stimulation, coupled with a significantly reduced ability to migrate toward the CCR7-ligand CCL21, although they were still able to stimulate allogeneic T cell proliferation in vitro. Through microRNA profiling we have identified 49 microRNAs, which were significantly enriched in MSC-EVs compared to their parent MSCs. MicroRNAs with known effect on DC maturation and functions, including miR-21-5p, miR-142-3p, miR-223-3p, and miR-126-3p, were detected within the top 10 most enriched miRNAs in MSC-EVs, with MiR-21-5p as the third highest expressed miRNA in MSC-EVs. In silico analysis revealed that miR-21-5p targets the CCR7 gene for degradation. To verify these observations, DCs were transfected with miR-21-5p mimics and analyzed for their ability to migrate toward the CCR7-ligand CCL21 in vitro. MiR-21-5p mimic transfected DCs showed a clear trend of reduced CCR7 expression and a significantly decreased migratory ability toward the CCL21. Our findings suggest that MSC-EVs are able to recapitulate MSC mediated DC modulation and MSC-EV enclosed microRNAs may represent a novel mechanism through which MSCs modulate DC functions. As MSCs are currently used in clinical trials to treat numerous diseases associated with immune dysregulation, such as graft-versus-host disease and inflammatory bowel disease, our data provide novel evidence to inform potential future application of MSC-EVs as a cell-free therapeutic agent.

Dataset Information

AICAR and nicotinamide treatment synergistically augment the proliferation and attenuate senescence-associated changes in mesenchymal stromal cells.

Publications

AICAR and nicotinamide treatment synergistically augment the proliferation and attenuate senescence-associated changes in mesenchymal stromal cells.

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