To bridge the gap between limited number of acquirable stem cells from donors and the large quantity of stem cells that are needed for tissue engineering, it is necessary to apply long-term in-vitro passaging for cell large-scale production, especially in autologous MSC transplantation . However, accumulating evidence suggested that long-term passaging might cause cellular senescence, with unexpected alterations including anomalous morphology , hindered proliferation capacity , decreased differentiation potential including chondrogenesis and osteogenesis . To avoid the senescence-associated alterations, the passage 2 to passage 5 cells, which were believed to be possessed with analogical capacity in self-renewal and differentiation, were often chosen as objective of study in numbers of studies. What’s more, one research suggested that the stemness of MSCs could only be retained up to 6 passages, and significantly declined after 10 passages . Another study suggested that the proliferation, differentiation and immunomodulation functions of mouse bone marrow-derived MSCs decreased after 15 passages . Here, we chose P2 cells as young group, P7 and P15 cells as senescent group to study the senescence-associated alteration along with long-term in-vitro expansion. In consistent with previous investigations, our study reported that in PDLSCs, long-term in-vitro expansion led to senescence evidenced by increased SA-β-gal activity, elevated expression of senescence-associated proteins p16, p21 p53 and γ-H2AX, which symbolized arrested cell cycle and DNA damage.
In order to overcome the challenge of accompanied senescence during in-vitro passaging, it’s prerequisite to find the mechanism in lone-term expansion-caused senescence. It has been revealed that hindered autophagy was concomitant with age-related disorders [38, 39], organismal aging , and also senescent stem cells [12–13]. Furthermore, inhibition of autophagy could accelerate neurodegenerative disease by accumulating disease-causing aggregate-prone proteins  and also induce kidney damage in aging mice . These results indicted indivisible relationship between autophagy and senescence. To our knowledge, the autophagy level of senescent stem cells after long-term passaging has hardly been tested before. In consistent with physiological or disease-related organismal and cellular senescence, a decline in basal autophagy was firstly found in long-term in-vitro expansion-caused cellular senescence in our study, as evidenced by impeded autophagic flux, prohibited expression of autophagy-related proteins including Atg7, Beclin-1 and LC3-II and accumulated autophagy substrate p62. To further verify the connection between autophagy alteration and cellular senescence, autophagy inhibitor 3-MA was supplemented. We found that 3-MA-mediated autophagy inhibition could deteriorate the accumulation of senescence, evidenced by elevated SA-β-gal capacity, worsened DNA damage and arrested cell cycle, suggesting hindered autophagy could deteriorate cellular senescence. On the contrary, previous study has found that the restored autophagy could alleviate the senescent status and reach functionally improvement. Pharmaceutical treatment with urolithin A to induce autophagy could improve the age-related declined muscle function and enhance exercise capacity in old rodents . The restored autophagy mediated by caloric restriction could decrease the cellular ROS level and enhance the regenerative ability of aging stem cells . Stimulation of lysosomal activity to promote autophagy in the aged neural stem cells significantly enhanced their ability for neural regeneration . These results collectively indicated that the regulation of autophagy could be a potential strategy for functional maintenance of senescent stem cells.
Up to now, researchers have made great effort to restore the functionality of senescent stem cells. The strategies to reach optimum cultivation for restoring or promoting capacity of stem cells can be generally divided into three categories. One is to mimic the in-vivo microenvironment and induce prior expression of cytoprotective and stemness-associated genes, including but not limited to providing an extracellular matrix (ECM)-simulated environment, and a short-term hypoxia preconditioning. Another is to utilize exogenous molecules to activate extracellular or intracellular signaling for resisting the harsh in situ microenvironment caused by ischemia, nutrition-deprivation and oxidative damage, such as pretreatments using exogenous cytokines, growth factors and other small molecule drugs. In addition, genetic strategy modulates the stem cells to acquire superior capacity in self-protection, proliferation and differentiation. Although progress has been made using former strategies, further problems have arisen. Hypoxic preconditioning was reported to give rise to arrested cell cycle and prohibited cell proliferation [46, 47]. The utilization of exogenous signaling proteins seemed to have relative low efficiency and greater expense on account of their short half-life time . Genetic engineering may cause the risk of insertional mutagenesis and oncogene transaction. Thus, other strategies that simultaneously prossess features of high efficiency and secured application are needed in restoring the functionality of senescent stem cells. As confirmed by recent studies, MLT exhibits unique superiority in tissue engineering. As a pleiotropic endogenous hormone secreted by pineal and almost every other tissue, MLT was confirmed to have low toxicity during treatment by both animal and clinic studies [49, 50]. In several studies, MLT could enhance the survival stability by attenuating apoptosis, resisting oxidative stress and inflammation damage , improve the chondrogenesis and osteogenesis of MSCs , and also maintain the stemness of long-term passaged stem cells, resulting in preserved proliferation, osteogenic differentiation and immunomodulation capacity . Except from the well-known role in stem cell-based therapy as a probable free radical scavenger with excellent anti-inflammation and differentiation enhancement properties, recently, researchers also found that MLT could function as a regulator for autophagy. In some studies, MLT could promote the basal levels of autophagy under physiological conditions and maintain neuronal homeostasis and survival from a subarachnoid hemorrhage followed by brain injury , and also modulates autophagy to attenuate the cardiac ischemia/reperfusion injury [54, 55]. The property of MLT-mediated regulation of autophagy could also benefit in some age-related diseases. It was found that MLT in-vivo application ameliorated AD-induced cardiac atrophy , and enhanced the impeded cognitive function in tau-related AD rats  by restoring the autophagic flux. What’s more, MLT could improve degradation of damaged mitochondria by mitophagy during aging and under neurodegenerative conditions . Similar to the studies mentioned above, our study also confirmed that MLT could effectively restore the cellular autophagy not only by elevating the basal autophagy level in senescent P15 cells, but also by reinstating the autophagic level by enhancing the lysosome-dependent degradation of autophagosomes. Our study also found that restored autophagy was further accompanied with ameliorated senescence. To further clarify if it was the restored autophagy that benefited the rejuvenation effects, we found that the defective autophagy caused by the autophagy inhibitor 3-MA could consequently abrogate the MLT-mediated rejuvenation. Therefore, our study suggests that the restored autophagy alleviated cellular senescence, and the manipulation of autophagy could be an effective strategy to maintain functionality of senescent cells.
To investigate the underlying mechanism of MLT-mediated autophagy restoration, we first explored if the effect was dependent on MT. We supplemented MT-specified inhibitor LUZ into cultured cells. We found that LUZ could further exacerbate the impeded autophagy in senescent cells without MLT treatment, giving rise to aggravated cellular senescence. Even in senescent cells treated with MLT, LUZ could abrogate the inducible effect of autophagy mediated by MLT, and further inhibited the rejuvenation mediated by MLT. These results indicated that the MLT-mediated autophagy restoration was in an MT-dependent manner in senescent cells. What’s more, mTOR plays a role as central switch to autophagy. The inhibitory regulation of PI3K/AKT/mTOR pathway can activate autophagy and as reported, several studies chose to target PI3K/AKT/mTOR pathway for autophagy regulation in stem cells [57–59]. Accordingly, our study demonstrated that the expression of p-PI3K, p-AKT and p-mTOR significantly decreased after the treatment of MLT but was reversely induced to increase by LUZ treatment. We thus concluded that the MT-dependent MLT-mediated autophagy was possibly through the inhibitory regulation of the PI3K/AKT/mTOR pathway. This is in consistence with a current study, which confirmed that MLT could regulate the PI3K/AKT pathway in an inhibitory way .
To precisely confirm the MLT-mediated autophagy through MT/PI3K/AKT/mTOR pathway, we respectively applied LUZ, PI3K/AKT specific agonist SC79 and mTOR agonist MHY1485 with the MLT treatment, and found that the autophagy restoration and senescence attenuation effect mediated by MLT could be abrogated with blocked MT and activation of PI3K/AKT/mTOR pathway. However, our study didn’t determine the exact type of MT involving in the MLT-mediated autophagy restoration, which should be our main pursue in the future.
To sum up, our study revealed that impaired autophagy was one of the vital mechanisms of cellular senescence induced by long-term ex-vivo expansion, which can be restored by MLT through MT/ PI3K/AKT/mTOR pathway. This work innovatively purposes a strategy that MLT treatment targeting impeded autophagy can be utilized to rejuvenate stem cells during long-term passaging. The investigation focused on the mechanism that inhibiting the PI3K/AKT/mTOR pathway could enhance autophagy to attenuate cellular senescence, which may provide possible target for the research and even clinical application aiming at the rejuvenation of stem cells for cellular therapy.