The Effects And Mechanism Of Autophagy In Mesenchymal Stem Cells Stenrness Maitaining Injuryed By Irradiation

Stem cells are biological cells found in all multicellular organisms, that can divide (throughmitosis) and differentiate into diverse specialized cell types and can self-renew to producemore stem cells. Stem cells were characterised by their stemness: self-renewal andpluripotency. Self-renewal was describesd the proliferation of undifferentiated stem cellswithout lineage commitment, while pluripotency refers to the ability of undifferentiated stemcells to differentiate into a variety of cell lineages and tissues.Mesenchymal stem cell（sMSCs）are a unique type of adult stem cells that differentiate notonly into the same mesodermal-lineage such as bone, cartilage, and adipocytes but alsodifferentiate into other lineages of ectodermal and endodermal cells. MSCs have been provento be involved in hematopoietic support, immunoloregulation, tumorigenesis, in addition,MSCs chemoattracted to damaged sites and differentiated into cells specific to the tissue. Itwould lose self-renewal capacity and multi-differentiation potential, what resulting in tissueimpair, even senescence or tumorgenesis when the stemness of MSCs was injured.Autophagy is a catabolic pathway characterized by the formation of a double-membranevesicle, called the autophagosome, which engulfs cytoplasmic components and delivers themto lysosomes for degradation. Autophagy is a conserved proteolytic mechanism that degradescytoplasmic material, including cell organelles and is important in maintaining intracellularhomeostasis and keeping the cell healthy, which can be activated as adaptive response toadverse environmental conditions, such as deprivation of nutrients, hypoxia, and differenttypes of therapeutic stress. There is some evidence that autophagy is an important event instem cells maintaining stemness. Mortensen et al reported that autophagic mechanisms wereactivated in hematopoietic stem cells (HSCs). HSCs lacking autophagy-related gene ATG7,an essential autophagy protein, were unable to survive in vivo. These reports suggested thatautophagy was important for the maintenance and differentiation of the HSCs. Other studyshowed that under conditions of autophagy blockade with3-MA or ATG5-shRNA, epidermal (ESC), dermal (DSC), and HSC underwent. So we investigated the roleof autophagy in preserving self-renewal and multi-differentiation potential of the MSCsexposed irradiation.When cells are exposed to ionizing radiation, decomposition reactions occur and a varietyof ROS are generated. Various defense mechanisms have been developed to protect cellsagainst oxidative stress, such as up-regulation of antioxidants, removal of specific proteins bythe ubiquitin–proteasome system and removal of damaged proteins and organelles byautophagy. When ROS be at high cytotoxic levels, the possible role of autophagy isrestraining oxidative damage from reaching cytotoxic levels as well as maintaining energyhomeostasis. For instance, T lymphocytes deficient for the autophagy genes ATG3,5or7,show abnormal mitochondria accumulation and expanded endoplasmic reticulum due toimpaired organelle homeostasis. As a result, there is an increase in ROS production, affectingthe survival of T lymphocytes. The report showed that ATG7-/-hematopoietic stem andprogenitor cells (HSPC) significantly accumulates more aberrant mitochondria, show elevatedmitochondrial superoxide levels, DNA damage and an atypical myeloproliferation. Lackingautophagy, iPS lost of self-renewal capacity. So we assume that increasing ROS levelsinduced by irradiation lead to DNA damage, thus destroy the stemness and multipotential ofMSCs. Autophagy may degrade defective mitochondria, prevent excessive ROS generationthat could induce DNA damage and protect MSCs from irradiation injury.In order to evaluate the influence of autophagy on MSCs stemness maintaining inirradiation enviroment, the subject would be carry out from the following three aspects: First,the effect of ionizing radiation on MSCs maintaining stemness; Second, the role of autophagyinduced by starvation in MSCs stemness maintaining; Third, the mechanism of autophagy inMSCs stemness maintaining injuryed by irradiation.Part1The effect of ionizing radiation on MSCs maintaining stemnessIn this study, we investigated the effect of ionizing radiation on MSCs maintainingstemness. We first compared the specific surface markers of umbilical cord-derived MSCs invitro, irradiated or not. We showed that the expression of CD105, CD90and CD29in irradiated-MSCs appeared to be less than control group. Moreover, the CFU-F numbers andthe mean colony size were significantly smaller in irradiated-MSCs than control group. Wethen demonstrated that irradiated-MSCs produced the greatly decrease in transcription factorsNanog, Oct4and Sox2related with stmness expression compared with control group byReal-time PCR. The induced differentiation studies show that the osteogenic relatedmarkers ALPL and OGN and transcript factor RUNX2mRNA expression down-regulated inirradiated-MSCs. The calcium nodules number of irradiated-MSCs stained by Alizarin Redsignificantly reduced compared with the control group. The adipogenic related markers andtranscript factor LPL, CFD and PPAR-γmRNA expression also down-regulated inirradiated-MSCs. The number of lipid droplets formed by oil red O staining decreased inirradiated-MSCs compared with the control group. Our results suggest that irradiation injuredthe self-renewal and multi-differentiation potential of MSCs.Part2The role of autophagy induced by starvation in MSCs stemness maintainingIn this study, we investigated the mechanism of MSCs maintain stemness after irradiationinjury. MSCs were cultured in serum-free medium as exogenous stimulation inducedautophagy occurs. pretreatment of starvation, then exposed to X-ray irradiation, MSCs wereobserved. Then we found that the calculated efficiency for CFU-F of irradiated-MSCs waslower than those of starvation pretreated group. The mRNA expressions of pluripotenttranscription factors Nanog, Oct4and Sox2were up-regulated when irradiated-MSCs werepretreated with starvation. The induced differentiation experiments also observed the sameresults, starvation pretreatment of irradiated-MSCs, can be found in osteogenic, adipogenicdifferentiation potential increased. Autophagy as a defense mechanism to protect the damagedcells, so we studyed whether autophagy mediated reversing the injury of MSCs stemnessinduced by irradiation. Consistent with the expected results, autophagy occurred in thestarvaion pretreated MSCs. Furthermore, we bloked autophagy with pharmacal inhibitor3-MA and CQ, then detected irradiation damage to MSCs stemness maintaining afterautophagy inhibition. The results illustrated that when autophagy in irradiated-MSCs pretreated with starvation was deleted by autophagy inhibitor, the self-renewal capacity andpotential of MSCs osteogenesis and adipogenesis obviously decreased. Our results suggestthat the autophagy induced by starvation can reverse the self-renewal capacity andmulti-differatation potential of MSCs injury by irradiation.Part3The mechanism of autophagy in MSCs stemness maintaining injuryed byirradiationIt is well documented that exposured to irradiation causes cells to generate ROS andinduces single-strand and double-strand DNA broken. Most ROS were generated in cells bythe mitochondrial respiratory chain. Autophagy has a known role in mitochondrial qualitycontrol by degrading damaged mitochondria, the reduction in mitochondrial membranepotential could reflect an accumulation of damaged mitochondria. Thus, we investigatewhether autophagy played a protective role in the MSCs during exposed ionizing radiationthrough decreasing ROS generation and reducing cell DNA damage. We access the ROS level,frequency DNA damage, the mitochondrial ROS and mitochondrial membrane potential inirradiated-MSCs pretreated with starvation, the results demonstrated induction autophagy inirradiated-MSCs decreased mitochondrial ROS generation, maintained mitochondrialmembrane potential and reduced cell DNA damage. Inhibition autophagy in irradiated-MSCsincreased ROS generation and aggravated cell DNA damage. These results indicate thatstarvation inducing autophagy in MSCs cleared of mitochondria damaged by ionizingradiation, deleted ROS generation and reduced cell DNA damage, so MSCs maintained theirstemness after irradiation sion injury..Conclusions：1. Ionizing radiation not only damage the self-renewal capacity of MSCs, but also damagethe multi-differentiation potentia, so that the loss of MSCs stemness partly.2. Autophagy induced by starvation reversed the damage of radiation to MSCs self-renewaland differentiation potential, and keep them stemness. 3. Autophagy induced by starvation in MSCs cleared of mitochondria damaged by ionizingradiation, deleted ROS generation and reduced cell DNA damage, so MSCs maintained theirstemness after irradiation injury.