Adipose-derived Stem Cells Modified With PGC-1α Resists Apoptosis Role In A Diabetic Microenvironment And Its Mechanism

Background:Diabetes mellitus is a group of metabolic disease characterized by chronichyperglycaemia associated with a series of vascular and neural complications, which hassome features with high morbidity, large hazard, hard conventional therapy and expensivecost. The more effective therapeutic options are need badly to develop; stem cell therapyprovides new hope for diabetes and its complications in recent years. However, effect ofhyperglycaemia and deprivation of nutrient and oxygen in the diabetic regionmicroenvironment, the apoptosis or necrosis of a large number of transplanted stem cells isa critical issue that limits the therapeutic efficacy of stem cells. Therefore, how to increasethe survival rates of the stem cells and enchance angiogenesis potential has been an urgentresolved problem in the treatment of the diabetes and its complications.Peroxisome proliferator-activated receptor-gamma coactivator1alpha (PGC-1α) beinga key transcriptional coactivator involved in the regulation of mitochondrial function andenergy metabolism was found in1998, which binds with many receptors and plays animportant role in mitochondrial biogenesis, glucose metabolism, fatty acid oxidation, celldifferentiation and angiogenesis. In the meantime, many studies have shown that PGC-1αinvolves in anti-apoptosis in recent years. Our recent study suggested that PGC-1α plays animportant role in anti-apoptosis and mediated angiogenesis in mesenchymal stem cells indiabetic hindlimb ischaemia by inducing an increase in hypoxia inducible factor-1α(Hif-1α), a higher ratio of B-celllymphoma/leukaemia-2(Bcl-2)/Bcl-2-associated X protein(Bax) in bone marrow derived mesenchymal stem cells (BMSCs).Recently, adipose-derived stem cells (ASCs) have become the focus of attention in thefield of stem cells transplantation because of their ease of isolation, relative abundance, andrapidity of growth. Compared with BMSCs, the rate of stem cells derived from of fat tissueis higher than that of bone marrow. There is no influence of cryopreservation on cell growthand phenotype. ASCs have the potential of immune privilege. The method of harvest ASCs is simple and safe, which can be performed by liposuction. Emerging evidence suggests thatASCs will be an ideal source of stem cells in regenerative medicine and tissue engineering.Therefore, in this study, ASCs were isolated and cultured, whether the overexpressionof PGC-1α protects ASCs from apoptosis by reducing ROS production and ameliorating themitochondrial damage induced by a diabetic environment were examined, which maycontribute to the development of new approaches and provide target cells for preventingcomplications from diabetes.Materials and methods：1. Isolation, culture and identification of Sprague-Dawley (SD) rat ASCsASCs were isolated and cultured with the method of collagenase digestion; cells wereexamined as follow after three passages.1.1The cell morphology was observed by inverted microscope.1.2The cell surface antigens CD90-FITC, CD105-FITC, CD34-PE and CD45-PE weredetected by flow cytometry.1.3Multilineage differentiation were accomplished using a special kit to determinewhether the culture cells were capable of adipogenic differentiation and osteogenicdifferentiation by oil red O staining and staining calcium deposits with alizarin red.1.4Trypan blue exclusion test was used to determine viability of cells.2. Overexpression of PGC-1α in ASCs using adenoviral vector encoding PGC-1α andGFP (Ad-GFP-PGC-1α or PGC-1α-ASC).2.1Adenovirus amplification and examination of the titer of the adenovirus.2.1.1HEK293cells were used to amplify the adenovirus.2.1.2The virus titer was determined by tissue culture infectious dose50(TCID50)assay.2.2ASCs transfection of adenovirus-mediated gene.2.2.1Multiplicity of infection (MOI) with high efficiency and low toxicity wasdetected by flow cytometry, and the ideal MOI was selected for the following experiments.2.2.2Western blotting (WB) was used to examine the protein levels of PGC-1α.2.3Resistant-apoptosis effect of ASCs with overexpression of PGC-1α under highglucose, hypoxia and serum deprivation conditions.2.3.1After being infected for48h, the apoptosis of ASCs modified with PGC-1α orGFP was induced by culture conditions of high glucose concentration (30mmol/L), hypoxia (5%O2) and serum deprivation. At0h,6h and24h after culture, the survival ratesof ASCs were measured using an Annexin V-APC/7-Amino-Actinomycin (7-AAD)apoptosis detection kit.2.3.2After being incubated GFP-ASC and PGC-1α-ASC under high glucose, hypoxiaand serum deprivation conditions for24h, the level of cellular ROS was deteced byfluorescent probe dihydroethidium (DHE) labelling.2.3.3Mitochondrial ROS production was detected by MitoTracker Red CM-H2XRos.2.3.4Transmission electron microscopy was used to observe the morphology andstructure of ASCs modified with PGC-1α or GFP under culture conditions of high glucose,hypoxia and serum deprivation for24h.Results:1. Isolation, culture and identification of rat ASCs1.1The multipotent stem cells being isolated from inguinal subcutaneous fat of SDrats could be culture and stable multiplication in vitro. The cells were able to grow adheringto the plastic wall and the morphology is obvious long fusiform shape like fibroblasts,which was present in regular arrange with certain polarity as school of fish. When beinggenerated to two or three passages, the cells morphology became big and flat.1.2After being generated third passage, the cell surface antigens were examined by theflow cytometry, the results showed that94.26%and95.83%ASCs were CD90and CD105positive respectively; however, only0.16%and0.09%ASCs were CD34and CD45antigenpositive respectively.1.3Multilineage differentiation was accomplished using a special kit to determinewhether the cultures were capable of adipogenic differentiation and osteogenicdifferentiation. The results indicated that the cultured rat ASCs could differentiate intoadipocytes, which could be stained by oil red O. ASCs could also differentiate intoadipocytes, the calcium deposits could be stained with alizarin red. But these of the controlwere all negative.1.4Trypan blue exclusion test was used to determine viability of cells; the resultshowed that the viability of cells was97%. The dead cells were few and could be stained bytrypan blue. The live cells were clear and could not be stained by trypan blue.2. Overexpression of PGC-1α in ASCs using adenoviral vector encoding PGC-1α andGFP (Ad-GFP-PGC-1α or PGC-1α-ASC) 2.1Adenovirus amplification and examination of the titer of the adenovirus2.1.1HEK293cells were used to amplify the adenovirus.95%HEK293cellsexpressed GFP at24h after being infected with Ad-PGC-1α under fluorescence microscope,the cells appeared obvious cytopathic effect at48-72h after infection. The cells floatinglike string-of-bead grape could be harvested at96h after infection.2.1.2The virus titer was determined by TCID50assay. The virus titer ofAd-PGC-1α-GFP is2.0×108PFU/ml. The virus titer of Ad-GFP is2.5×108PFU/ml.2.2ASCs transfection of adenovirus-mediated gene2.2.1An ideal MOI of200with high efficiency and low toxicity was selected totransfect, the transfection efficiency of Ad-GFP-PGC-1α was95.3%and Ad-GFP was95.5%at48h after transfection, as being quantified to detect the number of GFP-positivecells by flow cytometry.2.2.2Expression of PGC-1α protein were2.8-fold higher in PGC-1α-transfected ASCsthan that in Ad-GFP transfected ASCs (P<0.01).2.3Resistant-apoptosis effect of ASCs with overexpression of PGC-1α under highglucose, hypoxia and serum deprivation conditions2.3.1Apoptosis of ASCs modified with PGC-1α or GFP was measured using anAnnexin V-APC/7-Amino-Actinomycin (7-AAD) apoptosis detection kit after beinginduced by culture conditions of high glucose concentration (30mmol/L), hypoxia (5%O2)and serum deprivation. At24h under conditions of high glucose, hypoxia and serumdeprivation, the apoptosis rate of the ASC group (21.01±3.03%) and the GFP-ASC group(21.04±2.79%) were significantly higher than that of the PGC-1α-ASC group(14.73±1.57%)(P<0.01). The survival rate of the PGC-1α-ASC group was significantlyhigher those of the ASC group and the GFP-ASC group at6h and24h of culture underthese conditions (P<0.01). There was no difference in ASC group and the GFP-ASC group(P>0.05).2.3.2GFP-ASCs and PGC-1α-ASC being incubated under high glucose, hypoxia andserum deprivation conditions for24h, the level of cellular ROS was estimated byfluorescent probe DHE labelling. The results showed that DHE-associated fluorescencesignificantly increased in GFP-ASCs (853.44±76.72) compared with PGC-1α-ASC(688.67±43.98)(P<0.01).2.3.3Mitochondrial ROS production being detected by MitoTracker Red CM-H2XRos also showed a reduction in red fluorescence accumulation in the mitochondria of thePGC-1α-ASC group.2.3.4Transmission electron microscopy was used to observe the morphology andstructure of ASCs modified with PGC-1α or GFP under culture conditions of high glucose,hypoxia and serum deprivation for24h. The results showed that The most striking changeswas abnomality of morphology and structure of mitochondria, obvious mitochondrialautophagia in GFP-ASC, but which relieved significantly in PGC-1α-ASC.Conclusions：ASCs can be harvested from inguinal subcutaneous fat pats of SD rats with the methodof collagenase digestion, the ASCs have a high live rate and can multiply in vitro. Themethod of isolation, culture and identification of ASCs is consistent with the standards ofdomestic and international standard, which establish a basis for the treatment of diabetesand its complications based on stem cells. Our data suggest that overexpression PGC-1αmay protect ASCs from apoptosis by reducing the overproduction of intracellular andintramitochondrial ROS, which can induce mitochondrial dysfunction under conditions ofhyperglycaemia and deprivation of nutrients and oxygen. These results have importantimplications because they indicate that PGC-1α is a potential target in the stem celltreatment of diabetes and its complications. PGC-1α transferred to ASCs can be useful stemcells in the treatment of diabetes and its complications.