Study On The Profile Of Surface Markers Expressed On Human Adipose-derived Stem Cells

Objective:Adipose-derived stem cells(ADSCs)are adult stem cells derived from adipose tissue,with self-replication and multiple differentiation potentials.ADSCs have potentially a broad range of clinical applications.ADSCs play a role depending largely on the molecules expressed on cell surfaces to interact with the extracellular matrix as well as the adjacent cells and immunmodulate body's functions.Previous studies on surface molecules of ADSCs mostly focus on one or a few cell surface molecules.The disadvantage is that they were not suitable to find key molecules with important functions and new cell subsets with distinct phenotypes and functions.The expression of surface markers of ADSCs involved in migration,homing and immune regulation has not been reported.The mechanism of ADSCs's immunomodulatory role is not fully elucidated.The current study aimed at the detection of 86 kinds of surface markers on ADSCs by flow cytometry after in vitro culture of ADSCs followed by phenotypic identification and differentiation assay.Novel key molecules involved in the immunmodulation of ADSCs will be found and their expressions and influencing factors will be analyzed to explore the mechanism of immunomodulation of ADSCs and provide scientific basis for the clinical application of human ADSCs in tissue engineering and cellular immunotherapy.Methods:1 Adipose tissue sample sources.All of the 28 donors were females.The informed consent was approved by the ethics committee and obtained from the donors.The adipose tissue was about 10 ml in volume and the sampling locations were abdomen and breast.2.In vitro culture of ADSCs.After digested with collagenase,adipose tissue was cultured in low sugar DMEM medium containing 10%FBS.When the cells were more than 80%confluent,they were digested and passaged.3.The phenotypic characterization of ADSCs.Fourth generations of adherent cells were harvested and stained with anti-CD34,CD31,CD45,CD10,CD13,CD29,CD49d,CD90 fluorescent antibodies and fluorescent labeled isotypes followed by analyzing by flow cytometry.4.Differentiation of ADSCs.Adipogenic,osteogenic and chondrogenic differentiation potentials of ADSCs cultured in their respective media were assessed by staining with oil red O at 7~thh day,alizarin red S and toluidine blue at 14~thh day,respectively.5.Detection of the expression profile of surface markers on ADSCs.A total of 86 antibodies labeled by 6kinds of fluorescence were applied to measure using flow cytometry and analyzed by FlowJo software in 10 cases of ADSCs.6.Statistical analysis.SPSS 19.0 and Graph prism 5 were used to analyze the data.The two groups were compared with independent sample t test.Multivariate linear regression was used to analyze the factors that affect the expression of PD-L1 and Gal-9 on ADSCs.P<0.05 is statistically significant.Results:1.Phenotype characterization.Cultured ADSCs did not express CD34,CD31and CD45,but expressed a high level of CD10,CD13,CD49d,CD29 and CD90,all of which are in consistence with the characteristics of ADSCs surface markers.2.ADSCs differentiation potential test.After adipogenic induction and staining,the red lipid granules in variable sizes were observed inside the cytoplasm.After osteogenesis induction and staining,orange red mineralized nodules were observed.After chondrogenic induction and staining,blue nuclei and cytoplasm as well as dark blue metachromatic granules in cytoplasm were observed.The results showed that adherent cells had the ability to differentiate into adipocytes,osteoblasts and chondrocytes in consistence with international standards.3.The expression profile of the surface markers on ADSCs.Results by flow cytometry showed that ADSCs expressed a variety of surface markers related to immunmodulation.3.1 the expression of HLA related antigens.ADSCs did not express HLA-DR,but expressed a low level of HLA-G.66.35%of ADSCs expressed HLA-A B C.3.2 the expression of the cytokine receptors.ADSCs expressed a low level of CD123,CD127,CD25 and CD122,but did not express CD124 and CDw210.60%-80%of ADSCs expressed CD119.3.3 the expression of the chemokine receptors.ADSCs did not express CD192,CD194,CX3CR1,CD181 and CD182,but expressed a low level of CD195 and CD183 and a high level of CD184.3.4.the expression of the adhesion molecules.ADSCs expressed CD54,CD226 and CD56,and did not express CD206,CD209 and CD205.3.5.the expression of immunoinhibitory molecules.ADSCs expressed a low level of Gal-9,as well as PD-L1 and PD-1 at the same time.ADSCs did not express TIM-3 and CD152.3.6.the expression of immunostimulatory molecules.ADSCs did not express CD80,CD86,CD28 and CD40L,but expressed a low level of CD40.4.The factors affecting the expression of PD-L1 in ADSCs.The expression of PD-L1 in ADSCs of breast adipose tissue(37.24±8.2%)was significantly higher than that of ADSCs in abdominal adipose tissue(28.8±8.59%)(P=0.018).Multivvariate linear regression analysis showed that the PD-L1 expression of ADSCs was significantly affected by age and location,and BMI was not a factor affecting the expression of PD-L1onADSCs.ThelinearregressionequationwasPD-L1~+ADSCs(%)=34.437+8.58×location(abdomen=0,breast=1)+(-0.385×age).The expression of PD-L1 decreased by 0.385%with every increase of one year.5.The factors affecting the expression of Gal-9 in ADSCs.The expression of Gal-9 in ADSCs of breast adipose tissue(4.41±2.65%)was significantly higher than that of ADSCs in abdominal adipose tissue(2.51±1.39%)(P=0.039).Multivariate linear regression analysis showed that collection sites were independent risk factors for Gal-9 expression in ADSCs.Age and BMI were not independent risk factors for Gal-9 expression in ADSCs.The linear regression equation was Gal-9~+ADSCs(%)=0.623+1.894×location(abdomen=0,breast=1).Conclusion:1.Both the abdomen and breast are the reliable sources of human ADSCs.2.Human ADSCs express a variety of adhesion molecules,chemokine receptors and cytokine receptors,as well as immunoregulatory molecules and do not express or express a lower level of multiple costimulatory molecules.3.Human ADSCs express important immunosuppressor molecule PD-L1 and Gal-9.Age and location of harvest sites significantly affect the expression of PD-L1 and Gal-9 on ADSCs.