| Objective:Cell migration is a basic biological phenomenon in the process of body growth and development,and is involved in various physiological and pathological processes such as human embryonic development,tissue regeneration,wound healing and tumor metastasis.The ability of cells to undergo directional migration guided by an electrical gradient is called "electrotaxis" or "galvanotaxis".Studies have confirmed that electrical signals,as one of the signals guiding cells for directional migration,have priority over other coexisting signals and have been the focus of scholars in various countries.Adipose-derived stem cells(ADSCs)are adult stem cells isolated from adipose tissue.ADSCs can be induced to differentiate into adipocytes,osteoblasts and chondrocytes.Through paracrine,ADSCs can promote the generation of blood vessels and lymphatic vessels,inhibit the formation of pathological scars,and show immunosuppressive and anti-inflammatory properties,which has a good therapeutic application prospect.However,whether ADSCs can achieve ideal clinical therapeutic effects depends on the cellular function of ADSCs themselves,particularly the functional changes associated with aging.In the context of an aging population,the proportion of the elderly population is rising as a potential beneficiary of autologous stem cell-related therapies.However,the effect of aging on the function of ADSCs is still not comprehensively understood,especially regarding ADSCs galvanotaxis,which has not been reported in studies.The purpose of this study was to analyze the electrotaxis and directional migration ability of ADSCs derived from old and young donors by applying different intensities of direct current electric fields(DCEF)stimulation,and to explore the related mechanisms by gene sequencing and protein expression differences,providing new clues for the optimization of clinical treatment related to ADSCs.Method:The sample for this study was derived from abdominal adipose tissue of six healthy women:three healthy old women(age range 62.33±4.04 years),and three healthy young women(age range 27±4.58 years).1.hADSCs were extracted by collagenase digestion and centrifugation and then cultured.The characteristic surface markers CD29,CD44,CD90 and CD105 of h AD SC s were identified by flow cytometry.HADSCs were subjected to differentiation induction for osteogenesis,adipogenesis and chondrogenesis to verify their differentiation ability.2.Senescence-associated β-galactosidase staining was performed on hADSCs,and the expression levels of senescence markers p16 and p21 and proliferation marker ki67 were detected with immunofluorescence of hADSCs.3.Use culture dishes,sterile glass sheets and vacuum silicone to make a electrotaxis chamber.Steinberg’s solution were configured and then mixed with agar.After heating,the mixture were injected into a special glass tube and forming an agar salt bridge after cooling.A electrotaxis chamber,silver wire,agar salt bridge,power supply,and glass sheet seeded with hADSCs were assembled as a electrotaxis chamber device which’s stability will be examined later.4.Turn on the power supply,apply 0,100 and 200 mV/mm exogenous DCEF stimulation to hADSCs using a electrotaxis chamber,perform time-lapse photography of cells under four random fields by inverted microscope and Zeiss software,process the images obtained by imaging using ImageJ(ImageJ 1.37v,National Institutes of Health,Bethesda,MD,USA)and Chemotaxis tool(v.1.01,distributed by ibidi GmbH,Munchen,Germany)and manually track the cell trajectory,draw the cell migration coordinate diagram,and calculate the relevant movement parameters including Directedness,Accumulated distance,Track speed,Euclidean distance and Displacement speed.5.Quantify the morphological changes of hADSCs after exogenous DCEF stimulation using ImageJ,and calculate the long axis,vertical length and verticality of hADSCs.6.RNA-seq sequencing analysis was performed after applying 200 mV/mm exogenous DCEF to young female donor-derived hADSCs and old female donor-derived hADSCs,GO enrichment analysis and KEGG enrichment analysis were performed for differential genes.Differential genes related to cell migration were further examined by Western blotting for protein level expression.Result:1.The hADSCs extracted in this experiment have typical characteristics of stem cell surface markers(positive for CD29,CD44,CD90 and CD 105,and negative for CD34 and CD45)and have multilineage differentiation potential(adipogenesis,osteogenesis and chondrogenesis).2.HADSCs migrate directionally toward the anode under the stimulation of exogenous DCEF.Under exogenous DCEF stimulation at 0,100,and 200 mV/mm intensities,the Directedness of hADSC directed migration was statistically proportional to the electric field intensity.However,there was no significant correlation between Accumulated distance,Track speed,Euclidean distance and Displacement speed.3.HADSCs underwent significant morphological changes induced by exogenous DCEF.Compared with the control group at 0 mV/mm intensity,hADSCs’ verticality(p<0.01;p<0.001),vertical length(p<0.001;p<0.001),and long axis were significantly increased(p<0.01;p<0.001)under exogenous DCEF stimulation at 100 and 200 mV/mm intensities.4.Compared with hADSCs derived from young female donors,the activity of SA-β-gal,the expression of senescence markers p16 and p21 were increased in hADSCs derived from old female donors.The expression of proliferation marker ki67 were decreased in hADSCs derived from old female donors.5.Under exogenous DCEF stimulation at an intensity of 100 mV/mm,hADSCs derived from old female donors had lower Directedness of directional migration than hADSCs derived from young female donors(p<0.05),but there was no significant difference in Accumulated distance,Track speed,Euclidean distance and Displacement speed;under exogenous DCEF stimulation at an intensity of 200 mV/mm,hADSCs derived from old female donors had lower Directedness,Euclidean distance and Displacement speed of directional migration than hADSCs derived from young female donors(p<0.05;p<0.05;p<0.05).However,there was no significant difference in Track speed and Accumulated distance.6.Under exogenous DCEF stimulation at an intensity of 100 mV/mm,the verticality of hADSCs derived from old female donors was shorter than that of hADSCs derived from young female donors(p<0.01),but there was no significant difference in vertical length and long axis;under exogenous DCEF stimulation at an intensity of 200 mV/mm,the verticality,vertical length,and long axis of hADSCs derived from old female donors were shorter than those derived from young female donors(p<0.05;p<0.01;p<0.01).7.There were significant differences in gene expression between hADSCs derived from old female donors and hADSCs derived from young female donors after 200 mV/mm DCEF stimulation,with 747 genes up-regulated and 624 genes down-regulated.8.The results of GO enrichment analysis showed that the expression of genes related to the migration ability of hADSCs derived from old female donors after 200 mV/mm DCEF stimulation was down-regulated compared with that of hADSCs derived from young female donors after 200 mV/mm DCEF stimulation,including 20 genes like PIK3CD,MLCK and ADRA2A.9.Western blot verified the RNA sequencing results of hADSCs derived from old female donors and hADSCs derived from young female donors after 200 mV/mm DCEF stimulation.The KIAA1199,MLCK,PIK3CD,SMO,Smad3 and ADARA2A protein expression levels in the old group were lower than those in the young group.Conclusion:1.Under the stimulation of exogenous DCEF,hADSCs migrate directionally towards the anode with electrotaxis,and the Directedness of directional migration is directly proportional to the electric field intensity.2.After the stimulation of exogenous DCEF,hADSCs undergo morphological changes and rearrange.HAD SC s’ long axis increases and are perpendicular to the direction of electric field vector3.Aging reduce the Directedness of directional migration of hADSCs to the anode in exogenous DCEF.Aging affected the morphological changes and rearrangement of hADSCs after stimulation with exogenous DCEF.4.There are significant differences in gene expression between hADSCs derived from old female donors and hADSCs derived from young female donors after exogenous DCEF stimulation.The expression of genes related to cell migration ability was significantly down-regulated in hADSCs derived from old female donors.5.Aging may reduce PIP3 production and Akt signals by down-regulating PIK3CD,which in turn inhibits the eletrotaxis migration of hADSCs.ADRA2A and MLCK may also be involved in the negative regulation of electrotaxis migration of hADSCs by aging factors,and the in-depth mechanism needs further study and validation. |
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