Comparison Of Surface Markers And Multidirectional Differentiation Between Rat Bone Marrow-derived Stem Cells And Cardiac Fibroblasts

BackgroundBone marrow-derived stem cells(BMSCs)are widely used in basic research and clinical application for their advantages of rapid self-renewal,multidirectional differentiation,immune regulation and easy access.When myocardial injury occurs,compensatory proliferation of cardiac fibroblasts(CFs)can easily lead to ventricular remodeling,scar formation,diastolic dysfunction and other complications.At present,bone marrow-derived stem cell transplantation has become the main seed cells of cell therapy for myocardial injury.BMSCs and CFs are the main repair cells for myocardial injury.There is no literature about the difference between the expression of surface markers and differentiative capacity of BMSCs and CFs.ObjectiveTo explore the differences of expression of BMSCs and CFs surface markers Nanog,Sox2,Oct4,Sca-1 and C-Kit and multidisciplinary differentiation ability,and to provide experimental basis and theoretical reference for basic research and clinical application of CFs.Methods1.Resuscitate rat BMSCs,culture and passage;take the heart of newborn rats for 0-3 days,isolate and culture cardiac fibroblasts.The expression of CD29,CD44,CD45 and CD73 on the surface of BMSCs was detected by immunofluorescence staining,and the expression of Vimentin and DDR2 on the surface of CFs was detected by immunofluorescence staining.2.BMSCs and CFs of rats were cultured to the third generation.The expression of nanog,Sox 2,Oct 4,Sca-1 and C-Kit was detected by immunofluorescence staining,immunohistochemical staining and Western blotting.3.P3 BMSCs and P3 CFs were inoculated into 96-well plate,and the growth curves of BMSCs and CFs were compared by MTT staining.4.P3 BMSCs and P3 CFs were inoculated into 24-well plates for 7-9 days,respectively.The differentiation was detected by oil red O staining;the differentiation ability was detected by alizarin red staining after 21 days of induction of osteoblast;and the differentiation ability was detected by immunofluorescence after 21 days of induction of cardiomyocyte.Results1.Rat BMSCs grew adherently in long shuttle,round,triangle,polygon and other shapes.Rat CFs grew adherently with long shuttle and round cells.The cultured BMSCs expressed CD29,CD44 and CD73,but not CD45.The cultured CFs expressed Vimentin and DDR2,which accorded with the characteristics of bone marrow-derived mesenchymal stem cells and cardiac fibroblasts.2.Rat BMSCs and CFs expressed nanog,Sox 2,Oct 4,Sca-1 and C-Kit;BMSCs and CFs had an average optical density of 0.030±0.006 and 0.031±0.003;Sox 2 had an average optical density of 0.034±0.004 and 0.142±0.017;Oct 4 had an average optical density of 0.074±0.008 and 0.100±0.04;and Sea-1 had an average optical density of 0.084±0.009 and 0.366±0.025 respectively.They were 0.114±0.058 and 0.074±0.004 respectively.Western blotting results showed that the relative protein levels of BMSCs and CFs were 0.97±0.04 and 1.07±0.16,Sox 2 was 1.69±0.17 and 2.05±0.37,Oct 4 was 1.29±0.16 and 1.65±0.21,Sca-1 was 1.48±0.11 and 1.78±0.26,and C-Kit was 1.20±0.23 and 0.96±0.07,respectively Immunofluorescence and WB results were consistent,BMSCs and CFs had the same expression level of anog,while Sox2,Oct4 and Sca-1 were lower than CFs,and C-Kit was higher than CFs.3.BMSCs and CFs grew faster than CFs 24 hours after adherence,2-4 days after adherence,and 4-6 days after adherence,the growth rate of BMSCs decreased,the munber of cells decreased,while the growth rate of CFs decreased slightly,and the number of cells continued to rise.It can be seen that the growth rate of BMSCs and CFs is different at different time points.4.BMSCs and CFs have the ability of adipogenesis,osteogenesis and myocardium formation after induction of multidirectional differentiation.The adipogenesis rate of BMSCs and CFs is about 18%,7%,23%and 21%respectively.The positive rate of cTnT in BMSCs and CFs is about 73%and 30%respectively.The difference of BMSCs and CFs in adipogenic rate and myocardial formation rate showed P<0.05 by statistical analysis,while the difference of BMSCs and CFs in adipogenic rate showed P>0.05 by statistical analysis.In conclusion,BMSCs have higher adipogenic and myocardial capacity than CFs,but there is no significmit difference between BMSCs and CFs.Conclusions1.Early embryonic transcription factors are more expressed in rat CFs than BMSCs,suggesting that CFs have more stem cell characteristics.2.There were differences in the rapid growth period of rat BMSCs and CFs cultured in vitro.BMSCs were earlier than CFs,aad the grovth curve of the former decreased earlier than that of the latter.3.Rat BMSCs and CFs have multidirectional differentiation ability,but BMSCs have higher adipogenic and myocardial capacity than CFs,but there is no significant difference in their osteogenic capacity.