A Preliminary Study On The Biological Characterics Of Buffalo Adipose-derived Stem Cells And Their Epigenetic Modification

Buffalo inguinal tissues were digested in collagenase I solutions.The isolated cells were cultured and passaged in Dulbecco’s modified Eagle’s medium low glucose(LG-DMEM)and α modified Eagle’s medium(a-MEM)respectively.The growth curve of the bufflao adipose-derived stem cells(ASCs)was checked.Pluripotency markers Oct4,Sox2,Nanog,c-Myc and klf4 were also examined by reverse transcription polymerase chain reaction(RT-PCR).Multiple antibodies were used to identify surface antigens of buffalo ASCs by flow cytometry.Histochemistry staining and RT-PCR analysis were used to evaluate the multipotential differentiation.Buffalo ASCs epigenetic modifications were detected by quantitive real time PCR(qRT-PCR)in order to explore the feasibility of ASCs as donor cells to improve the efficiency of nuclear transfer.The results were as follows.(1)The primary buffalo ASCs were floating and had morphology bigger than red blood cells under inverted microscope.Buffalo ASCs begun to adhere the culture dish after 4h,and extended into long spindles,formed polygons 48h later.The growth of buffalo ASCs with fibroblast-like morphology was stable and reached to 80%-90%confluence after 7-8d culturing.The tenth passage of ASC had a regular karyotype during in vitro culture,and the buffalo ASCs were positive for the pluripotency related gene like Oct4,Sox2,Nanog,c-Myc and klf4.These results proved that buffalo ASCs had the ASCs general biological character:istics.According to the growth curve,LG-DMEM was better to ASCs for proliferation,and the population doubling time(PDT)was 2.071d.(2)Multiple antibodies(CD31-FITC,CD45-FITC,CD13-FITC,CD29-PE and CD44-FITC)were used to identify surface antigens of buffalo ASCs by flow cytometry.They expressed mesenchymal stem cells(MSC)markers such as CD 13,CD29 and CD44,and didnot express haematopoietic markers such as CD31 and CD45.After induced into adipocyte,osteocyte and chondrocyte,the cell lines were positive to PPAR γ,OCN and collagen I,and the histological staining results also proved the success of induced differentiation.(3)The expression of HD AC 1,Dnmtl of buffalo ASCs and buffalo fetal fibroblasts(BFF)were detected by qRT-PCR.The results showed that all the tested buffalo ASCs had significantly lower level of Dnmtl than this of the BFF of fourth passage(P<0.01).The buffalo ASCs of the second,fifth,ninth and eleventh passages had lower level of HDAC1 than this of the BFF of fourth passage(P<0.05).But there was no difference in the level of HD AC 1 between the ASCs of fourth and seventh passages and the BFF of fourth passage(P>0.05).These results meant that buffalo ASC might have a higer level of histone acetylation and a lower DNA methylation level.So,buffalo ASC may be more suitable as a donor cell than BFF in nuclear transfer and may be more advantageous to the nuclear reprogramming process.In conclusions,buffalo ASCs can be isolated from adipose by collagenase digestion,and has the ASCs general biological characteristics.Compare to a-MEM,LG-DMEM is more suitable for buffalo ASCs proli-feration.Buffalo ASCs express pluripotent related genes such as Oct4,Sox2,c-Myc,Nanog and klf4.They express mesenchymal stem cells(MSC)markers such as CD13,CD29 and CD44,but do not express haematopoietic markers such as CD31 and CD45.Buffalo ASCs has the ability to differentiate into adipocyte,osteocyte and chondrocyte.Compare to BFF,the buffalo ASC has a higher level of histone acetylation and a lower level of DNA methylation,which means ASC may be more suitable as a donor cell in nuclear transfer.