A Preliminary Study On The Isolation,Culture,and Identification Of Bama Minipig Ascs And Bmscs,and Using Them For The Production Of Handmade Cloned Embryos

Guangxi Bama minipig inguinal tissues were resected and adipose-derived stem cells(ASCs)were isolated in vitro by collagenase I digestion.Bone marrow mesenchymal stem cells(BMSCs)were isolated from minipig leg.Pig Ear Fibroblasts(PEFs)were isolated from minipig ear tissue.The isolated cells were cultured and passaged with Dulbecco’s modified Eagle’s medium low glucose DMEM.Three cells growth characteristics and genetic stability were detected by their growth curve,colony and karyotype analysis.Pluripotency markers Oct4,Sox2 and Nanog were also detected by immunofluorescence.Multiple antibodies were used to identify surface antigens of Bama minipig ASCs and BMSCs by flow cytometry.Immuno-fluorescence staining and RT-PCR analysis were used to evaluate the vitro multipotential differentiation.We compared the developmental ability with handmade cloning(HMC)reconstructed embryos from Bama minipig ASCs and BMSCs of the fifth and tenth generations.Expression of embryonic development related gene(Oct4、Sox2、Nanog and DNMT1)on the HMC reconstructed embryos were detected by quantitive real time PCR(QRT-PCR)technology.The preliminary study was to explore the feasibility of ASCs and BMSCs as HMC donor cells to improve the efficiency of nuclear transfer.The results were as follows.(1)The primary Bama minipig ASCs and BMSCs reached to 90%confluence and stop growing after 7-9d culturing.Subculture cells appeared as fibroblast-like,some of were in "vortex" growth.With the increase of the passages their growth rates were slow down.The cell growth curve were "S"shaped.Minipig ASCs,BMSCs and PEFs of the fifth generation had more than 90%of normal karyotypes rate.The normal karyotypes rate presented decline of the different degree as the passage number increased.Except for PEFs,Minipig ASCs and BMSCs expressed pluripotent related genes such as Oct4,Sox2 and c-Myc,which indicated both ASCs and BMSCs had stem cell pluripotency.(2)Multiple antibodies were used to identify surface antigens of Bama minipig ASCs and BMSCs by flow cytometry.The results showed that they expressed mesenchymal stem cells surface markers such as CD29,CD44,CD90 and CD 105,while did not express the hematopoietic stem cell surface markers such as CD31,CD45 and HLA-DR.Bama minipig ASCs and BMSCs could differentiate into fat cells,bone cells and cartilage cells after in vitro induction.And the histological staining results also proved the success of induced differentiation.(3)There was no significant difference in the cleavage and blastocyst development rate of reconstituted embryos among Bama minipig ASCs,BMSCs and PEFs of the fifth and tenth generations(P>0.05).The cell numbers of blastocyst also showed no significant difference among three groups(P>0.05).But the relative abundance of Oct4,Sox2 and Nanog in ASCs were significantly higher than that in BMSCs and PEFs(P>0.05).The relative abundance of DNMT1 in ASCs was significantly higher than PEFs(P>0.05),the relative abundance of and DNMT1 in the ASCs and BMSCs showed no significant difference(P>0.05).The relative abundance of Oct4,Sox2 and DNMT1 in BMSCs was higher than that in PEFs(P<0.05),and the relative abundance of and Nanog in the BMSCs and PEFs showed no significant difference(P>0.05).In conclusions,Bama miniature pig ASCs and BMSCs have the potential pluripotency and differentiation;nuclear transfer embryos constructed from Bama miniature ASCs may have a high growth potential.