Study On Isolation, Culture And Directed Differentiation Of Embryonic Stem Cells

Now,the work of Embryonic stem(ES)cells mainly focused on isolation and purification,passage and enlarge,inducement and differentiation. ES cells were research objective in this study,the research work included two parts:One part of the research was isolating and culturing porcine embryonic germ(EG)cells.We had got porcine EG cells when plating PGCs on STO feeder cells and adding differentiation inhibitory activities (DIA)in the medium. To find optimization culture system and get successive EG cells lines,this part was composed of three experiments to research the culture condition of porcine EG cells in.vitro.The first experiment discussed the effect of condition medium(CM)on isolating and culturing porcine EG cells.When isolating the PGCs,the porcine embryonic fibroblas(tPEF)and embryonic heart fibroblast(EHF)could be got. After culturing and purifying,we extracted RNA from two cells,then reverse transcripted them to cDNA. The results of RT-PCR showed that LIF gene expressed both in PEF and EHF. So, we collected the cell mediums and mixed them with DMEM to get CM. We cultured porcine EG cells using CM only or together with STO feeder cells. The results indicated porcine EG cells could be got only while using CM together with STO feeder cells.The second experiment discussed the effect of knockout medium on isolating and culturing porcine EG cells. The experiment group I was composed of Knockout DMEM and Knockout SR, the group II was Knockout DMEM/F-12+15%FCS,the control group was DMEM+15%FCS. The growth factors were supplemented in three groups. The results indicated that group I and control group could get the porcine EG cells,group I was better than control group;but group II could not maintain the EG cells. So group I could be used to isolate and culture porcine EG cells.The third experiment was isolating PGCs in co-culture with homologous fetal fibroblast to get porcine EG cells. The EG colonies were AKP-positive, reacted with SSEA-1 antibody and their immunity fluorescence staining of Oct-4 was positive. The EG cells had correct karyotype. The colonies placed in suspension culture in medium without growth factors differentiated into simple embryoid bodies(EBs)in vitro. The EG cells were adhesive by observing them using the scanning and transmission electron microscope. So we confirmed these cells have many characteristics of embryonic stem cells as described previously and they were EG cells. So porcine EG cells could be obtained by isolating PGCs in co-culture with homologous fetal fibroblast.Another part of the research included three experiments. It briefly discussed the effect of different inducers and different method on inducing mouse ES cells to insulin-producing cells(IPCs). The first experiment discussed the function of glucagons-like peptide-1(GLP-1)to inducing MES cells to IPCs. After one week,the DTZ-positive cells was little. Two weeks later,we extracted RNA from cells,the results of RT-PCR showed that GLP-1 improved the expression of Pdx1,but it could not induce MES to IPCs in two weeks.The second experiment discussed the function of bFGF to induce MES cells to IPCs. We extracted RNA from cells after inducing and checking the related genes expression of IPCs using RT-PCR. The results indicated,in this condition,MES could not be induced to IPCs when only adding bFGF into medium.The third experiment used a method of gene transfection to induce MES cells differentiation. Pdx1 gene and Isl1 gene were transfected to MES cells. About 24h later,Pdx1 gene and Isl1 genes overexpessed in MES cells,Pdx1 gene improved obviously,but other relational genes of IPCs could not be checked by RT-PCR.The results showed transfection time was not enough and the gene overexpressed for 24h in MES cells could not induce the cells differentiation. But the effect of gene overexpression was visibly. So the results maybe better,if we added the inducer into medium more and cultured the cells continuly.