Isolation, Cloning And Differentiation Of Chicken Embryonic Stem Cells In Buffalo Rat Liver Cells Conditioned Medium

Chicken Embryonic stem cells(CES cells) are undifferentiated cells capable of proliferation and self renewal and have the potential to differentiate into all somatic cells and the germ line.They provide a model of early embryonic development and differentiation in vitro and an efficient method for genome manipulation to get transgenic chicken,which can produce therapic protein or anti disease naturally.The purpose of this study is to develop novel culture system for isolation and culturing Shouguang black chicken embryonic stem cells from the blastoderm cells of stage X embryos in Buffalo rat liver 3 A cells conditioned medium (BRL-CM),and futher studied the identification and differentiation of CES cells.In this study, dispersed cells from the pellucida area of stage X embryos were seed onto mouse or chicken fibroblast feeder layers and cultured in 80% BRL-CM.One day after seeding, the chick cells have attached to the feeder layer in small clumps. Clones appeared and grew bigger gradually. Clonies looked like birds' nest form or sunflowers et al and were visible consisting of cells with large nucleus, little cytoplasm, and obvious nucleolus. Clones cultured in this culture system were positive with AKP staining and had the capacity of differentiation into multi-type of cells and chimeric chicks were generated from grafting CES cells of 2 and 3 generation into Hailan White eggs. These data indicated that ES-like cells generated in this system and 80% BRL-CM can be used to isolate and culture the CES cells and keep them in undifferentiate state.BRL-CM conditioned for 1 day, 2 days, 3 days, 4 days differently were used to isolate and culture CES cells.3-day of them for BRL-CM was the best. BRL-CM can be collected every 3 days for many times successively, however,CM of the forepart 5 times was better than the rest. In this study, ES basal medium was added FBS(v/v) with 0%, 10%. 15%, 20% respectively to confect 80% BRL-CM and found that 15%(v/v)FBS was the most suitable for cloning of CES cells.STO, PMEF, PCEF were used as feeders and found clones on feeders formed more quickly and many more than that grown without feeders. STO, PMEF feeders were significantly better for that than PCEF(P<0.01), and there was no apparent difference between STO and PMEF(P>0.05).CES cells differented into multiplacate type of cells,such as fibroblast-like,fat-like,muscle-like,neurocytes cells, spontaneously if the CES clones weren't passaged on time or cultured in the medium in the absent of differatiation inhibitory factors and feeder layers. When cultured in suspension,CES cells successfully formed blasrula-like structure. CES cells were passaged by picking up clones with a glass straw and treated by 0.25 % Trypsin-0.02%EDTA.CES cells differated into neuron like cells when induced by all-trans retinoic acid(RA) and Dimethylsulfoxide(DMSO) or by Gastrodin. Cell bodies of some dispersed cells contracted and turned to be round after adding of RA and DMSO for six hours. Some neuron like cells with one, two or more processes were present on the first day. Almost all of the clones lost undifferentiated state in 2 days. Large amount of neuron like cells appeared with the formation of neuron-like networks. When induced by Gastrodin,CES cells differented into mature neurocytes more quickly than those induced by retinoic acid and DMSO. Neuron like networks were formed after 7 days' inducation and 70% of the CES cells differented into glial cells which was identified by anti-GFAP antibody to be positiveAnother important technique that leades to make full use of CES cells isthe ability to culture manipulated embryos to hatch.In this study, the method was employed as follows:Plate the eggs blunt-end-up at 4°C for 5 days before manipulation,cut the eggshell into a round hole(diameter was 1.0cm),and dropped some PBS onto the undermembrance before pinched a little piece of shell membrance out.Their primary eggshells were used to culture the embryo after micro injection. A little egg shells were used to cover the window,and melted paraffin wax used to sealed around it.During the first 3 days,the eggs were cultured at 38.2°C, RH50%,rurn 90° once an hour;then 37.8°C ^ RH60% ^ 30° till hatch.The optimal hatching rate was 45%.