Isolation, Culture And In Vitro Neural-Like Cell Differentiation Of Mouse, Rabbit And Bovine Embryonic Stem Cells

Embryonic stem cells (ES cells) are cell lines which can maintain undifferentiated state, normal diploid karyotype, unlimited propagation ability, form chimaeric animal when injected into blastocyte and differentiate into all kind of cell lines derived from three germs. ES cells have been widely used for production of transgenic animal and clonal animal, for construction of medical animal model and for study of the human gene's function. Furthermore, we can use ES cells to study expression and regulation of gene, the mechanism of cell differentiation and cytothesis, and to study the transplantation of cells, tissues and organs. Research and application of ES cells have become one of the hotspot fields in the life science research. At present establishing ES cells line has many problems, for example single specie, low efficiency, which has serious influence to study and application of ES cells. In the study effect on isolation and culture of ES cells derived from different species, embryo age, getting material, isolation, passage way, digestion medium and culture medium were compared, and all kind of effect factor to establish ES cell lines were studied for perfect method and increase efficiency of establish ES cell lines of mouse, rabbit and bovine. Furthermore, a method direct induce mouse EG cells directional differentiate into neuron not by EBs phase was estalished, which can offer reference for mouse EG cells neuron differentiation in vitro.1.The method isolating 5dpc embryo epiblast have significantly improved efficiency of establish ES cell lines compared to traditional isolating ICM method to isolation and culture of KunMing mouse ES cells(12%&3.3%). It had significantly improved F1 passage rate(52%&36%), extramely significantly improved F2 passage rate (40%&16%)of isolated ICM when the medium of rat cardiomyocyte conditioned medium used for ES cells compared with routine medium added to LIF. Continous digestion with 0.125% tripsin+0.02%EDTA digestion medium used to ES cells isolation and passage improved the rate of ICM forming ES clones to 50% and the rate passaged to 5 passages to 20%. Both KunMingBai/str. mice and BALB/C/str. mice are suitable to get ES cells clones and passages without significant difference in embryo attaching rate, ICM attaching rate, getting F1 and F2 passage rate. Culture ES cells with ES medium added 10ng/mL LIF on MEF feeder layer treated 1 h with Mitomycine C can efficiently restrain ES cells differentiation. It has been shown that KunMing mouse ES cell line can be efficiently established with improved method.2.Mouse EG cell line passaged continuely 9～11 passages were got derived from 11.5～12.5 dpc KunMing mouse embryonic genital ridge PGCs. The method repetitious digestion to genital redge with 0.125% tripsin+0.02%EDTA digestion medium can efficiently preserve energy of mouse PGCs. Isolated PGCs with different speed attaching method and co-culture PGCs with embryonic fibroblast have got more PGCs clone and percentage of F1 EG cells signicantly increase compared with isolation method of co-culture back 1/3 embryonic tissue and puncture the genital ridges (21%, 30%&7%, 16%). Two EG cell lines passage to 11～13 passages with AKP+ in 10 passage and ability of differentiation into multi-type cells in 5 passage.3. After protein enzyme digestion and embryo dissection, isolated rabbit blastocyst ICM cultured on MEF feeder layer improved ICM attaching rate and got more ES cells clone(60%), but no ES cell clone having passage culture ability was got with intact embryo or dispersed embryo culture. Rat cardiomyocyte conditioned medium used for ES cells increased ICM attaching rate(59%)and efficiently restrained ES cells differentiation. It has been shown after embryo dissection isolated rabbit blastocyst ICM cultured on MEF feeder layer with rat cardiomyocyte conditioned medium efficiently established rabbit ES-like cell line.4. Isolated PGCs derived from 14～18 dpc rabbit embryonic genital ridge and around tissue by enzyme digestion and mechanical beating upon can get PGCs with proliferation ability. Isolation and passage to rabbit EG-like cells with 0.125% tripsin+0.02%EDTA digestion medium got more EG cell clones and higher passage number than high concentration digestion medium group. The method with co-culture PGCs with embryonic fibroblast adapted to isolation and culture of rabbit EG-like cells. Rabbit EG cell line with continuely passage ability were got with the method of together digestion to rabbit EG cells clone and embryonic fibroblast feeder layer.5. After sublimated by percoll centrifugal and different speed attach, PGCs clone derived from 5～13 week age bovine embryo genital ridge have no difference compared with puncture the genital ridges and enzyme digestion the genital ridges groups, but EG cell passage number is higher than the latter(13, 7 & 5, 3). Rat cardiomyocyte conditioned medium used for bovine EG cells got more PGCs clones and higher EG cell passage number than routine EG medium added to LIF (13&7). Bovine embryonic fibroblast feeder can maintain bovine EG cell proliferation and efficiently restrain EG cells differentiation. Freeze preserve have no effect on bovine EG cells energy. EG cells multipotential has been proved by in vitro differentiatin and AKP dye. It has show that sublimated bovine PGCs cultured on bovine embryonic fibroblast feeder layer with rat cardiomyocyte conditioned medium can efficiently establish bovine EG cell line.6. When mouse EG cells cultivated to high density for long extended periods without replacement of a feeder layer and not by EBs phase promoted neuron differentiation, got higher rate of neural-like cell clones (62.9%(78/124)), and produced TH+ neuron by ulteriorly inducement of RA. Out layer cells of EG cell-derived neural cell like clones and slightness cells transfer from neural cell like clones expressed nestin marker of neural precursor cells. Dispersed neural cell like clone by high concentration RA inducement produced 2.3±0.2% TH+ neurol. When dispersed neural cell like clone by different concentration RA inducement, rate of TH+ neuron increased with RA concentration increase and differentiation time length in a certain range. Rate of alive neural precursor cells after freeze and thaw was 83.2% and neural precursor cells enegy did not changed because they had still proliferation and differentiation ability. When mouse EG cells cultivated to high density for long extended periods without replacement of a feeder layer, a simple and efficient method of neuron differentiation in vitro was established and TH+ neuron were efficiently produced.