Study On Culture Of Chicken Spermatogonial Stem Cells And Its Pluripotency

Spermatogonial stem cells were located at seminiferous tubule which not only can maintain the number of themselves but also can differentiate to spermatocyte,are crucial for related stem cells research. This study investigate the vegetative character of spermatogonial stem cells(SSCs) and effect of different feeder when chicken SSCs were cultured in vitro, chicken SSCs were respectively cultured on chicken live cells feeder, chicken CEL feeder and sertoli cells of testicle, explore the capability of SSCs to differentiated into osteoblasts and neuron-like cells under different experimental conditions in vitro. We attempted to prepare the ground for the establishment of chicken SSCs lines and offer cell model for growth and differentiation of cells.The main results were as follows:1. In order to investigate the vegetative character of spermatogonial stem cells(SSCs) and effect of different feeder when chicken SSCs were cultured in vitro, chicken SSCs were respectively cultured on chicken live cells feeder, chicken CEL feeder and sertoli cells of testicle. The result showed: SSCs growth well and the vegetative character of them had no any change when they were subcultured on chicken CEL feeder and chicken live cells . the 4 passage rates of AKP positive colony respectively was 45%,40%,36% and 21% when SSCs cultured on chicken CEF and 40%,32% and 22% when SSCs cultured with testicle sertoli . No colony were observed when SSCs were cultured on chicken live cells feeder.2. SSCs were induced into osteoblasts by desamethason, glycerol 2-phosphate disodium salt hydrate and vitamin C and were examined with Von Kossa's stain,cytochemistry staining and immunohistochemistry stain. The differentiated cells were positive for ALP, the Von Kossa's stain was positive and immunohistochemistry stain.The result showed that the inductionⅡcombined withⅢshowed best results, about 85% positive cells formed 15～21d later. Compared to single use of inductionⅠ,Ⅱ,ⅢandⅣ, The results showed that the chicken SSCs could be differentiated into osteoblasts ,which showed the SSCs have the capability of multipotential differentiation in vitro.3. SSCs were induced into neuron-like cells in the medium containing RA and IBMX, the differentiated cells were identified by toluidine blue stain. The result showed that the inductionⅠandⅡshowds best results and the differentiation rates were 83% and 81% respectively.There was no significant difference between inductionⅠandⅡ(P﹥0.05).SSCs were identified by toluidint blue stain 3～7d later.That showed the SSCs have the capability of multipotential differentiation in vitro.