Reprogramming Of Somatic Cells From Sanhuang Chicken And Optimization Of Culture System For CiPSC

Termially differentiated cells could be reversed to pluripotent status, a cell type termed induced pluripotent stem cells (iPSC), by introduction of exogenouse transcription factors into cells and triggering the endogenous pluripotent net work. iPSC were capable of differentiating into various types of cells and forming three germ layers, with charateristics similar to ESC. iPSC has advantages over ESC in its unlimited cell source as well as circumvent the ethic contraversial. In the field of animal science, iPSC provides useful tool for research in genetic conservation and breeding. In the current study, we focused on isolation and reprogramming of somatic cells from Sanhuang chicken and optimization of the culture conditions for chicken iPSC.The main findings are as follows:1. Isolation and culture of chicken somatic cell. For chicken feather pulp cells, we optimized the isolation conditions by comparing effects of different culture medium, different concentrations of serum in culture medium and different trypsin treatment. More feather pulp cells were recovered with trypsin digest for ten minutes in DMEM/F12 medium, but cell failed to proliferate and started to die after passage. Somatic cell form chicken heart, liver, muscle tissue and chicken embryo were successfully isolated and maintained in DMEM/F12 culture system.2. Reprogramming of somatic cells from Sanhuang chicken. Transcription factors Oct4?Sox2?Klf4?Lin28 and c-Myc contructed in piggyBac transposon were first introduced and reprogram the CEF cells, no iPSC-like clone emerged after ten days. Whereas iPSC-like colonies were obtained from CEFs underwent lentiviral transduction with viruses containing the human stem cell genes Sox2?Lin28?Oct?Nanog or Sox2?Oct4?c-Myc?Klf4. These cell were strong positive for alkaline phosphatase.3. Optimization of culture conditions for chicken iPSC.We optimized the culture condition by comparing effects of different concentrations of FGF?LIF? BRL conditioned medium, of different feeder layers, different feeder cells BRL?STO?MEF, different source of MEF and the inactivation treament by mitomycin C and radiation, The results demonstrate that MEF isolated from ICR mice and inactivated by mitomycin C treament is the most optimal feeder system for the growth of chicken iPSC, while 4-10ng/mlLIF. need to supplemented in to the culture system with feeder cell inativated by gamma radiation. MEF isolated from KunMing mice is not suitable to culture iPSC in current culture system.In summary, we successfully isolated and reprogrammed somatice cells from Sanhuang chicken and optimized the culture system for chicken iPSC for better maintainence of the proliferation and pluripotency. The results of the current study lay the ground for the genetic conservation and transgenic animal production and native birds by using iPS techology..