Four Recombinant Factors Maintained The CESCs Pluripotency And Self-renewal In Vitro And The Regulating Of Lin28on CESCs Chromatin Remolding And Cell Cycle Progress

Embryonic stem cell (ESC) has been of big importance in basic biology research, medicine and drug production, and agriculture technology. But except for mouse ESC (mESC) and human ESC (hESC), the progress of ESC research was rarely slow in domestic animal and poultry, especially chicken embryonic stem cell (cESC), in which there has not yet been a whole and stable method to culture cESC in vitro. With its unique physiological characters, chicken has long time occupied a tremendous value in medicinal animal model production, animal evolution, medical protein production, and basic biological research. The iPSC (induced pluripotency stem cell) brought ESC research to a new direction. And brought a new train of thought in deeper understanding and explain the molecular mechanism of pluripotency and self-renewal maintaining in ESC. Pluripotency transcriptional factors which take a key role in dedifferentiation and reprogramming were introduced in this research to maintain cESC pluripotency in vitro culturing. The CDSs of chicken pluripotency factors, POUV, Sox-2, Nanog, and, Lin28, were foreign expressed as a recombinant protein form, with a series of continued9arginines conjunct at C-terminal as a protein transfer dominant (PTD). Expressed recombinant pluripotency proteins were brought to cESC culturing without feeder cells. cESC could in vitro generated for7passages, and cultured cESCs exhibited typical characteristics of pluripotency, including positive staining for stage-specific embryonic antigen(SSEA-I), and alkaline phosphatase(AKP). Expression levels of the pluripotency markers, POUV, Nanog, C-Myc, Sox-2and Lin28were the same as in uncultured stag X blastoderm cells, and most significantly, the formation of embryoid body (EB) and chimaera chicken by6th generation cESCs confirmed the ability of these cultured cESCs to differentiate into cells of all the three embryonic germ layers. Thus, with nine consecutive arginines as a kind of PTD, pluripotency proteins could be used to culture cESCs and maintain their pluripotency at least six generations. The pluripotency and self-renewal characters of ESC were viewed closely related with its unique fast cell cycle and plastic chromatin structure. And Lin28was found as an important factor participated in ESC cell cycle and chromatin reconstructive regulation. Based on the cESC in vitro culture method and the foreign expressed protein deliver way in this research, some functions of Lin28in cESC were researched. When the recombinant Lin28protein were added into the culture system of cESCs amplified its dose, cESCs could generated for10passages, while maintaining their pluripotency characters, including special ESC superficial antigens and internal enzymes expression, no weaken expression of pluripotency marker genes, and the ability to form EB. In a quantity way Lin28could remarkably changed the expression of some cell cycle related factors like CyclinAs, CyclinDs, CDC2, CDK2, and CDK4and some chromatin reconstructure related factors like HMGAs, DNMTs, and H2A. As these results showed, based on these recombinant pluripotency proteins with PTD, a new culture system was developed to in vitro culture cESC feeder cells free with rapid proliferation and maintained pluripotency, and Lin28could conduct an important regulation on cESC cell cycle program and chromatin reconstructure, through which an essential and unique role was taken, respect from other pluripotency factors in cESC.