The Biological Characteristics Of Mouse-porcine Interspecies Fusion Cells

Cell fusion has begun in 1969,and cell fusion is an important means to study cell pluripotency,fate and gene regulation.However,the current methods of cell fusion are inefficient,less than 1%,in previous studies,especially in interspecies cell fusion.Cell fusion can change the biological characteristics of cells.Pigs have many similarities with human in their physical structure,morphology and immunology.Therefore,pigs are often used as animal disease models in clinical studies of human diseases.However,pig embryonic stem cells have not yet been established successfully.The characteristics of the fusion cells contribute to the research work on the establishment of pig embryonic stem cell.The purpose of the study is:(1)to improve the efficiency of cell fusion;(2)to characterize the mouse-porcine interspecies fusion cells;(3)to explore the dependence of mouse-porcine interspecies fusion cells on growth factor.We established a single cell fusion method by one-to-one fusion of porcine cells with mouse embryonic stem cells.The fusion efficiency reached to~ 50%.The fused cells were cultured under the culture conditions of mouse embryonic stem cells.The fused cells could be cryopreserved and recovered,The cells could be passaged to about 30 passages.The interspecies fusion cells express both the porcine-specific gene Stat3 and the mouse genus-specific genes Wdr59 and Akt1.We found that the cells were alkaline phosphatase positive and with higher expression level of Sox2 compring to porcine fibroblasts.We screened the related factors of AKT,BMP,TGF,FGF,LIF and WNT signaling pathways and found that the fusion cells were more similar to the mouse embryonic stem cells expression,in which Bmp4,Tgf?2,Fgfr1 were highly expressed.The chromosomes from pig were lost badly in the fusion cells.From the results,we concluded that:(1)We successfully established a single cell fusion system to optimize the fusion efficiency.This method is suitable for intraspecies and interspecies cell fusion.The method is efficient,and easy to perform.(2)We successfully obtained 2 intraspecies fusion cell lines and 4 interspecies fusion cell lines,the fusion cells was AP positive;expressing pluripotent gene;with the ability to differenciate into three germ layers.(3)The expressions of Bmp4,Tgf?2 and Fgfr1 in the four interspecies fusion cell lines were higher.(4)The mouse chromosomes are stable in fusion cell lines,however the porcine chromosomes are unstable in fusion cell lines.