Culture And Identification Of Induced Pluripotent Stem Cell And The Detection Of Expression Level Of Its Pluripotent Genes-Oct4?Nanog?Lin28?Fgf4

Objectives:Our resrerch mainly observes the culture and biological characteristics of human induced pluripotent stem cells in vitro and detects the expression of the pluripotent genes of human induced pluripotent stem cells by real-time quantitative PCR technology,in order to set up an experimental foundation of inducing differentiation of induced pluripotent stem cells into the epidermal ? like stem cells,as well as providing the possibility that become a new source of seed cells in tissue engineering.Methods:First of all,human induced pluripotent stem cells were inoculated on the feeder cells,which were CF-1 mouse embryonic fibroblast irradiated by ultraviolet light,and cultured by human embryonic stem cells medium consisted of basic fibroblast growth factors.At this time,these cells should be devide into two groups--A1 and A2,A1 group cells were immediately detected by alkaline phosphatase staining,and we used Trizon lysate lysed cells for extracting the total RNA of cells,then by means of real-time quantitative RNA technology to analysize the expression of pluripotent genes;A2 group cells were taken by collagenase ? digestion method to passage and observed under Leica invert microscope,when passaged to 15 th generation,these cells were detected by the same method to A1 group cells.Results:The human induced pluripotent stem cells we cultured were stable to growth,the characteristics were similar to the human embryonic stem cells,both of A1 and A2 group cells were positive to alkaline phosphatase staining;the results of genes expression level from real-time quantitative RNA detection showed that in two groups four genes expression level was no significant difference and there was no statistical significance.Conclusions:The human induced pluripotent stem cells we cultured were stable to growth,the characteristics were similar to human embryonic stem cells.With the increase of thepassage of human induced pluripotent stem cells,the expression level of pluripotent genes were still same to the former cells and do not decrease.Building an experimental foundation of inducing differentiation of induced pluripotent stem cells into the epidermal?like stem cells,as well as providing the possibility that become a new source of seed cells in tissue engineering.