The Establishment Of Induced Pluripotent Stem Cells(iPSCs)and Immortalized Cells From Human Peripheral Blood Mononuclear Cells

Induced pluripotent stem cells(iPSCs),were first successfully established in Japan in 2006.It is a technology that reprogramming the genes of highly differentiated somatic cells through induction.Eventually the cells return to a pluripotent state similar to that of embryonic stem cells with the ability to self-renew and replicate and with a strong differentiation potential.Induced pluripotent stem cells have a wide range of applications,including disease models,regenerative medicine and drug development.Along with the development,the technology is also making progress and breakthroughs.From the initial integrated gene method to relatively safe and reliable non-integrated gene method,from feeder culture method to feeder-free culture method,induced pluripotent stem cell technology tends to be simplified and programmed gradually.Hayflick limit theory points out that the number of cell divisions is limited,but in some special cases,cells can escape the limit and be immortal.Immortalized cells are those that form between normal cells and cancer cells.Cells can proliferate indefinitely without signs of deterioration.Further study of immortalized cells will help us understand the physiological and functional changes between normal cells and tumor cells,as well as explore the physiological mechanism of cellular aging.Immortalized cells can also be collected as human genetic resources,especially some disease-specific genes,and be stored to build a biobank.In this study,human peripheral blood mononuclear cells were used as the cell source.Sampling is convenient and safe with little damage to human body.Sendai virus was used as vector to introduce 4 key transcription factors specifically expressed by embryonic stem cells into source cells for reprogramming.in order to construct induced pluripotent cell lines.The traditional feeder culture method with exogenous cells was abandoned and replaced by feeder-free cell culture method.At the same time,a small number of cells were extracted from the source cells and inoculated directly without further centrifugation purification,simplifying the traditional steps for the construction of immortalized B cell lines.The optimized cell lines were cultured,passed on and identified.The results showed that: 1.Obvious cell clones appeared on the 12 th day after culture,and the 18 th day became typical induced pluripotent stem cells.The cells closely contact with each other without obvious spacing.2.AP staining was positives,indicating that the cells had reprogrammed and returned to undifferentiated stem cell state.3.Immunofluorescence test showed positive for antigen Oct4 and SSEA4.4.The differentiation of the induced pluripotent stem cells into three germ layers was successfully identified,demonstrating the differentiation potential of the cells.5.The immortalized B cells grew in the form of clumps.CD19 positive cells were detected by flow cytometry,accounting for 89.92% of the total cells.The combination of induced pluripotent stem cells and immortalized B cells establishing method makes full use of the cell resources.The collection of immortalized B cells,especially some cells with rare genetic diseases,can provide a reserve of genetic resources for scientific research and exploration of cellular physiological mechanism in the future.Induced pluripotent stem cells have the possibility of future clinical application,which make up for the shortcomings of immortalized B cells' clinical inapplicability.In this paper,the optimization of technical details improves efficiency and saves time cost.The simplification of the procedure further advances the goal of fully automated cell culture in the future.