Establishment Of Induced Pluripotent Stem Cell And Its Differentiation Potency To Ectoderm

In 2006,Shinya Yamanaka used retroviruses to transfer four transcription factors into mouse fibroblasts,thereby obtaining a pluripotent cell similar to embryonic stem cells,called induced pluripotent stem cell(iPSC),and followed on the production of human iPSC.The emergence and development of iPSC gave people a new understanding of the regulation mechanism of pluripotency,and at the same time allowed people to see the great potential of iPSC in disease modeling.Among the many diseases that threaten human health,there is one disease called acute myelitis,which is a group of inflammatory diseases that cause spinal cord nerve damage,but its exact cause is still unclear.Therefore,the study of iPSC derived from patients with acute myelitis is important to clarify the pathogenic mechanism of acute myelitis.In this study,the human fetal fibroblasts and skin fibroblasts derived from a patient with acute myelitis were transferred into 8 exogenous factors,namely OCT4,SOX2,c-Myc,KLF4,NANOG,LIN28,RARG,and LRH1 by electrotransfection,and reprogrammed them to iPSCs.We then explored the biological characteristics of iPSCs from the two sources in terms of pluripotency,transcriptomic characteristics and differentiation in an ectodermal direction,in an attempt to find differences between normal and disease patients,in order to provide some theoretical basis for the study of the pathogenic mechanism of acute myelitis.The experimental results were showed as follows:1.Establishment of iPSC cell lines derived from human fetal fibroblasts(HEF)and patient skin fibroblasts with acute myelitis(P-HAF)In this experiment,we successfully established iPSCs derived from human fetal fibroblasts(iPS)and from a patient with acute myelitis(P-iPS)by using 8-factor electrotransfection.There was no obvious difference in the clonal morphology of the two types of cells,both were positive for alkaline phosphatase activity and had normal karyotypes,while endogenous pluripotent genes were also successfully activated.2.Analysis of molecular biological characteristics of iPS and P-iPSThe analysis of the transcriptome data once again verified that the two fibroblasts were successfully reprogrammed into iPSCs,and the data showed that there are differences between normal people and disease patients.The differential gene are mainly enriched in inflammation-related signaling pathways in HEF and P-HAF,and this difference continues to exist in derived iPS and P-iPS.3.Differentiation potency to ectoderm in iPS and P-iPSFirst,we use the human embryonic stem cell line(h ESC)to induce the neural crest(NC)and cranial placode(CP)for testing the induction process.Real-time quantitative PCR(q PCR)and immunofluorescence staining(IF)results showed that differentiated cells expressed NC and CP markers.Second,iPS and P-iPS were induced to differentiate to the direction of NC and CP.The results of q PCR and IF showed that iPS and P-iPS were induced to express NC and CP markers.The cell morphology of NC and CP and AP staining results were no significant difference in iPS and P-iPS.The q PCR results showed that regardless of NC or CP,the expressions of FOXG1,OTX2,and SOX2 were down-regulated in differentiated cells derived from iPS and PiPS.At the same time,the IF results showed that differentiated cells from both sources expressed NESTIN.Therefore,on the expression of marker genes,there is also no significant difference between iPS and P-iPS.