Establishment Of Malignant Transformation Models Of Nerve Cells Derived From IPS Cells Reprogrammed From Human Skin Fibroblasts

Objective: To establish malignant transformation models of nerve cells derived from iPS cells in order to investigate the cellular origin of malignant gliomas.Methods: Normal human skin fibroblasts were firstly isolated and cultured.After the DNA of the cells was extracted,exons 5,6,7 and 8 of p53 gene and exons 22,32,33,38,and 47 of NF1 gene were amplified by PCR for sequencing.Afterwards,skin fibroblasts were reprogrammed into iPS cells using the Cyto Tune?-iPS 2.0 Sendai Kit,then differentiated into neural stem cells,neurons,oligodendrocytes,and astrocytes,which were identified by immunocytochemistry.The homogeneous neuronal cell population was then purified by flow cytometry.Finally,the malignant transformation of astrocytes was induced using nitrosamines,and the cell growth rate and soft agar colony formation ability were measured to evaluate whether cells were malignantly transformed.Results: Normal human skin fibroblasts were spindle-shaped after isolation and culture in vitro.exon sequencing revealed that the samples did not carry NF1 and p53 mutants.After reprogramming fibroblasts for 14 days,small stem cell clones were visible under the microscope.Four cell clones were extracted and immunofluorescence staining showed that Nanog,SSEA4,OCT4,TRA1-60,Sox2,and TRA1-81 were expressed in cells,indicating that the iPS cell line was successful established.After neural differentiation of iPS cells,the results of immunofluorescence staining showed that the all kinds of cells expressed specific markers,suggesting that neural stem cells,neurons,oligodendrocytes and astrocytes were successfully differentiated from iPS cells.Preliminary experiments showed that after treatment of astrocytes with nitrosamines,the growth rate of cells was accelerated,and the clone formation ability in soft agar was enhanced.Conclusion: A model of malignant transformation of astrocytes derived from iPS cells was successfully established in vitro,which can provide a research model for investigating the cell origin of malignant gliomas,especially for the study of malignant transformation of neurons.