The Establishment And Identification Of Human Induced Pluripotent Stem Cells With Non-integrative Methods From Specific Burn Patients

Objective:The aim of the study is to search the feasibility and method that the fibroblasts from the burn patients skin are reprogrammed to the induced pluripotent stem cells(i PSCs),which lay the foundation for the application of individual specificity i PSCs in building the skin tissue engineering and repairing the scar and wound.Methods:(1)Harvest the residual skin grafts for skin grafting,which were digested by trypsin and EDTA before the epidermis and dermis were separated.The tissue blocks of the dermis were cultured by the normal culture of fibroblasts.When the primary cells reached 80%~90% confluence,it should subculture the cells.The cells were generated,cryopreserved,thawed as traditional methods.The cells were used for three to five generations in the study.(2)The fibroblasts from burn patients skin were transduced with Cyto Tune-i PS2.0 Sendai reprogramming vectors containing the human transcription factors NANOG,OCT4 and SOX2.And the cells were cultured with fibroblast complete medium.Picked and transferred the cell colonies onto fresh 1% Matrigel-coated dish with human PSCeasy medium,which were used for further amplification and analysis.(3)The AP staining and immunofluorescence staining were performed after harvesting the HSFs-i PSCs colonies,which aimed to detect the stemness and the expression of pluripotent markers including OCT4,NANOG,SOX2,TRA181,SSEA-4 and TRA-160.The karyotype analysis of HSFs-i PSCs was carried out to cells of passage 16.The HSFs-i PSCs were cultured in suspension and adherent culture in vitro,after that harvested the cells and measured the specific gene expression by PCR.The HSFs-i PSCs were injected subcutaneously into the hind legs of 4 week old male immunodeficiency mice and the tumors were formed after feeding.The formed tumors were dissected and were harvested and hematoxylin/eosin(HE)staining was used to detect the gene expression in the tumor.The methylation levelsof OCT4 and NANOG promoter in HSFs-i PSCs were detected by Kit.Results:(1)The HSFs came out from the small tissue pieces with cultured after 11 days.The cells exhibited the branch-shaped and spindle-shaped morphology.After 24 days cultured,the cells reached 90% confluence and then the cells were passaged.The vimentin of molecular marker on the surface of fibroblasts was detected at passage 3,which was positive.The results showed that fibroblasts were came out from the skin tissue of burn patients.(2)Eight days post transduction,several small ESCs-like colonies emerged under the inverted phase contrast microscope.Four weeks post transduction,the colonies have large size,large nucleoli and nucleus to cytoplasm ratio.And The cells of colonies packed tightly.The results of AP staining showed that we got the stem cells.The results of immunofluorescence staining showed that the HSFs-i PSCs strongly expressed not only the surface pluripotency marker such as TRA181,SSEA-4,TRA-160 but also intracelluar pluripotency marker such as OCT4,NANOG,SOX2.The HSFs-i PSCs were confirmed and exhibited a normal karyotype of 46 XY by chromosomal G-band analysis at passage 16.The chromosome structure was normal by using Video Tes T-Karyo 3.1 analysed.(3)The HSFs-i PSCs differentiated to EBs spontaneously in vitro.The specific gene OCT4 was upregulated in the HSFs-i PSCs,meanwhile the specific genes MSX1,GATA4 and SOX1 were upregulated in the EBs.These results suggested that the HSFs-i PSCs had the capable of differentiating into various cell types in vitro.The teratomas were formed after injected the HSFs-i PSCs.HE staining confirmed that the tumor contained the derivatives of all three germ layers including glands(endoderm),muscles(mesoderm),nervus(ectoderm).These results suggested that the HSFs-i PSCs had the capable of differentiating into various cell types in vivo.The results of methylation levels demonstrated that the promoter region of OCT4 and NANOG were highly demethylated.Conclusion:In this study,we obtained the fibroblasts from the skin tissue of burn patients with tissue block culture method.The individual specific i PSCs were successfullyconstructed with reprogrammed the fibroblasts from burn patient-derived skin using a non-integrative Sendai virus vectors,which didn’t interfere with genomic function.The i PSCs are similar with embryonic stem cells in the cloning of morphology,growth characteristics,surface markers and gene expression pattern and differentiation in vivo and vitro.The i PSCs as seed cells are expected to provide a wide application in skin tissue engineering and scar repair.