The Methylation Levels Of Induced Pluripotent Stem Cells

Objective: To study methylation levels of differentiated and induced pluripotent stem cells and TE-EMC.Methods: 1. The foreskin tissue was separated between the dermis and epidermis after treatment with neutral protease for two hours. The epidermal sheets were treated with 0.25% trypsin for 10 minutes to produce a suspension of individual epidermal cells. The cells were suspended in Medium254.When the cells reached 80% confluence, they were detached from the flask and seeded into other culture flasks in preparation for experimental procedure, then we to maintain and propagate the cell culture.As the same methods,we can obtained purified EMC. 2. The adults hair follicles were digested with 0.25% collagenase for two hours and 0.25% trypsin for 15 minutes,then added to PBS neutralization liquid which containing 10%FBS.The suspension was centrifuged and cultured,passaged. 3. The epidermis cells were suspended in 0.25% trypsin for four hours, then changed with Medium 254 cultured, passaged. 4. The passaged 3 melanocytes were infected with doxycycline-inducible lentiviral vectors expressing,or Oct4, c-myc and Klf4,according to the MOI values 15:1. After doxycycline withdrawal on 24 hours, these cells were seeded into feeder CF-1 and cultured with stem-cell medium, indicating that colony growth was independent of transgene expression. At almost day 20, ESC-like colonies were picked and expanded into stable i PSC lines. 5. Bisulfite sequencing was used to detect the methylation in Oct4 and Nanog promotor regions in epidermal melanocytes(EMC), trypsinization-enduring epidermal melanocytes(TE-EMC), keratinocytes from epidermis(EKC) or hair follicles(FKC), and the induced pluripotent stem cells(i PSCs) that generated from EMC by transfection of 3 factors(3F-i PSCs) or 4 factors(4F-i PSCs), respectively.Results: The methylation levels in Oct4 gene promoter in the EMC, TE-EMC,3F-i PSCs and 4F-i PSCs were 92.5%, 83.7%, 48.7%, 46.6%, in Nanog gene were 72.2%, 38.9%, 44.4%, 53.3%, respectively. There were significant difference. However, the methylation levels between 3F-i PSCs and 4F-i PSCs had not obviously difference. Moreover, the methylation levels between EKC and FKC had not difference in Oct4 locus, but had in Nanog locus.Conclusion:1. The methylation level was associated with cell type, passages and differentiation. 2. The methylation levels of 3F-i PSCs and 4F-i PSCs were similar.