Induction Of Pluripotent Stem Cells From Vitilogo Lesional Drmal Fibroblasts By Reprogramming

Objective To investigate the possibility of inducing pluripotent stem cells(i PS cells) from vitiligo lesional dermal fibroblasts(VLF) in vitro, and identify them in mrophology and epigenetics.Methods(1) In the tissue medium,to isolate the VLF from the vitiligo patient tissue throuh the adherent method. To package the retrovirus with transcription factors Oct4、Sox2、Klf4、c-Myc and infect the VLF by the packaged retrovirus, then observed the morphology of the infected cells by electron microscope. In the 12 th day of reprogramming, picked up the cell clones to passage cells;(2) Identify the inducted cells: alkaline phosphatase staining, tested the stemness proteins expression levels by immunofluorescence staining, q RT-PCR tested the endogenous pluripotent stem cell genes and exogenous four factors expression levels, demethylation analysis of the OCT4 promotor region, karyotypes analyze and teratoma formation analyze.Results 1. Reprogramming the VL-i PS cells(1) In the 4th day of reprogramming, we observed the ES cell-like clones, and they can group up by the induction days increased;(2) After passaging, the VP-i PS cells exhibited a more regular morphology which resemble ES cells, the nucleus were arragend tightness with high nucleo-cytoplasmic ratio. 2. Characterize the VP-i PS cells(1) AP staining showed the VP-i PS cells were undifferentiated;(2) The VP-i PS cells were positive of antibodies for OCT4、NANOG、TRA-1-60、SSEA4;(3) The q RT-PCR results explained the endogenous pluripotent stem cells genes Oct4、Sox2、Nanog、h TERT、Rex1、DNMT3B、GDF3 expression levels were high in VP-i PS cells compared with negative contol(P<0.05) and the exogenous four factors genes expression levels were low tin VP-i PS cells compared with positive contol(P<0.05);(4) The promoter region of OCT4 gene in VP-i PS cell was demethylation and in VLF was methylation by bisulphite squencing;(5) The karyotypes in VP-i PS cells were normal;(6) After infected the VP-i PS cells into NOD/SCID mice the teratomas could be observed which proved the VP-i PS cells could differentiate into three germ layer cells.Conclusions The vitiligo patient dermal fobroblasts can be induced into i PS cells in vitro and were equipped with characterization of stem cells. It can offer foudation for treating and studying vitiligo by stem cells.