Study On Induced Pluripotent Stem Cells Derived From Human Skin Keratinocytes

Background:Reprogramming of somatic cells can generate induced pluripotent stem cells(iPSCs).The resulting patient-specific stem cells can not only be used to simulate human disease processes,study pathogenesis,but also be used for drug development and screening,as well as provide new opportunities for personalized regenerative cell therapy.Objective:To explore a quick and easy method that does not damage cell viability,and can efficiently produce induced pluripotent stem cells(iPSCs),and evaluate the possibility of the obtained iPSCs to induce melanocytes in vitro,which will provide basis for the clinical treatment of pigmentary diseases.Methods:1.Normal foreskin was trimmed and was digested by dispase to obtain epidermal film.One part of the film was used for tissue culture,which was laid flat on culture plates that were precoated with matrix and cultured in a medium suitable for keratinocyte growth.Three weeks later,an immunofluorescent staining with antibody against keratin-15(K15)was performed on the cultured cell sheets.The other part of the epidermal film was further digested by trypsin to produce free cells for adherent culture in a six-well culture plate pre-coated with type IV collagen.Those fast-adhering cells that adhere to the plate within 15minutes were allowed to continue to culture for 24 h,then received immunofluorescent staining with antibodies against K15,SOX2 and OCT4 as well as staining with stem cell living cell dye CDy1.The unstained cells were further cultured for 1 week or until reaching70%cell confluence.Those lower-adhering cells that did not adhere to the plate within 15minutes were also incubated simultaneously as a control group.The extracted hair follicles were placed in matrigel-coated culture plates for in vitro culture.The levels of DNA methylation in the OCT4 and NANOG promoter regions of hair follicle KCs were analyzed and compared with KCs from the epidermis.2.Lentiviruses with OCT4,SOX2,KLF4 and c-MYC transcription factors were transfected into epidermal KCs to generate iPSCs.The morphology and time of the clone formation were observed and recorded.The clone cells were detected using some antibodies related to pluripotent markers,the staining of alkaline phosphatase and staining with CDy1dye for the living stem cell.DNA methylation levels of OCT4 and NANOG promoter regions were analyzed.Expressions of some pluripotent genes were detected by a method of RT-PCR.Different media were used to induce these iPSCs to differentiate in vitro into different cell types,including hepatocytes,adipocytes,neurons and melanocytes Hair follicle KCs was also transfected with lentivirus carrying four transcription factors,and the generation of iPSCs was observed.IPSCs was implanted subcutaneously into the hindlimb of SCID mice,and the formation of teratoma was observed.After the teratoma was obtained,histopathology and immunohistochemistry were performed.The cells were obtained from teratoma and cultured in keratinocyte medium,M254 medium and DMEM medium,respectively,and the cell morphology was observed.3.Normal foreskin was digested by dispase to obtain keratinocytes,melanocytes and fibroblasts by different culture methods.Keratinocytes and melanocytes were transfected with lentivirus carrying OCT4,SOX2,KLF4,and c-MYC transcription factors,respectively,and corresponding iPSCs were obtained.Fibroblasts obtained Muse cells through long-term trypsin digestion.The ultrastructural of KCs,MCs,iPSCs and Muse cells were compared using transmission electron microscopy.Results:1.The epidermal film was attached to the bottom of the matrigel-coated culture plate,from which many KCs growed outwardly.The size of cells in the central region was small.Towards the periphery of these sheets,the cells'volume gradually become larger and present as polygonal.The staining with an antibody against K15 showed that positive cells were dense in the central area of the cell sheet and were sparse at the peripheral areas,confirming the presence and approximate distribution of KSCs in the cultured epidermis.Other uncultured epidermal slices were digested by trypsin to obtain free cells,which were seeded in the type IV collagen-coated culture plate.Approximately 20-30%of the smaller cells adhered to the surface of type IV collagen within 15 minutes.After 24 hours,these rapid adherent cells had been firmly attached,some of them began to proliferate,showed positive staining with antibody against K15 and with stem cell CDy1 dye,and expressed weak positive for stem cells specific markers SOX2 and OCT4.After one week,the fast-adherent cells expanded many cell clones that were larger than those produced by slow-adherent cells under the same culture conditions.The extracted hair follicles were easily attached to the Matrigel.Many KCs migrated out of the outer root sheath.A lot of cell clones appeared in the primary culture.The levels of DNA methylation in the SOX2 and OCT4 promoters of subcultured hair follicle KCs were similar to that of epidermal KCs.2.Epidermal KSCs was transfected with lentivirus carrying four factors.The earliest clone appeared on day 7 after transfection,and many typical iPSCs clones appeared on the10^th day.The obtained cloned cells expressed the stem cell-specific markers SOX2,OCT4,NANOG and SSEA3,indicated positive CDy1 staining and showed purple-red staining with alkaline phosphatase.Compared to their parent cells,the obtained iPSCs showed DNA demethylation of OCT4 and NANOG promoters.The results of RT-PCR showed that the expression levels of SOX2,OCT4,NANOG in iPSCs were higher,while the KLF4 were lower,compared with their parent KCs.The embryoid body grew well on gelatin-coated culture plates and automatically differentiated into ectoderm,mesoderm and endoderm cells.By using different induced differentiation media,the iPSCs differentiated and produced hepatocytes,adipocytes,nerve cells and melanocytes,respectively,their corresponding positive stainings were:alpha-fetoprotein(+),oil red O staining(+),?III-tubulin(+),neurofilament(+),HMB45(+)and TYR(+).Hair follicle keratinocytes had also been reprogrammed by transfecting with four factors and successfully produced iPSCs.The iPSCs were injected subcutaneously in the hind limbs of 4-week-old SCID mice.At 8 weeks,the tumors had grown to a sufficient size,and teratoma tissues were excised for pathological and immunopathological examination.The results showed that Vimentin(+),CK(pan)(+)and KI-67(+)were positive,and GFAP and AFP staining were negative.The single cells obtained from teratoma tissues were grown in KCs medium for 3 weeks.The cells were small and round,with a large nucleus-to-plasma ratio.The cell fluorescence was still very strong.After 6 weeks of culture,the cells were closely arranged,with good cell viability and rapid proliferation.When grown in DMEM medium for 3 weeks,the cells were mainly round,with some protrusions.After 6 weeks of culture,the cells showed a flat shape,similar to the shape of fiber cells.When grown in M254 medium for 3 weeks is dominated by small round cells.Some cells had a small number of synapse-like structures.Cells cultured for 6 weeks showed elongated dendrites,similar to melanocytes.3.Under transmission electron microscopy,keratinocytes were roughly square,with a large nucleus and a nucleolus.MCs were elliptical and have poor nuclear chromatin aggregation.Melanosome encapsulated in membrane could be seen in the cytoplasm.The ultrastructure of the iPSCs generated from melanocytes and KCs were roughly the same.These cells were round,with multiple nucleoli,sparse cytoplasm,and high nuclear-to-plasmic ratio,showing dysplastic endoplasmic reticulum and Golgi complex.The fibroblast cells were fusiform or irregular,the nucleus were oval or round,with rich chromatin and many organelles.Muse cells had a high nucleus-cytoplasm ratio,more nucleus protrusions,larger nucleoli,and fewer organelles in the cytoplasm,which were relatively naive.Conclusions:1.The keratinocytes quickly attached to the type IV collagen surface within 15 minutes are rich in KSCs.A large amount of KCs can be obtained by plucked hairs.These cells are similar to epidermal-derived KCs and contain abundant KSCs.2.Human epidermal KSCs Transfected with lentivirus carrying four transcription factors could efficiently and quickly generate iPSCs.These cells are pluripotent and could be induced to differentiate into vary types of cells.Hair-derived KCs can also be reprogrammed to successfully generate iPSCs.The iPSCs were inoculated into SCID mice could produce teratomas,which showed different morphology in different media.3.Transmission electron microscopy showed that the ultrastructure of iPSCs produced by KCs reprogramming was similar to that of iPSCs derived from MCs and that of Muse cells.The ratio of nucleoplasm was high,there were many nucleoli and few organelles with immature development in their cytoplasm.