Wilasinee Promjantuek1, Nipha Chaicharoenaudomrung1, Ruchee Phonchai1, Phongsakorn Kunhorm1, Parinya Noisa2. 1. Laboratory of Cell-Based Assays and Innovations, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand. 2. Laboratory of Cell-Based Assays and Innovations, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand p.noisa@sut.ac.th.
Abstract
BACKGROUND/AIM: Human dermal fibroblasts (HDFs) are widely used as a skin model in cosmetic and pharmaceutical industry due their advantages for the cosmetic industry and medical aspects. Telomeres are key players in controlling cellular aging, in which telomeres and the telomerase enzyme (hTERT) can maintain proliferative capacity and prolong cellular senescence. The primary aim of the study was to elucidate the underlying mechanisms of hTERT/SV40 immortalization of human dermal fibroblasts. MATERIALS AND METHODS: Transgenic expression of hTERT and SV40 large antigen, as well as co-transfection of both factors was performed and their significance evaluated in terms of HDF immortalization efficiency. RESULTS: The results showed that the immortalized fibroblasts of all conditions can be cultured in over 60 passages and maintain their telomere length. Further, key markers of skin cells, such as COL1A1, KRT18 and ELASTIN, were up-regulated in immortalized cells. In addition, p53 expression was enhanced in all immortalized cells, in accordance with activation of the SIRT1 gene upon transgenic immortalization. The significant role of SIRT1 in fibroblast proliferation was assessed by shRNA-knockdown, and it was found that SIRT1 silencing led to loss of Ki67, a proliferation marker. Moreover, miR-93, a SIRT1-targeted miRNA, also had a significantly reduced expression in the co-transfected immortalized cells, highlighting the linkage of the miRNA and SIRT1 pathway in the immortalization of human dermal fibroblasts. CONCLUSION: This evidence from this study could benefit the efficient development of human skin cell lines for use in the cosmetic industry in the future.
BACKGROUND/AIM: Human dermal fibroblasts (HDFs) are widely used as a skin model in cosmetic and pharmaceutical industry due their advantages for the cosmetic industry and medical aspects. Telomeres are key players in controlling cellular aging, in which telomeres and the telomerase enzyme (hTERT) can maintain proliferative capacity and prolong cellular senescence. The primary aim of the study was to elucidate the underlying mechanisms of hTERT/SV40 immortalization of human dermal fibroblasts. MATERIALS AND METHODS: Transgenic expression of hTERT and SV40 large antigen, as well as co-transfection of both factors was performed and their significance evaluated in terms of HDF immortalization efficiency. RESULTS: The results showed that the immortalized fibroblasts of all conditions can be cultured in over 60 passages and maintain their telomere length. Further, key markers of skin cells, such as COL1A1, KRT18 and ELASTIN, were up-regulated in immortalized cells. In addition, p53 expression was enhanced in all immortalized cells, in accordance with activation of the SIRT1 gene upon transgenic immortalization. The significant role of SIRT1 in fibroblast proliferation was assessed by shRNA-knockdown, and it was found that SIRT1 silencing led to loss of Ki67, a proliferation marker. Moreover, miR-93, a SIRT1-targeted miRNA, also had a significantly reduced expression in the co-transfected immortalized cells, highlighting the linkage of the miRNA and SIRT1 pathway in the immortalization of human dermal fibroblasts. CONCLUSION: This evidence from this study could benefit the efficient development of human skin cell lines for use in the cosmetic industry in the future.
Authors: L R Beckenkamp; D M S da Fontoura; V G Korb; R P de Campos; G R Onzi; I C Iser; A P S Bertoni; J Sévigny; G Lenz; Márcia Rosângela Wink Journal: Stem Cell Rev Rep Date: 2020-08 Impact factor: 5.739
Authors: Bo Wang; Dongping Li; Igor Kovalchuk; Ingrid J Apel; Arul M Chinnaiyan; Rafał K Wóycicki; Charles R Cantor; Olga Kovalchuk Journal: Proc Natl Acad Sci U S A Date: 2018-06-25 Impact factor: 11.205