Reporter system controlled by the involucrin promoter as a tool to follow epidermal differentiation

Autor(es)

Fernandes, Myrian Thiago Pruschinski ; dos Santos, Jeniffer Farias ; Freitas, Bruna Leticia ; Reigado, Gustavo Roncoli ; Antunes, Fernanda ; Tessarollo, Nayara Gusmao ; Chambergo, Felipe Santiago ; Strauss, Bryan Eric ; Nunes, Viviane Abreu

• Tipo de documento: Artigo Científico
• Fonte: Biochimie; v. 201, p. 10-pg., 2022-07-07.

Resumo

Various approaches have been explored to study skin biology, including the use of stem cells. Mesen-chymal stem cells (MSCs) from the umbilical cord can be safely and easily obtained; however, a simple strategy to monitor their differentiation is essential. Involucrin is a marker of keratinocyte differentiation, and its promoter (pINV) directs stratum-specific expression of this protein. We designed a reporter system containing EGFP under the control of pINV to assess MSC transdifferentiation into keratinocytes. The functional sequence of pINV was inserted into a lentiviral vector, producing LeGO-GpINV. MSCs were transduced with LeGO-GpINV and induced to transdifferentiate into keratinocytes under cultivation with keratinocyte serum-free medium. MSC transdifferentiation was confirmed by morphological changes and by the expression of epidermal markers by flow cytometry, quantitative PCR, Western blot and the activity of epidermal kallikreins 5, 6 and 7. After 14 days of transdifferentiation, MSCs transduced with LeGO-GpINV showed an increase in EGFP fluorescence and expressed CK10, CK14, involucrin and filag-grin. There was also an increase in kallikrein activity. This reporter system allowed us to temporally assess epidermal differentiation, simultaneously with involucrin expression, opening possibilities for the in vivo study of skin biology and in regenerative medicine.(c) 2022 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved. (AU)

• Processo FAPESP: 16/14150-2 - Efeito da progesterona na expressão de genes envolvidos no estresse oxidativo e defesa antioxidante em células beta pancreáticas e a relação entre o estado redox no plasma de gestantes e desenvolvimento do diabetes gestacional
• Beneficiário: Viviane Abreu Nunes Cerqueira Dantas
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 19/11963-0 - Uso de células-tronco mesenquimais em modelos tridimensionais de epiderme: aplicabilidade na engenharia tecidual
• Beneficiário: Viviane Abreu Nunes Cerqueira Dantas
• Modalidade de apoio: Auxílio à Pesquisa - Regular