Reporter System Controlled by the Involucrin Promoter as a Tool to Follow the Epidermal Differentiation

Different approaches have been explored to study skin biology, including the use of stem cells. Mesenchymal stem cells (MSC) from umbilical cord can be safely and easily obtained, however a simple strategy to monitor their differentiation is essential. Involucrin is a marker of keratinocyte terminal differentiation, and its promoter (pINV) directs stratum-specific expression of this protein. We designed a reporter system containing EGFP under control of pINV to assess MSC differentiation into keratinocytes. The functional sequence of pINV was inserted into a lentiviral vector, originating LeGO-GpINV. MSC were transduced with the LeGO-GpINV and induced to differentiate into keratinocytes upon cultivation with Keratinocyte Serum Free Medium supplemented. MSC differentiation was confirmed by morphological changes and by the expression of epidermal markers, by flow cytometry, quantitative PCR and western blot. The activity of kallikreins 5, 6 and 7 was detected using fluorogenic substrates. After 14 days of differentiation, MSC transduced with LeGO-GpINV showed an increase in EGFP fluorescence and expressed CK10, CK14, involucrin and filaggrin. There was also an increase in the kallikrein activity. This reporter system allowed to temporally assess the epidermal differentiation, simultaneously with involucrin expression, opening perspectives for the in vivo study of skin biology and in regenerative medicine.