Mesenchymal stem cells express epidermal markers in an in vitro reconstructed human skin model

Abstract

Introduction: In skin traumas, such as burns, epidermal homeostasis is affected, often requiring clinical approaches. Different therapeutic strategies can be used including transplantation, besides the use of synthetic or natural materials with allogeneic cells. In this context, tissue engineering is an essential tool for skin regeneration, and using mesenchymal stem cells (MSC) from the umbilical cord appears to be a promising strategy in regenerative medicine due to its renewal and differentiation potential and hypo immunogenicity. We evaluated the transdifferentiation of MSC from umbilical cord into keratinocytes in three-dimensional (3D) in vitro skin models, using dermal equivalents composed by type I collagen with dermal fibroblasts and a commercial porcine skin decellularized matrix, both cultured at air-liquid interface (ALI).Methods: The expression of epidermal proteins cytokeratins (CK) 5, 14 and 10, involucrin and filaggrin was investigated by real-time PCR and immunofluorescence, in addition to the activity of epidermal kallikreins (KLK) on the hydrolysis of fluorogenic substrates.Results and discussion: The cultivation of MSCs with differentiation medium on these dermal supports resulted in organotypic cultures characterized by the expression of the epidermal markers CK5, CK14, CK10 and involucrin, mainly on the 7th day of culture, and filaggrin at 10th day in ALI. Also, there was a 3-fold increase in the KLK activity in the epidermal equivalents composed by MSC induced to differentiate into keratinocytes compared to the control (MSC cultivated in the proliferation medium). Specifically, the use of collagen and fibroblasts resulted in a more organized MSC-based organotypic culture in comparison to the decellularized matrix. Despite the non-typical epithelium structure formed by MSC onto dermal equivalents, the expression of important epidermal markers in addition to the paracrine effects of these cells in skin may indicate its potential use to produce skin-based substitutes.

CALU‐3 lung cells three‐dimensionally assembled onto CellFate® matrix present angiotensin‐converting enzyme‐2 activity

“…Samples of 40 μL were used with 50 μ: of 2x concentrated enzyme reaction buffer (200 mM Tris buffer p.H 7.5, 200 mM NaCl, and 10 mM ZnCl 2 ), in a final volume of 100 μL. Substrate hydrolysis (10 μM) was accompanied by the increase in fluorescence at λ ex = 320 nm and λ em = 420 nm in the Synergy HT plate reader (Biotek) according to Dos Santos et al (2023) Measurements were performed in duplicates, and ACE‐2 activity values were obtained as fluorescence arbitrary units (FAU). Assay specificity was demonstrated by the enzyme inhibition with 10 μM DX600 (Phoenix Pharmaceuticals), a specific ACE‐2 inhibitor.…”

Obesity and Wound Healing: Focus on Mesenchymal Stem Cells

“…Moreover, we demonstrated ADSC’s ability to promote re-epithelization in organotypic models through a direct action on basal stem keratinocytes [ 16 ]. Several authors also postulated that ADSCs themselves could differentiate into keratinocytes [ 158 , 159 ]. However, whether this is likely to happen in real-life remains to be determined, since basal and hair-follicle stem cells are considered to be physiologically more prone to contributing to re-epithelization [ 160 ] in the wound healing process.…”

Effect of zinc ions on the proliferation and differentiation of keratinocytes

“…The KLK7 is part of a family of enzymes that has been associated to the physiologic desquamation and skin renewal. KLK7 and KLK5 are the most active kallikreins (Santos et al, 2023) involved in epidermis homeostasis, due to their action such as primary regulators of the KLK cascade activation, in the deeper layers of stratum corneum.…”