Mesenchymal stem cells differentiate into keratinocytes and express epidermal kallikreins: Towards an in vitro model of human epidermis

Abstract

Epidermal differentiation is a complex process in which keratinocytes go through morphological and biochemical changes in approximately 15 to 30 days. Abnormal keratinocyte differentiation is involved in the pathophysiology of several skin diseases. In this scenario, mesenchymal stem cells (MSCs) emerge as a promising approach to study skin biology in both normal and pathological conditions. Herein, we have studied the differentiation of MSC from umbilical cord into keratinocytes. MSC were cultured in Dulbecco's modified Eagle's medium (DMEM) (proliferation medium) and, after characterization, differentiation was induced by culturing cells in a defined keratinocyte serum-free medium (KSFM) supplemented with epidermal growth factor (EGF) and calcium chloride ions.Cells cultivated in DMEM were used as control. Cultures were evaluated from day 1 to 23, based on the cell morphology, the expression of p63, involucrin and cytokeratins (KRTs) KRT5, KRT10 and KRT14, by quantitative polymerase chain reaction, Western blot analysis or immunofluorescence, and by the detection of epidermal kallikreins activity. In cells grown in keratinocyte serum-free medium with EGF and 1.8 mM calcium, KRT5 and KRT14 expression was shown at the first day, followed by the expression of p63 at the seventh day. KRT10 expression was detected from day seventh while involucrin was observed after this period.Data showed higher kallikrein (KLK) activity in KSFM-cultured cells from day 11th in comparison to control. These data indicate that MSC differentiated into keratinocytes similarly to that occurs in the human epidermis. KLK activity detection appears to be a good methodology for the monitoring the differentiation of MSC into the keratinocyte lineage, providing useful tools for the better understanding of the skin biology. K E Y W O R D S differentiation, epidermal markers, keratinocytes, mesenchymal stem cells, tissue kallikreins J Cell Biochem. 2019;120:13141-13155.wileyonlinelibrary.com/journal/jcb

Fabrication of chitosan-polyvinyl alcohol and silk electrospun fiber seeded with differentiated keratinocyte for skin tissue regeneration in animal wound model

“…Stained cells were rinsed with phosphate buffered saline (PBS; pH: 7.4) and were xed in 1% formaldehyde solution and studied using BD FACCS Calibur (BD bioscience San Jose, CA, USA). Furthermore, the differentiation potential of adherent broblastoid cells into keratinocyte lineage was performed as described previously [21,22]. For this, cells at density 1 × 10 5 were seeded in 6-well tissue plate and incubated in conditional DMEM media containing 2 mM calcium chloride, 5 µg/mL insulin and 10 ng/mL recombinant human epidermal growth factor (EGF) and keratinocyte growth factor (KGF) when changing culture media every 3 days until 18 days.…”

Development of Novel Microenvironments for Promoting Enhanced Wound Healing

“…Adult stem cells, which can be derived from hair follicle, epidermis, and mesenchymal stem cells (MSCs), are popular selections and have been shown to secrete various growth factors and cytokines that aid in the wound repair process [114,115]. These stem cell sources can differentiate into keratinocytes [116]. When delivered into the proper environment that has correct mechanical characteristics, temperature, pH, and oxygen concentration that recapitulate the skin microenvironment, stem cells can be induced to differentiate into target skin cells [4•].…”

Fabrication of Chitosan-Polyvinyl Alcohol and Silk Electrospun Fiber Seeded with Differentiated Keratinocyte for Skin Tissue Regeneration in Animal Wound Model

“…Stained cells were rinsed with phosphate buffered saline (PBS; pH: 7.4) and were xed in 1% formaldehyde solution and studied using BD FACCS Calibur (BD bioscience San Jose, CA, USA). Furthermore, the differentiation potential of adherent broblastoid cells into keratinocyte lineage was performed as described previously [20,21]. For this, cells at density 1 × 10 5 were seeded in 6-well tissue plate and incubated in conditional DMEM media containing 2 mM calcium chloride, 5 µg/mL insulin and 10 ng/mL recombinant human epidermal growth factor (EGF) and keratinocyte growth factor (KGF) when changing culture media every 3 days until 18 days.…”