Abstract
Skin regeneration is an important area of research in the field of tissue-engineering, especially for cases involving loss of massive areas of skin, where current treatments are not capable of inducing permanent satisfying replacements. Human adipose-derived stem cells (ASC) have been shown to differentiate in-vitro into both mesenchymal lineages and non-mesenchymal lineages, confirming their transdifferentiation ability. This versatile differentiation potential, coupled with their ease of harvest, places ASC at the advancing front of stem cell-based therapies. In this study, we hypothesized that ASC also have the capacity to transdifferentiate into keratinocyte-like cells and furthermore are able to engineer a stratified epidermis. ASC were successfully isolated from lipoaspirates and cell sorted (FACS). After sorting, ASC were either co-cultured with human keratinocytes or with keratinocyte conditioned media. After a 14-day incubation period, ASC developed a polygonal cobblestone shape characteristic of human keratinocytes. Western blot and q-PCR analysis showed the presence of specific keratinocyte markers including cytokeratin-5, involucrin, filaggrin and stratifin in these keratinocyte-like cells (KLC); these markers were absent in ASC. To further evaluate if KLC were capable of stratification akin to human keratinocytes, ASC were seeded on top of human decellularized dermis and cultured in the presence or absence of EGF and high Ca2+ concentrations. Histological analysis demonstrated a stratified structure similar to that observed in normal skin when cultured in the presence of EGF and high Ca2+. Furthermore, immunohistochemical analysis revealed the presence of keratinocyte markers such as involucrin, cytokeratin-5 and cytokeratin-10. In conclusion this study demonstrates for the first time that ASC have the capacity to transdifferentiate into KLC and engineer a stratified epidermis. This study suggests that adipose tissue is potentially a readily available and accessible source of keratinocytes, particularly for severe wounds encompassing large surface areas of the body and requiring prompt epithelialization.
Highlights
The ideal aim of skin regeneration is to find a means to replace or regenerate this complex organ with a normal appearance and with complete functionality [1,2,3]
We hypothesize that adipose-derived stem cells (ASC) might transdifferentiate into keratinocytes (which in this report we describe as keratinocyte-like cells (KLC)) and identify epidermal growth factor (EGF) as a critical cytokine responsible for keratinocytelike cells (KLC) differentiation; we demonstrate that the KLC can form a stratified epithelium under physiological conditions when seeded on a decellularized dermal matrix
Contrary of what described by some authors [30] and confirming what described by others [29,56], freshly isolated ASC did not express CD34
Summary
The ideal aim of skin regeneration is to find a means to replace or regenerate this complex organ with a normal appearance and with complete functionality [1,2,3] This process can be done in-vivo or in-vitro and may require cells, natural or synthetic cellsupporting scaffold materials, bioactive molecules, genetic manipulation, or combination of all of these [4]. Adult stem cells have unique features that might represent an effective way to meet the challenges of skin restoration These include such characteristics as their potential to provide an unlimited source of donor material for grafting, along with their ability to switch into a variety of cell phenotypes in vitro
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