Transdifferentiation of Adipose-Derived Stem Cells into Keratinocyte-Like Cells: Engineering a Stratified Epidermis

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.

Derivation of keratinocytes from chicken embryonic stem cells: Establishment and characterization of differentiated proliferative cell populations

“…K-cES cell populations all displayed the typical morphology of chicken keratinocytes, with large intracellular lipid droplet, desmosomes and tight junctions with abundant (keratin) tonofilaments (Vanhoutteghem et al, 2004). As in CPK, corneocytes could be observed in our cell culture system; however optimization of cell culture conditions will have to be conducted toward the generation of pluristratified reconstituted epidermis, possibly at the air-to-liquid interphase as reported for keratinocytes derived from mammalian ES cells (Aberdam, 2004;Chavez-Munoz et al, 2013;Coraux et al, 2003;Guenou et al, 2009;Lamb and Ambler, 2013;Metallo et al, 2010;Yoshida et al, 2011).…”

“…In our K-cES cells, these markers appeared at the same time, earlier than in the ES cell derived human keratinocytes. In reconstituted skin, the immunodetection of both proteins shows their association with the spinous/granulous or cornified (INV positive) layers (Chavez-Munoz et al, 2013;Guenou et al, 2009). As KRT10 is supposed to replace KRT5 intermediate filaments during differentiation of human skin keratinocytes (Candi et al, 2005), one cannot exclude an early upregulation of KRT10 in differentiating keratinocytes, before they reach the following stages that lead to their final cornification.…”

Regenerative Medicine: Charting a New Course in Wound Healing

“…79,80 Adipose-derived stem cells have also been induced to differentiate into keratinocytelike cells that form a stratified epidermis. 81 Stem cells derived from hair follicles are another key area of interest due to their ability to differentiate into multiple skin cell types, including melanocytes, and the ease with which they can be harvested from patients. 82 Yet another line of promising research is the transdifferentiation of terminally differentiated adult cells into other cell types using only drugbased induction, thereby avoiding the need for stem cells altogether.…”

New challenges on the production of oral mucosa using Tissue Engineering Techniques

“…Además de células madre de la gelatina de Wharton (4), se han utilizado diferentes fuentes de células madre para su diferenciación a células epiteliales, como por ejemplo las células madre de la médula ósea (20)(21), células madre del folículo piloso (22), células madre del estroma corneal (23), células madre embrionarias (24), células madre de la pulpa dental (25), células madre adiposas (26), las cuales también se podrían proponer para ser utilizadas en la generación de epitelio de la mucosa oral.…”