A Sequential Culturing System for Generating Epithelial-Like Stem Cells from Human Mesenchymal Stem Cells Derived from Adipose Tissue

Abstract

Skin burn repair requires efficient regeneration in order to achieve proper healing. We applied a sequential culturing system 2D1w/3D2w protocol for differentiating human mesenvhymal stem cell derived from adipose tissue (hMSC-AT) into epithelial-like stem cells (ELSC). hMSC-AT were cultured, characterized by immunophenotyping, mesenchymal differentiation and induced in a 2D culture using epithelial differentiation medium containing ATRA, ITS, dexamethasone, EGF and FGF-2 for 7 days (2D1w), followed by seeding in a 3D culture using chitosan hydrogel combined with the same cocktail (3D2w) for 2 weeks. The immunostaining of the cells was done at different stages of induction in order to characterize the differentiated cells using pan cytokeratin and anti-cytokeratins 14 and 18 antibodies. The expression of cytokeratins 5, 10, 14, 18 and 19 was evaluated by RT-PCR. The toxicity of chitosan hydrogel and proliferation of ELSC in 3D chitosan hydrogel were evaluated by MTT test. The viability of ELSC in 3D culture was evaluated by acridine orange/propidium iodide staining. The hMSC-AT were immunopositive to CD90, CD105, CD73, CD45 and CD34. The ELSC in the 2D culture expressed cytokeratin 19 after 7 days, while the other cytokeratins were not expressed. At the second week (3D culture), all of the above markers were expressed except cytokerstin10, moreover, the viability and proliferation were 98.02 ± 1.34 and 5.45 ± 0.36%, respectively. The cytotoxicity assay demonstrates the biocompatibility of chitosan hydrogel. The study reveals that hMSC-AT, seeded on chitosan hydrogel, can be induced into ELSC in the presence of other inducers.