title: Differentiation of porcine mesenchymal stem cells into epithelial cells as a potential therapeutic application to facilitate epithelial regeneration.
abstract: Epithelial denudation is one of the characteristics of chronic asthma. To restore its functions, the airway epithelium has to rapidly repair the injuries and regenerate its structure and integrity. Mesenchymal stem cells (MSCs) have the ability to differentiate into many cell lineages. However, the differentiation of MSCs into epithelial cells has not been fully studied. Here, we examined the differentiation of MSCs into epithelial cells using three different media compositions with various growth supplementations. The MSCs were isolated from porcine bone marrow by density gradient centrifugation. The isolated MSCs were CD11(-) CD34(-) CD45(-) CD44(+) CD90(+) and CD105(+) by immunostaining and flow cytometry. MSCs were stimulated with EpiGRO (Millipore), BEpiCM (ScienCell) and AECGM (PromoCell) media for 5 and 10 days, and epithelial differentiation was assessed by qPCR (keratin 14, 18 and EpCAM), fluorometry (cytokeratin 7-8, cytokeratin 14-15-16-19 and EpCAM), western blot analysis (pancytokeratin, EpCAM) and flow cytometry (cytokeratin 7-8, cytokeratin 14-15-16-19 and EpCAM). The functional marker MUC1 was also assessed after 10 days of air-liquid interface (ALI) culture in optimized media. Cells cultured in BEpiCM containing fibroblast growth factor and prostaglandin E2 showed the highest expression of the epithelial markers: CK7-8 (85.90%); CK-14-15-16-19 (10.14%); and EpCAM (64.61%). The cells also expressed functional marker MUC1 after ALI culture. The differentiated MSCs when cultured in BEpiCM medium ex vivo in a bioreactor on a decellularized trachea for 10 days retained the epithelial-like phenotype. In conclusion, porcine bone marrow-derived MSCs demonstrate commitment to the epithelial lineage and might be a potential therapy for facilitating the repair of denuded airway epithelium.
authors.affiliation: Center for Clinical and Translational Science and Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE, USA.
authors.forename: Kelsey
authors.initials: K
authors.lastname: Kokubun
authors.affiliation: Center for Clinical and Translational Science and Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE, USA.
authors.forename: Divya
authors.initials: D
authors.lastname: Pankajakshan
authors.affiliation: Center for Clinical and Translational Science and Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE, USA.
authors.forename: Min-Jung
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: Center for Clinical and Translational Science and Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE, USA.
authors.forename: Devendra K
authors.initials: DK
authors.lastname: Agrawal
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D000071858:Epithelial Cell Adhesion Molecule; D004847:Epithelial Cells / Q000166:cytology*; D004848:Epithelium / Q000502:physiology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005470:Fluorometry; D007633:Keratins / Q000378:metabolism; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D018396:Mucin-1 / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012038:Regeneration; D034421:Sus scrofa; D014132:Trachea / Q000166:cytology
chemical_list: D000951:Antigens, Neoplasm; D015415:Biomarkers; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D018396:Mucin-1; D012333:RNA, Messenger; D007633:Keratins
keywords: decellularization; differentiation; epithelial cells; epithelial repair; growth factors; lung injury; mesenchymal stem cells