title: Functional corneal endothelium derived from corneal stroma stem cells of neural crest origin by retinoic acid and Wnt/β-catenin signaling.
abstract: Corneal endothelial dysfunction remains a major indication for corneal transplantation. Both corneal endothelial cells and stromal cells originate from the neural crest, but have distinct phenotypes and function in the adult cornea. We previously reported that stem cells isolated from the adult corneal stroma [cornea-derived precursors (COPs)] show characteristics of multipotent neural crest-derived stem cells. In this study, we report the induction of functional tissue-engineered corneal endothelium (TECE) from mouse and human COPs. TECE was engineered from Wnt1-Cre/Floxed EGFP mouse COPs in a medium containing retinoic acid and glycogen synthase kinase (GSK) 3β inhibitor (activator of Wnt/β-catenin signaling). The expression levels of major markers characterizing corneal endothelial function (Atp1a1, Slc4a4, Car2, Col4a2, Col8a2, and Cdh2) were significantly upregulated. Both retinoic acid and GSK 3β inhibitor upregulated the expression of Pitx2, a homeobox gene involved in the development of the anterior segment of the eye. GSK 3β inhibitor increased Atp1a1 expression and Na,K-ATPase pump activity of TECE, which was significantly higher than COPs or control 3T3 cells, and 2.6-fold higher than cultured mouse corneal endothelial cells. Mouse TECE transplanted into rabbit corneas maintained transparency and corneal thickness, whereas control corneas without TECE showed marked edema and increased corneal thickness. Furthermore, we successfully induced TECE from human COPs, and human TECE transplanted into rabbit corneas also maintained corneal transparency and thickness. This protocol enables efficient production of corneal endothelium from corneal stromal stem cells by direct induction, which may lead to a novel stem cell therapy for corneal endothelial dysfunction.
authors.affiliation: Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Shin
authors.initials: S
authors.lastname: Hatou
authors.forename: Satoru
authors.initials: S
authors.lastname: Yoshida
authors.forename: Kazunari
authors.initials: K
authors.lastname: Higa
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Miyashita
authors.forename: Emi
authors.initials: E
authors.lastname: Inagaki
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Okano
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tsubota
authors.forename: Shigeto
authors.initials: S
authors.lastname: Shimmura
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis; D015820:Cadherins / Q000096:biosynthesis; D002450:Cell Communication; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000096:biosynthesis; D024102:Collagen Type VIII / Q000096:biosynthesis; D003319:Corneal Stroma / Q000166:cytology / Q000378:metabolism*; D016039:Corneal Transplantation; D004728:Endothelium, Corneal / Q000166:cytology* / Q000502:physiology*; D018398:Homeodomain Proteins / Q000096:biosynthesis; D006801:Humans; D051379:Mice; D039902:Multipotent Stem Cells; D009419:Nerve Tissue Proteins / Q000494:pharmacology; D009432:Neural Crest / Q000166:cytology*; D058953:Neural Stem Cells; D011817:Rabbits; D027982:Sodium-Bicarbonate Symporters / Q000096:biosynthesis; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis / Q000378:metabolism; D023822:Tissue Engineering / Q000379:methods*; D014157:Transcription Factors / Q000096:biosynthesis; D014212:Tretinoin / Q000494:pharmacology; D015854:Up-Regulation; D051153:Wnt Proteins / Q000378:metabolism; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000378:metabolism; D000097577:Homeobox Protein PITX2
chemical_list: D015703:Antigens, CD; D015820:Cadherins; D024141:Collagen Type IV; D024102:Collagen Type VIII; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D027982:Sodium-Bicarbonate Symporters; D000254:Sodium-Potassium-Exchanging ATPase; D014157:Transcription Factors; D014212:Tretinoin; D051153:Wnt Proteins; D051176:beta Catenin; D000097577:Homeobox Protein PITX2; C050361:CDH2 protein, human; C502625:COL4A2 protein, human; C443712:COL8A2 protein, human; C498898:SLC4A4 protein, human; C118136:TPPP protein, human; C499076:ATP1A1 protein, human