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.

Stem Cell-Based Therapy of Corneal Epithelial and Endothelial Diseases

“…A procedure where CECs were differentiated from mouse corneal stromal precursors by retinoic acid and activation of Wnt/(beta)catenin signaling was also reported, which confirmed the function of these CECs on a rabbit corneal disease model, and human CEC-like cells were acquired from differentiation of human corneal stromal precursors [52]. Corneal endothelial precursors were also found by sphere-forming assay [53] ( Table 2).…”

Identification of Cell Surface Markers Glypican-4 and CD200 That Differentiate Human Corneal Endothelium From Stromal Fibroblasts

“…9 To circumvent the shortage of corneal tissues and reliance on human donor material, development of suitable tissue-engineered constructs using cultured HCECs from cadaveric donors [10][11][12][13] or HCECs derived from multipotent progenitor cells are being explored by various groups as potential graft alternatives. [14][15][16] The isolation of HCECs from cadaveric donors involves peeling of the DM together with the corneal endothelium layer, and subsequent enzymatic digestion with collagenase to release the corneal endothelial cells from the DM. 11 However, in some cases excessive manipulation may result in tearing into and coisolation of small amounts of stromal tissue.…”

Targeted Transplantation of Human Umbilical Cord Blood Endothelial Progenitor Cells with Immunomagnetic Nanoparticles to Repair Corneal Endothelium Defect

“…When the cell density is reduced to below 400-500 cells per mm 2 , the CECs could no longer adequately function, resulting in corneal edema, the development of bullous keratopathy, and the loss of visual acuity [12]. With the increasing worldwide shortage of corneas, stem cells, such as corneal endothelial precursor cells [13], corneal stroma stem cells [14], human embryonic stem cells [15], and human UCB mesenchymal stem cells [16], have been investigated to replace CECs. Putative EPCs were first isolated from human peripheral blood by magnetic bead selection on the basis of cell surface expression of the CD34 antigen [17].…”