Transplanting embryonic stem cells onto damaged human corneal endothelium

Abstract

Aim: To investigate whether human embryonic stem cells (hESCs) could be made to attach, grow and differentiate on a human Descemet's membrane (DM).

Methods: Spontaneously differentiated hESCs were transferred onto a human corneal button with the endothelial layer removed using ocular sticks. The cells were cultured on a DM for up to 15 d. The genetically engineered hESC line expressed green fluorescent protein, which facilitated identification during the culture experiments, tissue preparation, and analysis. To detect any differentiation into human corneal endothelial-like cells, we analysed the transplanted cells by immunohistochemistry using specific antibodies.

Results: We found transplanted cells form a single layer of cells with a hexagonal shape in the periphery of the DM. The majority of the cells were negative for octamer-binding transcription factor 4 but positive for paired box 6 protein, sodium potassium adenosine triphosphatase (NaKATPase), and Zona Occludens protein 1. In four of the 18 trials, the transplanted cells were found to express CK3, which indicates that the stem cells differentiated into corneal epithelial cells in these cases.

Conclusion: It is possible to get cells originating from hESCs to become established on a human DM, where they grow and differentiate into corneal endothelial-like cells in vitro.

Keywords: Cornea; Descemet’s membrane; Embryonic stem cells; Endothelium; Immunohistochemistry.

Figure 1

Overview of embryonic stem cells transplanted onto Descemet’s membrane. A: Fluorescent cells derived from human embryonic stem cells; B: Outline of the cell walls of the same cells. Red stars mark the same cells in both photos.

Figure 2

Endothelial parts of three corneal sections: A-C, D-F, and G-I. A: Nuclei through a 4‘,6-diamidino-2-phenylindole (DAPI) filter; B: The same cells through a fluorescein isothiocyanate (FITC) filter [green fluorescent protein (GFP) fluorescence]; C: Expression of the tight junction protein Zona Occludens protein 1 (ZO-1) [tetramethylrhodamine (TRITC) filter]; D: Nuclei seen through a DAPI filter; E: GFP expression (using an FITC filter); F: Expression of sodium potassium ATPase (NaKATPase) (using a TRITC filter); G: Nuclei seen through a DAPI filter; H: GFP expression (FITC filter); I: Expression of the cornea-related protein paired box 6 (PAX-6).