Recent advances in corneal regeneration and possible application of embryonic stem cell-derived corneal epithelial cells

Abstract

The depletion of limbal stem cells due to various diseases leads to corneal opacification and visual loss. The unequivocal identification and isolation of limbal stem cells may be a considerable advantage because long-term, functional recovery of corneal epithelium is linked to graft constructs that retain viable stem cell populations. As specific markers of limbal stem cells, the ATP-binding cassette, sub-family G, member2 (ABCG2), a member of the multiple drug-resistance (MDR) family of membrane transporters which leads to a side population phenotype, and transcription factor p63 were proposed recently. Conventional corneal transplantation is not applicable for patients with limbal stem cells deficiency, because the conventional allograft lacks limbal stem cells. The introduction of limbal epithelial cell transplantation was a major advance in the therapeutic techniques for reconstruction of the corneal surface. Limbal epithelial cell transplantation is clinically conducted when cultured allografts as well as autografts are available; however, allografts have a risk of immunologic rejection and autografts are hardly available for patients with bilateral ocular surface disorders. Embryonic stem (ES) cells are characterized by their capacity to proliferate indefinitely and to differentiate into any cell type. We induced corneal epithelial cells from ES cells by culturing them on type IV collagen or alternatively, by introduction of the pax6 gene into ES cells. Recent advances in our study supports the possibility of their clinical use as a cell source for reconstruction of the damaged corneal surface. This review summarizes the recent advances in corneal regeneration therapies and the possible application of ES cell-derived corneal epithelial cells.

Keywords: corneal epithelial cell; embryonic stem cell; limbal stem cell; transplantation.

Figure 1

Schematic representation of corneal epithelial cells. The ocular surface is composed of three epithela, conjunctival, limbal and corneal. Limbal stem cells are located in the palisades of Vogt, the transitional zone between the cornea and the conjunctiva. Limbal stem cells are close to blood vessels. They generate transient amplifying cells that terminally differentiate after a discrete number of cell divisions to corneal epithelial cells and undergo both centripetal migration and vertical migration.

Figure 2

Schematic representation of the side population cells analyzed by flow cytometry. Rat limbal stem cells were treated with Hoechst 33342 dye and were analyzed by flow cytometry. A population of cells with low Hoechst 33342 blue/red fluorescence was isolated as side population cells in the limbus. Non-side population cells accompanying high Hoechst 33342 blue/red fluorescence formed a distinct cell population.

Figure 3

Corneal regeneration by using cell sheets made of cultured corneal epithelial cells. Several cell sources which include limbal stem cells are applicable to form epithelial cell sheets for treating patients with limbal stem cell deficiencies. Limbal stem cells are obtained from a healthy area of a patient’s injured cornea (autologous limbal epithelium). Allogeneic limbal epithelium and autologous limbal epithelium of a patient’s contralateral normal cornea are similarly applicable. After culturing on fibrin gel, the amniotic membrane and a temperature-sensitive cell culture surface, these cells formed cell sheets, which were then transplanted onto the damaged cornea. Successful application of a patient’s oral mucous membrane was reported. Oral epithelium is used as an alternative to the limbal epithelium when the patient has severe bilateral limbal stem cell deficiency.

Figure 4

Establishment of ES cells from fertilized egg. ES cells are derived from embryos developed from eggs that have been fertilized in vitro. Human ES cells are typically four or five days old and are a hollow microscopic ball of cells called a blastocyst. The inner cell mass of the blastocyst is collected and cultured on a feeder cell layer. After several passages, the cells are established as ES cells.

Figure 5

Differentiation of ES cells which give rise to several descendants. ES cells are pluripotent, and give rise during development to all derivatives of the three primary germ layers: endoderm, mesoderm and ectoderm.

Figure 6

Transplantation of ES cell-derived corneal epithelial cells onto injured cornea. Mouse cornea was denuded by n-heptanol treatment. ES cell-derived epithelial progenitor cells were transplanted to the injured cornea. Histologic analysis was conducted. (A) At 24 hours after transplantation, the eyes were enucleated. Cryostat sections were stained with hematoxylin and eosin staining. (B) Schematic representation of panel A.

Figure 7

Corneal epithelial progenitor cells differentiated from cynomolgus monkey ES cells in vitro. Inverted microscopic view. ES cells were cultured on cell culture dishes for 4 days to form embryoid bodies, and the embryoid bodies were cultured on type IV collagen for 4 more days. The adhering cells emerged from the embryoid bodies had an epithelial cell-like appearance.