Tissue engineered corneal epithelium derived from clinical-grade human embryonic stem cells

Highlights

• Clinical-grade human embryonic stem cells (hESCs) differentiated into corneal epithelial lineage by a stepwise protocol.

• Single cell RNA sequencing analyses monitored gene expression and phenotypic changes during hESCs differentiation.

• Tissue engineered epithelium generated from hESCs exhibit biosafety in nude mice after subcutaneous transplantation.

• Tissue engineered epithelium generated from hESCs successfully reconstructed ocular surface of a limbal stem cell-deficient rabbit.

Abstract

Purpose

To construct tissue engineered corneal epithelium from a clinical-grade human embryonic stem cells (hESCs) and investigate the dynamic gene profile and phenotypic transition in the process of differentiation.

Methods

A stepwise protocol was applied to induce differentiation of clinical-grade hESCs Q-CTS-hESC-1 and construct tissue engineered corneal epithelium. Single cell RNA sequencing (scRNA-seq) analysis was performed to monitor gene expression and phenotypic changes at different differentiation stages. Immunostaining, real-time quantitative PCR and Western blot analysis were conducted to detect gene and protein expressions. After subcutaneous transplantation into nude mice to test the biosafety, the epithelial construct was transplanted in a rabbit corneal limbal stem cell deficiency (LSCD) model and followed up for eight weeks.

Results

The hESCs were successfully induced into epithelial cells. scRNA-seq analysis revealed upregulation of ocular surface epithelial cell lineage related genes such as TP63, Pax6, KRT14, and activation of Wnt, Notch, Hippo, and Hedgehog signaling pathways during the differentiation process. Tissue engineered epithelial cell sheet derived from hESCs showed stratified structure and normal corneal epithelial phenotype with presence of clonogenic progenitor cells. Eight weeks after grafting the cell sheet onto the ocular surface of LSCD rabbit model, a full-thickness continuous corneal epithelium developed to fully cover the damaged areas with normal limbal and corneal epithelial phenotype.

Conclusion

The tissue engineered corneal epithelium generated from a clinical-grade hESCs may be feasible in the treatment of limbal stem cell deficiency.

Introduction

The transparent cornea is crucial for normal vision. One of the requirements for sustaining corneal transparency is preserving the structural integrity of the stratified epithelium on the surface layer of the cornea [1,2]. To meet this requirement, the corneal epithelium needs to maintain self-renewal during homeostasis, a property which is governed by corneal epithelial stem cells. It is well accepted that human corneal epithelial stem cells are located at the basal layer of corneal limbus, i.e., the palisades of Vogt at the junction between the cornea and the conjunctiva [[3], [4], [5], [6], [7]]. Various clinical conditions in the cornea, such as acute trauma, chemical or thermal injury, Stevens-Johnson syndrome and genetic disorders like aniridia, can cause partial or total limbal stem cell deficiency (LSCD), thereby compromising the homeostasis of the corneal epithelium to cause corneal conjunctivalization, opacification, and neovascularization, all of which can eventually cause vision loss or even blindness [[8], [9], [10], [11], [12], [13], [14], [15], [16]]. LSCD is one of the leading causes of irreversible vision loss in the world, especially in the developing countries.

Over the past two decades, different strategies have been developed to treat LSCD. Autologous limbal tissue transplantation has been applied to treat unilateral LSCD [17,18]. Conjunctival transplantation, limbal allograft transplantation, and tissue engineered corneal epithelial transplantation have been used in bilateral LSCD [[19], [20], [21], [22], [23]]. To construct tissue engineered corneal epithelium, different cell sources including hair follicle stem cells, oral mucosal epithelial cells, dental pulp stem cells, nasal mucosal epithelial cells, induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs) have been employed, and some have been successfully applied in the clinic [[24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34]].

General interest in using hESC-derived cells for cell therapy has been rising due to their relatively unlimited cell availability and the development of robust lineage differentiation protocols. The first hESC-derived retinal pigmented epithelial cells transplantation that proved to be safe was performed in 2012 in patients with Stargardt's macular dystrophy [35]. Another hESC-derived cardiac progenitors transplantation for severe heart failure provided symptomatic improvement without any complications [36]. For severe ocular surface diseases, mouse ESC-derived corneal epithelial progenitors were first used for epithelial reconstruction of damaged mouse corneal epithelium in 2004 [37]. While different protocols have been reported to induce the differentiation of hESCs into corneal epithelium-like cells [24,38,39], the cellular phenotypic changes underwent during the sequential differentiation procedures were not well defined, and their final cell fate after transplantation into animal models had remained elusive.

In this study, we applied a stepwise differentiation protocol to generate corneal epithelial cell sheets from the first Chinese clinical-grade hESC line Q-CTS-hESC-1 [40]. We profiled 29,812 single-cell transcriptomes at multiple time points throughout the entire differentiation procedure using single-cell RNA-sequencing (scRNA-seq) and reconstructed their cell fate trajectories. Moreover, various functional assays were performed to characterize the phenotype of the cells at different stages. Our protocol successfully generated corneal epithelial progenitor cells and tissue-engineered epithelium cell sheets using hESCs, to restore ocular function in a LSCD rabbit model[53].