Tissue engineered corneal epithelium derived from clinical-grade human embryonic stem cells
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].
Section snippets
Stepwise differentiation of hESC line
Q-CTS-hESC-1 is the first Chinese clinical-grade human embryonic cell line that meets the requirements of the China Food and Drug Administration [40]. This cell line is maintained in a serum-free and feeder cell-free culture system over the long-term. To generate corneal epithelial progenitor cells, the entire differentiation process is demarcated into four stages, stage I as the hESCs stage, stage II as the neural sphere culture stage (NP), stage III as the epithelial progenitor cells stage,
Discussion
In this study, we generated a corneal epithelial cell sheet derived from the first Chinese clinical grade embryonic stem cell line Q-CTS-hESC-1, and successfully transplanted this ES-CE cell sheet with a denuded amniotic membrane scaffold onto a LSCD rabbit model to reconstruct the ocular surface. Eight weeks after ES-CE cell sheet transplantation, the construct became transparent in the corneal area with well-stratified corneal terminal differentiation and peripheral neovascularization in the
Declaration of competing interest
The authors have declared that no competing interests exist.
Acknowledgments
This study was supported in part by the National Key R&D Program of China (2018YFA0107301 [to WL], 2018YFA0107304 [to ZL], 2013CB967003 [to WL]), the National Natural Science Foundation of China (NSFC, No.81770894, No.81470602 [to WL], and No.81330022 [to ZL]). The funders have no role in the study design, data collection and analysis, decision on publishing, or preparation of the manuscript.
The authors thank Jing-Ru Huang and Xiang You from the Biomedical Science Core Facility of Xiamen
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2022, Ocular SurfaceCitation Excerpt :Notably, the latter approach is less successful due to a higher risk of immune rejection and the likelihood of adverse effects developing from long-term immunosuppressive co-therapy [10,11]. Towards optimizing transplantation, the ex vivo expansion of SCs from the same source (e.g., limbus [12–14], or conjunctiva [15]), or alternative non-ocular sources (such as the hair follicle bulge [16], embryo [17] and oral or nasal mucosa [14,18,19]) has been an attractive strategy to address the limited supply of LESCs. Since LSCD is an uncommon disease, obtaining sufficient patient populations to validate novel interventions in large-scale clinical trials is a challenge.
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2021, Medical HypothesesCitation Excerpt :To understand the biological characteristics of LSC, the first work is to find accurate biomarkers. Studies on potential biomarkers of human LSC showed that PAX6 and ATP binding cassette subfamily G member 2 (ABCG2) have a significantly increased expression in the limbus, which could be used as potential LSC biomarkers [6,7]. Besides, previous observational study demonstrated that soft cellular stiffness was related with the stemness of LSC and this study highlighted the relationship between biomechanical properties and physiological functions of LSC.
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These authors contributed equally to this work thus should be considered as co-first authors.