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Jia He, Huimin Sun, Shangkun Ou, Xin He, Zhongyang Zhao, Yangluowa Qu, Vimalin Jeyalatha, Peter Sol Reinach, Zuguo Liu, Wei Li; Construction and application of tissue engineered corneal epithelium from human embryonic stem cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4157.
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© ARVO (1962-2015); The Authors (2016-present)
To construct corneal epithelial like tissue from human embryonic stem cells (hESCs) and determine the efficacy of this tissue in rabbit corneal epithelial stem cell deficiency model.
hESCs were seeded on low attachment plates containing a differentiation medium I. After 8 days, an intermediate neural progenitor cell (NPs) was obtained. Then dissociate the NPs into single cells and reseed on attachment plates followed by 4 to 7 days of exposure to differentiation medium II. Subsequently, the resulting cells were harvested and seeded onto a deepithelialized amniotic membrane (dAM) and exposed to differentiation medium II for another 7 days. Epithelial cell sheets (ES-CE) were obtained after 21 days, which served as bandages for covering bare corneal stroma in limbal stem cell deficiency (LSCD) model. The progression of each stage of this controlled differentiation process was monitored based on characterization of changes in cell-type biomarker expression pattern.
Human ESCs cultured in differentiation medium I for 8 days differentiated into NPs. P63 and Pax6 expression gradually increased and peaked after 8 days. Single cell enzymatic dissociation of these spherical intermediates and subsequent differentiation induction for 7 days by differentiation medium II on dAM resulted in generation of a stratified epithelium. H&E staining along with P63, E-cadherin, K14 and Ki67 gene expression and localization documented successful simulation of in-vivo structural integrity. 8 weeks after the ES-CE transplantation, corneal transparency was restored to an extent similar to that in the normal uninjured control. In contrast, epithelial stem cell deficient rabbits bandaged instead with dAM remained translucent. Limbal and central corneal epithelial morphology in the ES-CE group were similar to that in normal corneas and expressed corneal epithelial cell biomarker K3 / K12 duplex. However, cornea transplanted with dAM alone showed only 1-2 layers of epithelial cells. 8 weeks after transplantation, a large number of hESCs-derived cells were still present on the corneal surface based on human cell nuclear staining.
A defined novel protocol induces hESCs to rapidly differentiate into Pax6 positive corneal epithelial progenitor cells. CE-liked tissue derived from this protocol restores normal corneal tissue structural integrity and transparency in LSCD rabbit model.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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