Construction of a high cell density human corneal endothelial equivalent and its transplantation in primate models

Abstract

Background Recently, many patients with corneal blindness caused by endothelial dysfunction have no opportunity to receive keratoplasty therapy because of the extremely limited number of donor corneas. Corneal tissue engineering opens a new path for in vitro reconstruction of tissue‐engineered HCE which will cure the corneal endotheliopathy by clinical corneal transplantation. In this study, we construct a human corneal endothelium (HCE) equivalent with non‐transfected monoclonal HCE (mcHCE) cells and modified denuded amniotic membrane (mdAM), and evaluate its functions in monkey models. Methods Tissue‐engineered HCE (TE‐HCE) was constructed by culturing DiI‐labeled mcHCE cells on mdAMs in 20% fetal bovine serum‐containing DMEM/Ham’s Nutrient Mixture F12 (1:1) medium and 5% CO2 at 37°C on a 24‐well culture plate. The constructed TE‐HCE was transplanted into monkey corneas via penetrating keratoplasty with Descemet’s membrane and endothelium stripped. The corneal transparency, thickness, and intraocular pressure were monitored in vivo, and the corneal morphology and histological structure were examined ex vivo 181 days after surgery. Results The constructed TE‐HCE, with an average density of 3602.22 ± 45.22 cells/mm2, mimicked its natural counterpart both in morphology and histological structure. In vivo, corneal transparency was maintained, and the corneal thickness gradually decreased to 567.33 ± 72.77 μm at day 181 after TE‐HCE transplanted into monkey eyes, while intense corneal edema and turbid were found in mdAM‐transplanted eyes with their corneal thicknesses maintained over 1000 μm during the monitoring period. Ex vivo, a monolayer of corneal endothelium, consisting of mcHCE cells at a density of 2795.65 ± 156.83 cells/mm2, was reconstructed in transplanted monkey eyes. The cells in the transplanted area had the hexagonal or polygonal morphology and normal ultrastructure, and established plenty of cell‐cell and cell‐stromal matrix junctions. Besides, huge membrane‐bounded flat stacks with electric dense inclusions were found in mcHCE cells beneath the plasma membrane at the stromal side. Conclusions The constructed TE‐HCE has normal histological property and functions well in monkey models. The TE‐HCE could be used as a promising HCE equivalent in therapy of corneal endothelium dysfunction and corneal regenerative medicine.

Xenotransplantation literature update, July/August 2019

“…10 Zhao et al provide insight on the historic limitations of HCE grafts, generating excitement for recent advancements. 10 Primate models were used to test these grafts for functionality and compatibility; the success of this study presents likelihood of use in future clinical applications. 10 Screening of biomarkers in grafts for future corneal xenotransplantation studies could be used to reduce graft rejection.…”

“…10 Primate models were used to test these grafts for functionality and compatibility; the success of this study presents likelihood of use in future clinical applications. 10 Screening of biomarkers in grafts for future corneal xenotransplantation studies could be used to reduce graft rejection. Yoon et al provide details on several critical biomarkers for predicting graft failure; the candidates tested were given values for late rejection and survival; thus, their impact could be estimated.…”

“…Zheng et al provide much needed details on new factors discovered, such as pre‐surgical inflammation, and potential correlations with complications . Similarly, corneal blindness from endothelium dysfunction may be reversed with transplantation of tissue‐engineered human corneal endothelium (TE‐HCE) grafts . Zhao et al provide insight on the historic limitations of HCE grafts, generating excitement for recent advancements .…”

“…Similarly, corneal blindness from endothelium dysfunction may be reversed with transplantation of tissue‐engineered human corneal endothelium (TE‐HCE) grafts . Zhao et al provide insight on the historic limitations of HCE grafts, generating excitement for recent advancements . Primate models were used to test these grafts for functionality and compatibility; the success of this study presents likelihood of use in future clinical applications …”

“…In the previous issue of Xenotransplantation, Zhou et al provided an updated review of the complement system in xenograft rejection; Hundrieser et al discuss how porcine and human MHC‐II polymorphisms influence donor reactivity; Ladowski et al seek to eliminate all donor xenoantigens; Cooper et al review a 9 gene alteration of porcine donors for clinical xenotransplantation; Zhou et al present a strategy to monitor xenograft rejection with porcine cDNA biomarkers; Zafar et al provide further research into reducing porcine islet immunoreactivity; Li et al studied the cold storage of porcine hepatocyte spheroids; Iizuka et al deep dive into porcine C‐peptide pharmacokinetics; and, finally, Zheng et al, Zhao et al, and Yoon et al provide updates in corneal xenotransplantation.…”