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Pluripotent stem cell-derived corneal endothelial cells as an alternative to donor corneal endothelium in keratoplasty.

ABSTRACT: Here, we evaluate the efficacy of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs) to form a functional monolayer of corneal endothelium (CE) in rabbits and monkeys. We injected cryopreserved hESC-derived CECs into the anterior chamber of rabbits and monkeys either immediately after mechanical scraping of the central CE or a few days later when corneal edema developed. All preclinical models developed deturgesced and clear corneas following the injection of cryopreserved hESC-derived CECs and remained comparable to the corneas of the untreated eye. Confocal scanning microscopy confirmed an intact structure of hexagonal/polygonal cells and immunohistochemical analysis illustrated a monolayer expressing barrier and pump function proteins in the regenerated CE. The necropsy examination confirmed no remarkable change in multiple tissues assessed for teratoma formation. In conclusion, our data demonstrate the efficacy of cryopreserved hESC-derived CECs to form a functional CE on the denuded Descemet's membrane.

SUBMITTER: Ali M

PROVIDER: S-EPMC8452521 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Pluripotent stem cell-derived corneal endothelial cells as an alternative to donor corneal endothelium in keratoplasty.

Ali Muhammad M Khan Shahid Y SY Gottsch John D JD Hutchinson Eric K EK Khan Aisha A Riazuddin S Amer SA

Stem cell reports 20210805 9

Here, we evaluate the efficacy of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs) to form a functional monolayer of corneal endothelium (CE) in rabbits and monkeys. We injected cryopreserved hESC-derived CECs into the anterior chamber of rabbits and monkeys either immediately after mechanical scraping of the central CE or a few days later when corneal edema developed. All preclinical models developed deturgesced and clear corneas following the injection of ...[more]

PMID: 34358452

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Project description:Corneal endothelial cells (CECs) do not proliferate or recover after illness or injury, resulting in decreased cell density and loss of pump/barrier function. Considering the shortage of donor cornea, it is vital to establish robust methods to generate CECs from induced pluripotent stem cells (iPSCs). We investigated the efficacy and safety of transplantation of iPSC-derived CECs into a corneal endothelial dysfunction (CED) rabbit model. iPSCs were generated from human fibroblasts. We characterized iPSCs by demonstrating the gene expression of the PSC markers OCT4, SOX2, TRA-1-60, and NANOG, teratoma formation, and differentiation into three germ layers. Differentiation of iPSCs into CECs was induced via neural crest cell (NCC) induction. CEC markers were detected using immunofluorescence and gene expression was analyzed using quantitative real-time PCR (qRT-PCR). After culturing iPSC-derived NCCs, we found the expression of zona occludens-1 (ZO-1) and Na+/K+ ATPase and a hexagonal morphology. ATP1A1, COL8A1, and AQP1 mRNA expression was higher in iPSC-derived CECs than in iPSCs and NCCs. We performed an injection of iPSC-derived CECs into the anterior chamber of a CED rabbit model and found improved levels of corneal transparency. We also found increased numbers of ZO-1- and ATP1A1-positive cells in rabbit corneas in the iPSC-derived CEC transplantation group. Usage of the coating material vitronectin (VTN) and fasudil resulted in good levels of CEC marker expression, demonstrated with Western blotting and immunocytochemistry. Combination of the VTN coating material and fasudil, instead of FNC mixture and Y27632, afforded the best results in terms of CEC differentiation's in vitro and in vivo efficacy. Successful transplantation of CEC-like cells into a CED animal model confirms the therapeutic efficacy of these cells, demonstrated by the restoration of corneal clarity. Our results suggest that iPSC-derived CECs can be a promising cellular resource for the treatment of CED.

Project description:BackgroundHuman corneal endothelial cells lack regenerative capacity through cell division in vivo. Consequently, in the case of trauma or dystrophy, the only available treatment modality is corneal tissue or primary corneal endothelial cell transplantation from cadaveric donor which faces a high global shortage. Our ultimate goal is to use the state-of-the-art 3D-bioprint technology for automated production of human partial and full-thickness corneal tissues using human stem cells and functional bioinks. In this study, we explore the feasibility of bioprinting the corneal endothelium using human pluripotent stem cell derived corneal endothelial cells and hydrazone crosslinked hyaluronic acid bioink.MethodsCorneal endothelial cells differentiated from human pluripotent stem cells were bioprinted using optimized hydrazone crosslinked hyaluronic acid based bioink. Before the bioprinting process, the biocompatibility of the bioink with cells was first analyzed with transplantation on ex vivo denuded rat and porcine corneas as well as on denuded human Descemet membrane. Subsequently, the bioprinting was proceeded and the viability of human pluripotent stem cell derived corneal endothelial cells were verified with live/dead stainings. Histological and immunofluorescence stainings involving ZO1, Na+/K+-ATPase and CD166 were used to confirm corneal endothelial cell phenotype in all experiments. Additionally, STEM121 marker was used to identify human cells from the ex vivo rat and porcine corneas.ResultsThe bioink, modified for human pluripotent stem cell derived corneal endothelial cells successfully supported both the viability and printability of the cells. Following up to 10 days of ex vivo transplantations, STEM121 positive cells were confirmed on the Descemet membrane of rat and porcine cornea demonstrating the biocompatibility of the bioink. Furthermore, biocompatibility was validated on denuded human Descemet membrane showing corneal endothelial -like characteristics. Seven days post bioprinting, the corneal endothelial -like cells were viable and showed polygonal morphology with expression and native-like localization of ZO-1, Na+/K+-ATPase and CD166. However, mesenchymal-like cells were observed in certain areas of the cultures, spreading beneath the corneal endothelial-like cell layer.ConclusionsOur results demonstrate the successful printing of human pluripotent stem cell derived corneal endothelial cells using covalently crosslinked hyaluronic acid bioink. This approach not only holds promise for a corneal endothelium transplants but also presents potential applications in the broader mission of bioprinting the full-thickness human cornea.

Project description:BackgroundCorneal transplantation is the most effective clinical treatment for irreversible corneal endothelial decompensation. However, while visual rehabilitation can be achieved by corneal transplantation, transplant rejection, poor postoperative visual acuity, and lack of suitable donor tissue are currently the greatest obstacles to corneal transplantation. As a result, endothelial cell-based therapy has emerged as an alternative to corneal transplantation treatment. Human induced pluripotent stem cells (hiPSCs) were induced to differentiate into human corneal endothelial cell (hCEC)-like cells in our study, which aimed to provide an experimental basis for studying the clinical translation and application of induced PSC (iPSC) to corneal endothelial cell (CEC) differentiation.MethodsA two-step method to convert hiPSCs into hCEC-like cells was applied in the study. First, the transforming growth factor beta (TGF-β) and Wnt signaling pathways were regulated by adding SB4315542 and CHIR99021, and hiPSCs were induced to differentiate into neural crest cells (NCCs) by a chemically defined and serum-free in vitro induction method. Subsequently, NCCs were induced to differentiate into hCEC-like cells by adding B27, platelet-derived growth factor (PDGF)-BB, and XAV939 (Wnt pathway inhibitor) to CEC culture medium.ResultsIPSCs were directionally differentiated into NCCs. During the process of differentiation, iPSCs gradually lost the monoclonal morphology of PSCs. Moreover, β-catenin and SRY-related HMG-box gene 10 (SOX10) proteins were immunohistochemically expressed on the 7th day of differentiation. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) results demonstrated that the cells expressed SOX9, SOX10, nerve growth factor receptor (NGFR), human natural killer-1 (HNK-1), and β-catenin, indicating that they were successfully directionally differentiated from iPSCs to NCCs. After 5-7 days of differentiation, the cells demonstrated a hexagonal morphology of monolayer tight junctions. Immunofluorescence results demonstrated that the cells expressed CEC indicator zonula occludens-1 (ZO-1) protein. QRT-PCR results demonstrated that the cells expressed collagen type IV alpha 1 (COL4A1), COL8A2, COL8A1, and ZO-1, which indicated that they were successfully directionally differentiated from NCC to CEC.ConclusionsOur study successfully provided a simple and efficient method with clear chemical composition and serum-free media to directionally differentiate hiPSCs into hCEC-like cells, thereby advancing the results of previous studies on directional transformation into epithelial cells.

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Pluripotent stem cell-derived corneal endothelial cells as an alternative to donor corneal endothelium in keratoplasty.

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Pluripotent stem cell-derived corneal endothelial cells as an alternative to donor corneal endothelium in keratoplasty.

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