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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 mammals (rabbits) and non-human primates (monkeys). We injected cryopreserved hESC-derived CECs in rabbits and monkeys either immediately after removing 8 mm of the central portion of the CE or a few days later when corneal edema developed. All clinical models developed deturgesced and clear corneas 2-3 weeks following the CEC injection and remained comparable to the CE 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 examined 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE173692 | GEO | 2021/09/29

REPOSITORIES: GEO

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Project description:Corneal endothelial cells (CECs) are critical to maintaining clarity of the cornea. This study was initiated to develop peripheral blood mononuclear cells (PBMC)-originated induced pluripotent stem cells (iPSCs)-derived CECs. We isolated PBMC and programmed the mononuclear cells to generate iPSCs. Subsequently, the PBMC-originated iPSCs were differentiated to CECs. The morphology of differentiating iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotent, and CECs-associated markers was investigated by quantitative real-time PCR (qRT-PCR). The molecular architecture of the iPSCs-derived CECs and human corneal endothelium (CE) were examined by mass spectrometry-based proteome sequencing. The PBMC-originated iPSCs expressed pluripotent-specific markers at levels similar to expression in H9 human embryonic stem cells (hESCs). Phase contrast microscopy illustrated that iPSCs-derived CECs are tightly adherent, exhibiting a hexagonal-like shape, one of the cardinal characteristics of CECs. The CECs-associated markers were expressed at many orders of magnitude higher in iPSCs-derived CECs at days 13, 20, and 30 compared to their respective levels in iPSCs. Importantly, only residual expression levels of pluripotency markers were detected in iPSCs-derived CECs. Mass spectrometry-based proteome profiling identified 10,575 proteins in iPSCs-derived CECs. In parallel, we completed proteome profiling of the human CE identifying 6345 proteins. Of these, 5763 proteins were identified in the iPSCs-derived CECs suggesting a 90.82% overlap between the iPSCs-derived CECs and human CE proteomes. Importantly, cryopreservation of iPSCs-derived CECs did not affect the tight adherence of CECs, and their hexagonal-like shape while expressing high levels of CECs-associated markers. We have successfully developed a personalized approach to generate CECs that closely mimic the molecular architecture of the human CE. To the best of our knowledge, this is the first report describing the development of PBMC-originated, iPSCs-derived CECs.

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

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