Long-Term Observation and Sequencing Analysis of SKPs-Derived Corneal Endothelial Cell-Like Cells for Treating Corneal Endothelial Dysfunction

Abstract

Corneal endothelial dysfunction is a principal cause of visual deficiency. Corneal transplantation is the most effective treatment for corneal endothelial dysfunction. However, a severe shortage of available donor corneas or human corneal endothelial cells (HCECs) remains a global challenge. Previously, we acquired corneal endothelial cell-like cells (CEC-like cells) derived from human skin-derived precursors (SKPs). CEC-like cells were injected into rabbit and monkey corneal endothelial dysfunction models and exerted excellent therapeutic effect. In this study, we prolonged the clinical observation in the monkey experiment for 2 years. Polymerase chain reaction (PCR) and DNA sequencing were carried out to confirm the existence of CEC-like cells. Histological examinations were carried out to show the corneal morphology. Further transcriptome sequencing was also carried out on HCEC, CEC-like cells before transplantation and after transplantation. We found that the monkeys cornea remained transparent and normal thickness. The total endothelial cell density decreased gradually, but tended to be stable and remained in a normal range during 2-year observation. The CEC-like cells persist during observation and could adapt to the microenvironment after transplantation. The gene expression pattern of CEC-like cells was similar to HCEC and changed slightly after transplantation. In conclusion, this study presented a brand-new insight into CEC-like cells and further provided a promising prospect of cell-based therapy for corneal endothelial dysfunction. The renewable cell source, novel derivation method and simple treatment strategy may be clinically applied in regenerative medicine in the future.

Keywords: cell-based therapy; corneal endothelial cell-like cells; corneal endothelial dysfunction; skin-derived precursors; transcriptome sequencing.

Figure 1.

Cell culture. (A) Human SKPs were small floating spheres. CEC-like cells displayed hexagonal or polygonal shapes and formed a mosaic monolayer, which were similar to HCEC. (B) CEC-like cells expressed Na⁺/K⁺ ATPase and ZO-1. Scar bars 100 μm.

Figure 2.

Clinical observation of the experimental group and control group during 2 years. (A) In the experimental group, the corneas remained transparent, and almost no keratic precipitates or corneal neovascularization appeared. (B) In the control group, the corneas remained opaque with edema and as a consequence the iris could not be seen. Monkey 1 and 2 are shown here. The other three monkeys are shown in Supplemental figure 2.

Figure 3.

Ophthalmic examinations of the experimental group and control group during 2-year observation. (A) Visante OCT showed significant corneal thickness differences in the experimental group and the control group (Monkey 1 and 2). (B) Non-contact specular microscopy showed that cells after transplantation were in the form of a polygonal monolayer (Monkey 1). (C) The changes of corneal thickness in the experimental group and control group. (D) The changes of cell density in the experimental group. (E) Chamber angle examination. (F) B-ultrasonography. (G) Fundus imaging. (E–G: Monkey 1 at 2-year time is shown)

Figure 4.

DNA-sequencing and immunofluorescent staining after transplantation. (A) Polymerase chain reaction of HCEC, MON cells, CEC-like cells, EXP1 cells, EXP2 cells. (B) DNA-sequencing of EXP2 456 bp gene fragment products. (C) Immunofluorescent staining of the EXP1, EXP2, MON and control group. Scar bars 100 μm.

Figure 5.

HE staining of the EXP1, EXP2, MON and control group. The morphology of EXP1 and EXP2 group were all similar to the normal group. However, in the control group, the cornea was very thick and edematous. The arrow points to bare Descemet’s membrane in control group. Scar bars 100 μm.

Figure 6.

Functional pattern of gene expression in HCEC, CEC-like cells, EXP1 cells, and EXP2 cells. (A) Venn analysis of CEC-like cells and HCEC; (B) GO annotation analysis of CEC-like cells and HCEC. (C) Venn analysis of CEC-like cells, EXP1 cells, EXP2 cells. (D) GO annotation analysis of CEC-like cells, EXP1, EXP2. (E) Venn analysis of HCEC, EXP1 cells, EXP2 cells. (F) GO annotation analysis of HCEC, EXP1 cells, EXP2 cells. (G) The correlation analysis of CEC-like cells, HCEC, EXP1 cells, EXP2 cells.

Figure 7.

Differential expression gene analysis of CEC-like cells, EXP1 cells, and EXP2 cells. (A) Heapmap of CEC-like cells versus EXP1 cells versus EXP2 cells. (B) Volcano of CEC-like cells versus EXP1 cells. (C) GO enrichment analysis of CEC-like cells versus EXP1 cells. (D) Volcano of CEC-like cells versus EXP2 cells. (E) GO enrichment analysis of CEC-like cells versus EXP2 cells.