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Arresting Proliferation Improves the Cell Identity of Corneal Endothelial Cells in the New Zealand Rabbit

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Arresting Proliferation Improves the Cell Identity of Corneal Endothelial Cells in the New Zealand Rabbit

Carlos-Alberto Rodríguez-Barrientos et al. Mol Vis.

Abstract

Purpose: Corneal endothelium engineering aims to reduce the tissue shortage for corneal grafts. We investigated the impact of mitogenic and resting culture systems on the identity of corneal endothelial cells (CECs) for tissue engineering purposes.

Methods: Rabbit CECs were cultured in growth factor-supplemented media (MitoM) until confluence. At the first passage, the CECs were divided into two populations: P1 remained cultured in MitoM, and P2 was cultured in a basal medium (RestM) for another passage. Morphologic changes in the CECs were analyzed, and RNA was isolated for transcriptome analysis. Quantitative PCR and immunocytochemistry validation of selected differentially expressed markers were performed.

Results: The CECs in MitoM showed fibroblastic morphology, whereas the CECs in RestM exhibited polygonal morphology. Circularity analysis showed similar values in human (0.75±0.056), rabbit basal (before cultured; 0.77±0.063), and CECs in RestM (0.73±0.09), while MitoM showed lower circularities (0.41±0.19). Genes related to collagen type IV and the extracellular matrix, along with the adult CEC markers ATP1A1, ATP1B1, COL8A2, GPC4, and TJP1, were highly expressed in RestM. Conversely, the IL-6, F3, and ITGB3 genes and the non-adult CEC markers CD44, CNTN3, and CD166 were more expressed in MitoM. Overall, from the transcriptome, we identified 832 differentially expressed probes. A functional analysis of the 308 human annotated differentially expressed genes revealed around 13 functional clusters related to important biological terms, such as extracellular matrix, collagen type 4, immune responses, cell proliferation, and wound healing. Quantitative PCR and immunocytochemistry confirmed the overexpression of ATP1A1, TJP1, and GPC4 in CECs in RestM.

Conclusions: The addition of a stabilization step during CEC culture improves the cells' morphology and molecular identity, which agrees with transcriptome data. This suggests that stabilization is useful for studying the plasticity of the corneal endothelium's morphology, and stabilization is proposed as a necessary step in corneal endothelium engineering.

Figures

Figure 1
Figure 1
CECs in MitoM and RestM culture conditions. A:Corneal endothelial cells (CECs) in MitoM culture conditions at P0 before the subculture (10X). B: CECs in MitoM at P1 (10X); and (C) CECs in RestM at P1 (10X). D: CECs in MitoM at P2 (10X); and (E) CECs in RestM at P2 (20X). G: Cellular circularity of human, rabbit basal, MitoM, RestM passage 1 (RestM P1), and RestM passage 2 (RestM P2) CECs. H: Cellular yield analysis of CECs obtained after the first passage in MitoM and RestM.
Figure 2
Figure 2
Overall gene expression comparison among the biological replicates 1, 2, 3, and 4. A: Hierarchical clustering comparing all probes in the microarray. B: First and second principal components. The percentage of the total explained variability is shown in the axis labels. PC1 seems to be associated with treatment. C: Hierarchical clustering using 5% of the probes with the highest variability.
Figure 3
Figure 3
Gene expression comparison and functional analysis. A: Differential expressed corneal endothelial cell (CEC) markers between cells cultured in RestM and MitoM. The molecular markers are grouped by type. Only markers close to p=0.05 are shown. Relative expression is estimated in the Z-score (standard deviations from the mean). B: Functional analysis of differentially expressed genes. The heat map shows the genes (horizontal axis) contained within functional biological terms (vertical axis). The color represents the fold change in gene expression (cyan is used to represent those genes that were more greatly expressed in MitoM, while purple represents those genes more greatly expressed in RestM). Genes whose t test p values were less than 0.01, and which demonstrated a fold change greater than 1, were used. Only over-represented biological terms with a p value of less than 0.01, a false discovery rate of less than 0.25, and those that contained more than three genes were used in this analysis. Details of this figure, including the genes and biological terms used, are provided in the Appendix 1 and Appendix 2. C: The relative expression of selected functional terms. The color represents the fold change in gene expression (cyan is used to represent those genes that are more expressed in MitoM, while purple is used for those genes more greatly expressed in RestM). Genes with a t test p value less than 0.01 and a fold change greater than 1 were used. The dashed lines mark twofold expression.
Figure 4
Figure 4
Comparison of protein and transcript expression. A: TJP1 / ZO-1 specific surface marker. 20X, immunostaining on flat-mounted cornea, corneal endothelium, basal condition respectively. B: 40X, immunocytochemistry of second passage cultured CECs upon a two-phase culture system. Specific surface markers were assessed upon a two-phase culture system. In RestM, tight junction zig-zag characteristic configuration is observed and well stablished between cells. In MitoM, weak fluorescent signal and lack of protein location. In control no primary antibody; exposure normalized for each antibody set. C: qPCR of markers in rabbit CECs (basal expression levels), MitoM condition or RestM condition. Ct values were normalized using GAPDH. ΔCT represent the difference in Ct values between GAPDH and the gene shown.

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