Novel Identity and Functional Markers for Human Corneal Endothelial Cells

Abstract

Purpose: Human corneal endothelial cell (HCEC) density decreases with age, surgical complications, or disease, leading to vision impairment. Such endothelial dysfunction is an indication for corneal transplantation, although there is a worldwide shortage of transplant-grade tissue. To overcome the current poor donor availability, here we isolate, expand, and characterize HCECs in vitro as a step toward cell therapy.

Methods: Human corneal endothelial cells were isolated from cadaveric corneas and expanded in vitro. Cell identity was evaluated based on morphology and immunocytochemistry, and gene expression analysis and flow cytometry were used to identify novel HCEC-specific markers. The functional ability of HCEC to form barriers was assessed by transendothelial electrical resistance (TEER) assays.

Results: Cultured HCECs demonstrated canonical morphology for up to four passages and later underwent endothelial-to-mesenchymal transition (EnMT). Quality of donor tissue influenced cell measures in culture including proliferation rate. Cultured HCECs expressed identity markers, and microarray analysis revealed novel endothelial-specific markers that were validated by flow cytometry. Finally, canonical HCECs expressed higher levels of CD56, which correlated with higher TEER than fibroblastic HCECs.

Conclusions: In vitro expansion of HCECs from cadaveric donor corneas yields functional cells identifiable by morphology and a panel of novel markers. Markers described correlated with function in culture, suggesting a basis for cell therapy for corneal endothelial dysfunction.

Figure 1

Human corneal endothelial cells isolation and culture. (A) Outline of the HCEC isolation and primary culture. (B–G) Bright-field micrographs of cultured HCECs at different passage (P) numbers. Primary cultures of HCECs often demonstrated the distinctive cobblestone-like morphology until P3 or P4 (B–E); at later passages (F) fibroblastic conversion was common. (G) An exceptional culture maintained canonical morphology to P10, but by P12 showed senescent characteristics including lengthened cells and slowed growth rate. Scale bars: 50 μm. (H) Cell yields after expansion of HCECs from corneas of young donors for three or four passages. (I) Scatter plot of the time to confluency in relation to donor age: HCECs cultured from younger donors (average age: 22 years old; range, 0–34 years; n = 35) showed significantly greater proliferation rates (***P < 0.0001) compared with older donors (average age: 50 years old; range, 35–77 years; n = 20). (J) There is a weak correlation between HCEC density and in vitro proliferation (r² = 24%), but the correlation is statistically significant (P = 0.0002). (K) There was a statistically significant difference between corneal endothelial density measured before enucleation in younger donors (average endothelial cell density: 3181.6 mm²; range, 2571–4425 mm²; n = 30) compared with older donors (average endothelial cell density: 2761.5 mm²; range, 1969–2865 mm²; n = 11; P = 0.02).

Figure 2

Cultured HCECs express characteristic tight-junction-associated markers. (A) Confocal micrographs of cultured HCECs immunostained for ZO-1 and Na/K-ATPase; nuclei were counter-stained with DAPI. Scale bar: 50 μm. (B) Bar graph showing no difference between canonical and fibroblastic HCECs in Na/K-ATPase expression by flow cytometry (N = 3). Histograms of one representative flow cytometry run show no difference in Na/K-ATPase expression between canonical and fibroblastic cells. (C) Human corneal endothelial cells function assessed by TEER measurements from different HCEC cultures. A bovine corneal endothelial cell line (BCEC-line) was used as positive control (N = 4). P values resulting from the statistical analysis of TEER measured on canonical HCECs compared with mixed 1, mixed 2, and fibroblastic HCECs are presented in the Table below the graph.

Figure 3

Transcriptome analysis of corneal layers and cultured HCECs. (A) Venn diagram representing the probes expressed in the microarray dataset in the three corneal layers and freshly cultured HCECs. (B) Principal component analysis revealed distinct clusters per sample type as labeled; biological replicates (dots with the same color) clustered together. (C) Pearson correlation (r²) was higher within biological replicates than across different tissues (N = 3). (D) Hierarchical clustering demonstrated that HCECs and endothelium were more closely related than epithelium and stroma.

Figure 4

(A) Graphical outline of the gating process. (B) Representative 2D dot plots showing no difference in size and internal complexity between unstained canonical and unstained fibroblastic HCECs. (C) Representative histogram showing no difference in baseline fluorescence between unstained canonical and fibroblastic HCECs.

Figure 5

Flow cytometry analysis of surface markers expression differentiates HCEC subpopulations in culture. (A–C) Three morphologically distinct HCEC cultures, canonical, mixed, and fibroblastic as marked, were carried forward for flow cytometry. (D–G) Surface marker expression by flow cytometry. Flow cytometry histograms are representative illustrations of each antibody expression profile. For quantification, a threshold at the top 1% of negative control cells was set to identify positive cells throughout all the independent experiments. Quantification of the percentage of positive cells for each marker showed that CD56 (D), CD248 (E), and CAR (F) expressions are low in a fibroblastic culture, while CD109 (G) is high. Data is representative of three or more independent experiments from separate corneal cultures (CD56: N = 10; CD109: N = 6; CD248: N = 3; CAR: N = 4; P values: #0.1; *<0.05; **<0.001; ***<0.0001).

Figure 6

(A–C) Flow cytometry analysis by dual-color fluorescent dot plot histograms for the canonical (A), mixed (B), and fibroblastic (C) HCEC cultures show shift in the expression of CD56, CD248, CAR, and CD109 surface markers. Each graph is divided into four quadrants determined by the autofluorescence of unstained control cells as in Figure 4, and gated to include 99% of unstained cells in the lower left quadrant (all negative markers). (D–F) Quantification of the percentage of canonical, fibroblastic, and mixed cell populations expressing markers tested in pairs, as marked. (G) Quantification of flow cytometry experiments showing no difference between canonical and fibroblastic cells in CD166/ALCAM, CD73, CD9, CD90, and β₁Na⁺/K⁺ ATPase expression. (H) Transendothelial electrical resistance assay using in vitro expanded HCECs whose CD56 expression had been determined by flow cytometry, demonstrating the greater ability of canonical CD56^high cells than fibroblastic CD56^low to form a barrier.