A hierarchy of endothelial colony-forming cell activity displayed by bovine corneal endothelial cells

Abstract

Purpose: To test the hypothesis that the robust expansion of bovine corneal endothelial cells (BCECs) in vitro is due to the presence of individual endothelial cells with various levels of proliferative potential.

Methods: BCECs and bovine vascular endothelial cells (ECs) derived from aorta, coronary artery, and pulmonary artery were cultivated in optimized medium. These cell populations were confirmed by morphologic features, functional assays, and gene expression profiles. Moreover, ECs were plated in a single-cell clonogenic assay to evaluate colony-forming ability.

Results: Both corneal and vascular ECs were confirmed to be pure populations of endothelium uncontaminated with hematopoietic cells. A complete hierarchy of endothelial colony-forming cells (ECFCs) was identified in BCECs by a single-cell clonogenic assay. The distribution of the various types of ECFCs was similar to the control ECs removed from the systemic vessels.

Conclusions: Cultured BCECs display clonal proliferative properties similar to those of vascular ECs.

Figure 1.

The morphology of cultured bovine vessel wall–derived ECs and BCECs. Bovine vascular ECs derived from aorta (BAECs), coronary artery (BCAECs), and pulmonary artery (BPAECs) displayed a spindle-shaped morphology and BCECs displayed a hexagonal morphology within the cell monolayer. Magnification, ×10; scale bar, 100 μm.

Figure 2.

Phenotypic and functional characterization of bovine vessel wall–derived ECs and BCECs. (A) Immunophenotyping of bovine peripheral blood MNCs and the monolayers derived from BAECs, BCAECs, BPAECs, and BCECs, determined by fluorescence flow cytometry. BAECs, BCAECs, BPAECs, and BCECs bound to the lectins GSL I and LEL but did not express the common leukocyte antigen CD45. Green: negative control cells. (B) Incorporation of 488-AcLDL in bovine vessel wall–derived ECs and corneal ECs. BAECs, BCAECs, and BPAECs were able to ingest 488-AcLDL (green), but BCECs failed to bind the material. Blue: DAPI-stained nuclei. (C) LDLR and CD36 expression in bovine vascular and corneal ECs identified by RT-PCR. LDLR was expressed in all types of ECs, whereas CD36 was detectable only in bovine vascular ECs. (D) Formation of capillary-like structures when bovine vascular ECs and BCECs were plated in synthetic basement membrane. Three independent experiments showed similar results. (B, D) Magnification, ×40; scale bar, 100 μm.

Figure 3.

RT-PCR analysis of gene expression in bovine vessel wall–derived ECs and bovine corneal ECs. PECAM1, VE-cadherin, and eNOS were specifically expressed in bovine vascular ECs but not in BCECs. S100B transcripts were detectable in BCECs but not in vessel wall–derived ECs. Enolase2 was also detected in BCECs with variable expression level. Three independent experiments showed similar results.

Figure 4.

Quantitation of the clonogenic and proliferative potential of single endothelial cells derived from bovine vascular endothelium and bovine corneal endothelium. (A) The percentage of single BAECs, BCAECs, BPAECs, and BCECs that divided at least once after 14 days in culture. There were significantly fewer BCECs undergoing division compared with bovine vessel wall–derived ECs. (B) The distribution of different sizes of colonies derived from plated single ECs in an individual well after 14 days of culture. There was a significantly higher percentage of HPP-ECFC than LPP-ECFC colonies and endothelial clusters in BCEC24s and BAEC samples than was observed in the BCAEC and BPAEC samples. *P < 0.05, **P < 0.01, ***P < 0.001 by parametric ANOVA (n = 3).

Figure 5.

Telomerase activity of HPP-ECFCs derived from bovine vessel wall–derived ECs and BCECs. The presence and intensity of a ladder of PCR products with six base increments indicates the level of telomerase activity in the cells. Thus, the progeny of HPP-ECFCs isolated from BCECs displayed telomerase activity similar to that of the progeny of HPP-ECFCs derived from BAECs, BCAECs, and BPAECs. P, telomerase activity in the Hela cells (positive control); N, negative control; IC, 36-bp internal control sample provided by the manufacturer. Three independent experiments showed similar results.

Figure 6.

The formation of a sphere colony by bovine corneal endothelial cells. (A) Representative photograph of a sphere derived from plated BCECs. (B) The distribution of different-sized colonies derived from single BCECs dissociated from corneal endothelial sphere colonies in an individual well after 14 days of culture. The complete hierarchy of ECFC is present in BCECs residing in the spheres. Scale bar, 100 μm.