Icated as template copy number was measured in cell extracts using the telomeric repeat amplification protocol (TRAPeze RT). Heat-inactivated extracts served as negative controls. No specific telomerase HIV-RT inhibitor 1 custom synthesis activity was detected in 21M primary cells, while HCEnC-21T cell lysates contained strongly elevated telomerase activity. Note that HCEnC-21 intrinsically upregulated endogenous telomerase activity. (C) Cell doubling time of earlier (19?4) and later (32?1) passages of HCEnC-21 and HCEnC-21T. Cells were plated in 12-well plates at a density of 50,000 cells/well and counted after 2, 3, and 4 days using a hemocytometer. HCEnC-21T showed a higher proliferation rate compared to HCEnC-21 in long-term cultures. Error bars indicate mean 6 SEM. *, P,0.05. doi:10.1371/journal.pone.0051427.gHCEnC-21 and HCEnC-21T Synthesize Characteristic Corneal Endothelial Cell MarkersCell-cell contacts between corneal endothelial cells are the fundamental basis for the barrier function of HCEn and consist of tight junctions as well as cadherin-based adherens junctions [23]. ZO-1, an essential component of tight junctions and one of the key markers for HCEn, was detected by immunofluorescence staining of HCEnC-21 and HCEnC-21T monolayers. Primary stromal fibroblasts were used as a biological negative control. Immunostaining with ZO-1 was detected in both HCEnC-21 (passage 13, Figure 4A) and HCEnC-21T (passage 14, Figure 4B) cells and localized to the cell-cell junctions, whereas no staining of stromal fibroblasts was detected (Figure 4C). Similar staining with ZO-1 was observed in later passages of HCEnC-21 (passage 32) and HCEnC-21T (passage 46) cells (Figure S3). The ZO-1 immunostaining demonstrates the typical polygonal morphology of HCEnC-21 and HCEnC-21T cells and suggests the formation of tight junctions between neighboring cells. Additionally, Ncadherin, a key component of adherens junctions, was detected in cell extracts from earlier (,25) and later (.45) passages of HCEnC-21 and HCEnC-21T cells by Western blotting (Figure 5A). No N-cadherin was present in lysates from stromal fibroblasts. Collagen type 8 is buy ML 240 synthesized by HCEnCs and is the major component of Descemets membrane, the corneal endothelial basement membrane [24]. Figure 5A shows that the collagen type 8 a2 chain is detected in corneal endothelial primary cells as well as in HCEnC-21 and HCEnC-21T cells, but not in stromal fibroblasts, demonstrating the presence of another major marker for HCEn in HCEnC-21 and HCEnC-21T cells.HCEnC-21 and HCEnC-21T Express Typical Corneal Endothelial Ion TransportersTo further study the corneal endothelial-specific phenotype of HCEnC-21 and HCEnC-21T cells, expression of 14 different ion transporter genes was investigated (Figure 5B ). Na/K ATPaseis one of the most important ion transporters responsible for creating an ionic gradient across the corneal endothelial basolateral membrane and is composed of a and b subunits [25,26]. Both a and b subunits exist in different isoforms that are synthesized in a tissue-specific manner [27]. HCEn is known to distinctly express the a1 and a3 isoforms, which are responsible for hydrolyzing ATP and ion transportation [28]. Real-time PCR showed similar expression of the Na/K ATPase a1 subunit (Figure 5B) and upregulation of the Na/K ATPase a3 subunit (Figure 5C) in HCEnC-21 and HCEnC-21T cells compared to 21M primary cells. Immunofluorescence staining confirmed that the Na/K ATPase a1 subunit was highly abundant in HCEnC-21.Icated as template copy number was measured in cell extracts using the telomeric repeat amplification protocol (TRAPeze RT). Heat-inactivated extracts served as negative controls. No specific telomerase activity was detected in 21M primary cells, while HCEnC-21T cell lysates contained strongly elevated telomerase activity. Note that HCEnC-21 intrinsically upregulated endogenous telomerase activity. (C) Cell doubling time of earlier (19?4) and later (32?1) passages of HCEnC-21 and HCEnC-21T. Cells were plated in 12-well plates at a density of 50,000 cells/well and counted after 2, 3, and 4 days using a hemocytometer. HCEnC-21T showed a higher proliferation rate compared to HCEnC-21 in long-term cultures. Error bars indicate mean 6 SEM. *, P,0.05. doi:10.1371/journal.pone.0051427.gHCEnC-21 and HCEnC-21T Synthesize Characteristic Corneal Endothelial Cell MarkersCell-cell contacts between corneal endothelial cells are the fundamental basis for the barrier function of HCEn and consist of tight junctions as well as cadherin-based adherens junctions [23]. ZO-1, an essential component of tight junctions and one of the key markers for HCEn, was detected by immunofluorescence staining of HCEnC-21 and HCEnC-21T monolayers. Primary stromal fibroblasts were used as a biological negative control. Immunostaining with ZO-1 was detected in both HCEnC-21 (passage 13, Figure 4A) and HCEnC-21T (passage 14, Figure 4B) cells and localized to the cell-cell junctions, whereas no staining of stromal fibroblasts was detected (Figure 4C). Similar staining with ZO-1 was observed in later passages of HCEnC-21 (passage 32) and HCEnC-21T (passage 46) cells (Figure S3). The ZO-1 immunostaining demonstrates the typical polygonal morphology of HCEnC-21 and HCEnC-21T cells and suggests the formation of tight junctions between neighboring cells. Additionally, Ncadherin, a key component of adherens junctions, was detected in cell extracts from earlier (,25) and later (.45) passages of HCEnC-21 and HCEnC-21T cells by Western blotting (Figure 5A). No N-cadherin was present in lysates from stromal fibroblasts. Collagen type 8 is synthesized by HCEnCs and is the major component of Descemets membrane, the corneal endothelial basement membrane [24]. Figure 5A shows that the collagen type 8 a2 chain is detected in corneal endothelial primary cells as well as in HCEnC-21 and HCEnC-21T cells, but not in stromal fibroblasts, demonstrating the presence of another major marker for HCEn in HCEnC-21 and HCEnC-21T cells.HCEnC-21 and HCEnC-21T Express Typical Corneal Endothelial Ion TransportersTo further study the corneal endothelial-specific phenotype of HCEnC-21 and HCEnC-21T cells, expression of 14 different ion transporter genes was investigated (Figure 5B ). Na/K ATPaseis one of the most important ion transporters responsible for creating an ionic gradient across the corneal endothelial basolateral membrane and is composed of a and b subunits [25,26]. Both a and b subunits exist in different isoforms that are synthesized in a tissue-specific manner [27]. HCEn is known to distinctly express the a1 and a3 isoforms, which are responsible for hydrolyzing ATP and ion transportation [28]. Real-time PCR showed similar expression of the Na/K ATPase a1 subunit (Figure 5B) and upregulation of the Na/K ATPase a3 subunit (Figure 5C) in HCEnC-21 and HCEnC-21T cells compared to 21M primary cells. Immunofluorescence staining confirmed that the Na/K ATPase a1 subunit was highly abundant in HCEnC-21.