Identification of Cell Surface Markers Glypican-4 and CD200 That Differentiate Human Corneal Endothelium From Stromal Fibroblasts

Cheong, Yuen Kuen; Ngoh, Zi Xian; Peh, Gary S L; Ang, Heng-Pei; Seah, Xin-Yi; Chng, Zhenzhi; Colman, Alan; Mehta, Jodhbir S.; Sun, William

doi:10.1167/iovs.13-11754

Cited by 64 publications

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“…10 To date, no HCEC-specific cell surface markers have been elucidated, although two groups have reported the expression of CD markers. 11,12 The effector cell did not show any sign of surface CD200. Okumura et al 11 have reported that CD340, CD166, and CD98 are elevated in HCECs of nonfibroblastic phenotype, while CD9, CD49e, CD44, and CD73 are the indices of fibroblastic phenotypes.…”

Section: Discussionmentioning

confidence: 93%

“…In fact, cHCECs are heterogeneous from culture to culture in their morphology and in their surface markers, such as cluster of differentiation (CD) antigens. [10][11][12] Studies, [13][14][15][16][17][18][19][20] either directly or indirectly, and including those from Joyce et al, have shown that heterogeneity is present in HCEC cultures. Of particular and striking interest is the finding by Miyai et al 10 of the presence of frequent chromosomal aneuploidy in cHCECs, thus indicating the presence of heterogeneous subpopulations (SPs) with or without aneuploidy.…”

mentioning

confidence: 99%

“…Glypican-4 and CD200 have been proposed as HCEC markers to distinguish HCECs from corneal stromal fibroblasts. 12 However, the practical problem associated with HCEC culture is the potential contamination with vulnerable transformed cHCECs.…”

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confidence: 99%

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Cell Homogeneity Indispensable for Regenerative Medicine by Cultured Human Corneal Endothelial Cells

Hamuro

Toda

Asada

et al. 2016

Invest. Ophthalmol. Vis. Sci.

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Citation: Hamuro J, Toda M, Asada K, et al. Cell homogeneity indispensable for regenerative medicine by cultured human corneal endothelial cells. Invest Ophthalmol Vis Sci. 2016;57:4749-4761. DOI:10.1167/ iovs.16-19770 PURPOSE. To identify the subpopulation (SP) among heterogeneous cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition applicable for cell-based therapy.METHODS. Subpopulation presence in cHCECs was confirmed via surface CD-marker expression level by flow cytometry. CD markers effective for distinguishing distinct SPs were selected by analyzing those on established cHCECs with a small cell area and high cell density. Contrasting features among three typical cHCEC SPs was confirmed by PCR array for extracellular matrix (ECM). Combined analysis of CD markers was performed to identify the SP (effector cells) applicable for therapy. ZO-1 and Na þ /K þ ATPase, CD200, and HLA expression were compared among heterogeneous SPs. RESULTS. Flow cytometry analysis identified the effector cell expressing CD166 and the presence of other SPs with CD166þþþ (CD26 and CD24, either þ or À) was confirmed. PCR array revealed three distinct ECM expression profiles. Some SPs expressed ZO-1 and Naþ ATPase at comparable levels with effector cells, while only one SP expressed CD200, but not on effector cells. Human leukocyte antigen expression was most reduced in the effector SP. The proportion of effector cells (E-ratio) inversely paralleled donor age and decreased during prolonged culture passages. The presence of Rho-associated protein kinase (ROCK) inhibitor increased the E-ratio in cHCECs. The average area of effector cells was approximately 200~220 lm 2 , and the density of cHCECs exceeded 2500 cells/mm 2 . CONCLUSIONS.A specified cultured effector cell population sharing the surface phenotypes with mature HCECs in corneal tissues may serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction.

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Cell Homogeneity Indispensable for Regenerative Medicine by Cultured Human Corneal Endothelial Cells

Hamuro

Toda

Asada

et al. 2016

Invest. Ophthalmol. Vis. Sci.

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“…n = 4, *p < 0.01, and **p < 0.05. Primary CECs were cultivated using the dual media approach to the third passage and characterized for their expression of known markers indicative of the corneal endothelium such as (B) sodium-potassiumtransporting adenosine triphosphatase (Na and expressed higher levels of corneal endothelium-specific genes ATP1A1, SLC4A11 and COL8A1, as well as reported cellular markers of the corneal endothelium (11,44).…”

Section: Characterization Of Cultivated Human Corneal Endothelial Celmentioning

confidence: 99%

Propagation of Human Corneal Endothelial Cells: A Novel Dual Media Approach

et al. 2015

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Corneal endothelium-associated corneal blindness is the most common indication for corneal transplantation. Restorative corneal transplant surgery is the only option to reverse the blindness, but a global shortage of donor material remains an issue. There are immense clinical interests in the development of alternative treatment strategies to alleviate current reliance on donor materials. For such endeavors, ex vivo propagation of human corneal endothelial cells (hCECs) is required, but current methodology lacks consistency, with expanded hCECs losing cellular morphology to a mesenchymal-like transformation. In this study, we describe a novel dual media culture approach for the in vitro expansion of primary hCECs. Initial characterization included analysis of growth dynamics of hCECs grown in either proliferative (M4) or maintenance (M5) medium. Subsequent comparisons were performed on isolated hCECs cultured in M4 alone against cells expanded using the dual media approach. Further characterizations were performed using immunocytochemistry, quantitative real-time PCR, and gene expression microarray. At the third passage, results showed that hCECs propagated using the dual media approach were homogeneous in appearance, retained their unique polygonal cellular morphology, and expressed higher levels of corneal endothelium-associated markers in comparison to hCECs cultured in M4 alone, which were heterogeneous and fibroblastic in appearance. Finally, for hCECs cultured using the dual media approach, global gene expression and pathway analysis between confluent hCECs before and after 7-day exposure to M5 exhibited differential gene expression associated predominately with cell proliferation and wound healing. These findings showed that the propagation of primary hCECs using the novel dual media approach presented in this study is a consistent method to obtain bona fide hCECs. This, in turn, will elicit greater confidence in facilitating downstream development of alternative corneal endothelium replacement using tissue-engineered graft materials or cell injection therapy.

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“…In fact, cHCECs are known to be heterogeneous from culture to culture in their morphology and in their surface markers, such as cluster-ofdifferentiation (CD) antigens. [3][4][5] Studies, either directly or indirectly, and including those from Joyce et al, [6][7][8][9][10][11][12][13] have shown that heterogeneity is present in HCEC cultures. Of particular and striking interest is the finding by Miyai et al 3 of the presence of frequent chromosomal aneuploidy in cHCECs, directly indicating the presence of heterogeneous subpopulations (SPs) with or without aneuploidy.…”

mentioning

confidence: 99%

MicroRNA Profiles Qualify Phenotypic Features of Cultured Human Corneal Endothelial Cells

Ueno

Asada

Toda

et al. 2016

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. To elucidate a noninvasive method to qualify and identify cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition and adaptable for cell-based therapy.METHODS. The variations of cHCECs in their composition of heterogeneous subpopulations (SPs) were verified in relation to their surface cluster-of-differentiation (CD) markers and their morphology. The profiles of microRNA (miRNA) in cultured cells or supernatants were detected by 3D-Gene Human microRNA Chips (Toray Industries, Inc.). The profiles were also analyzed for fresh corneal tissues with distinct endothelial cell densities (ECD) with or without gutatta. To validate the 3D-Gene results, quantitative real-time polymerase chain reaction (PCR) was performed. RNAs were extracted from cHCECs transfected with selected miRNA, and target genes were presumed by PCR array (Qiagen).RESULTS. Among a variety of morphologically different cHCECs, miRNA expression profiles were distinctively revealed. The one miRNA capable of discriminating CD44 À SP from SPs with CD44 þþ~C D44 þþþ phenotypes was identified as miR34a. The downregulation of miRNAs in the 378 family paralleled the upregulation of surface CD44 on cHCECs. Interestingly, upregulated miRNAs in the 378 family in corneal endothelium dramatically decreased in the tissues with lower ECD with advanced gutatta, providing new insight on the pathogenesis of Fuchs' endothelial corneal dystrophy. CONCLUSIONS. The specified cultured SPs sharing the CD44À surface phenotypes with matured HCECs showed the highest expression of miR-378. Conversely, SPs with upregulated CD44 þþþ showed a reduction of miR-378. Thus, miRNA in cultured cells may serve as an alternative method to qualify cHCECs.Keywords: heterogeneity of cultured corneal endothelial cells, CD44, miR, gutatta, epithelium-mesenchymal transition (EMT) A lthough human corneal endothelial cells (HCECs) have the ability to proliferate in vitro, 1 culturing HCECs for an extended period of time is known to be extremely difficult.2 To date, most researchers have conceptualized cultured HCECs (cHCECs) only from the aspect that they are derived from corneal endothelium tissue, and disregarded details pertaining to the refinement of the biochemical features. In fact, cHCECs are known to be heterogeneous from culture to culture in their morphology and in their surface markers, such as cluster-ofdifferentiation (CD) antigens.3-5 Studies, either directly or indirectly, and including those from Joyce et al., [6][7][8][9][10][11][12][13] have shown that heterogeneity is present in HCEC cultures. Of particular and striking interest is the finding by Miyai et al.3 of the presence of frequent chromosomal aneuploidy in cHCECs, directly indicating the presence of heterogeneous subpopulations (SPs) with or without aneuploidy.Cultured HCECs have an inclination toward cell-state transition (CST) into a senescence phenotype, epitheliummesenchymal transition (EMT), and fibroblastic cell morphology. In addition, the plasticity of metabolic profiles of cH...

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Identification of Cell Surface Markers Glypican-4 and CD200 That Differentiate Human Corneal Endothelium From Stromal Fibroblasts

Abstract: Taken together, the anti-GPC4 and anti-CD200 antibodies can be useful for purification and identification of HCECs in cultures containing stromal fibroblasts.

Cited by 64 publications

References 29 publications

Cell Homogeneity Indispensable for Regenerative Medicine by Cultured Human Corneal Endothelial Cells

Cell Homogeneity Indispensable for Regenerative Medicine by Cultured Human Corneal Endothelial Cells

Propagation of Human Corneal Endothelial Cells: A Novel Dual Media Approach

MicroRNA Profiles Qualify Phenotypic Features of Cultured Human Corneal Endothelial Cells

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