Investigation of K14/K5 as a Stem Cell Marker in the Limbal Region of the Bovine Cornea

Chen, Bo; Mi, Shengli; Wright, Bernice; Connon, Che J.

doi:10.1371/journal.pone.0013192

Cited by 25 publications

(18 citation statements)

References 30 publications

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“…Indeed, a larger part of cells in G0 phase was made up with CD200 + cell population which in itself contained 79% of the SP cells, corroborating previously published findings by Umemoto et al that limbal epithelial SP are quiescent and do not demonstrate proliferative capabilities in ex vivo culture conditions . We also observed a consistently higher expression of putative LSC markers including WNT7A , PAX6 , ABCB5 , CDH3 , CK14 , and CK15 in the CD200 + subpopulation.…”

Section: Discussionsupporting

confidence: 91%

CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability

et al. 2018

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One of the main challenges in limbal stem cell (LSC) biology and transplantation is the lack of definitive cell surface markers which can be used to identify and enrich viable LSCs. In this study, expression of 361 cell surface proteins was assessed in ex vivo expanded limbal epithelial cells. One marker, CD200 was selected for further characterization based on expression in a small subset of limbal epithelial cells (2.25% ± 0.69%) and reduced expression through consecutive passaging and calcium induced differentiation. CD200 was localized to a small population of cells at the basal layer of the human and mouse limbal epithelium. CD200 cells were slow cycling and contained the majority of side population (SP) and all the holoclone forming progenitors. CD200 cells displayed higher expression of LSCs markers including PAX6, WNT7A, CDH3, CK14, CK15, and ABCB5 and lower expression of Ki67 when compared to CD200 . Downregulation of CD200 abrogated the ability of limbal epithelial cells to form holoclones, suggesting an important function for CD200 in the maintenance and/or self-renewal of LSCs. A second marker, CD109, which was expressed in 56.29% ± 13.96% of limbal epithelial cells, was also found to co-localize with ΔNp63 in both human and mouse cornea, albeit more abundantly than CD200. CD109 expression decreased slowly through calcium induced cell differentiation and CD109 cells were characterized by higher expression of Ki67, when compared to CD109 subpopulation. Together our data suggest that CD200 expression marks a quiescent population of LSCs with holoclone forming potential, while CD109 expression is associated with a proliferative progenitor phenotype. Stem Cells 2018;36:1723-1735.

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Section: Discussionsupporting

confidence: 91%

CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability

et al. 2018

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“…CKs have been used as markers for cornea‐specific epithelial differentiation and SCs characterization. Our results of CK 14, CK 19, and CK 3 expression in the equine cornea are in accordance with previous research on cornea . In the equine corneal epithelium, CK 14 and 19 expression reflect epithelial differentiation but are no suitable marker for exclusive SC detection.…”

Section: Discussionsupporting

confidence: 84%

Morphological and immunohistochemical characteristics of the equine corneal epithelium

Kammergruber

Rahn

Nell

et al. 2019

Veterinary Ophthalmology

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ObjectiveThe morphology of the corneal epithelium in two age groups of horses is described. Distribution patterns of proliferation‐, differentiation‐, stem cell‐associated markers and cell junction proteins were assessed.MethodsCorneal samples from 12 horses (six foals and six adult horses) were analyzed after H&E staining and immunohistochemistry using the following antibodies: E‐cadherin, β‐catenin, Connexin 43 (Cx43), tight junction protein 1 (TJP1), cytokeratin (CK) 14, CK 19, CK 3, CK 10, vimentin, Ki67, p63, nerve growth factor (NGF), ABCG2, and epithelial growth factor receptor. Semiquantitative analysis of crypt, limbal, peripheral, and central zone was performed. Semithin and ultrathin sections were used for ultrastructural evaluation of the epithelium.ResultsThe height of the epithelium varied between age groups and crypts were consistently present. In the peripheral and central epithelium, three types of basal cells resembling a pseudostratified epithelium were characterized. Potential stem cell markers (CK 14, p63, NGF, and ABCG2) were present in all zones with decreasing frequency toward the center. Cornea‐specific differentiation marker CK 3 was not expressed in the most basal cell layer of the limbal epithelium. E‐cadherin, β‐catenin, and Cx43 revealed a similar apico‐lateral signal pattern throughout the entire epithelium; only TJP1 was additionally seen at the basal surface.ConclusionsThis study presents a systematic semiquantitative evaluation of the equine corneal epithelium, showing the presence of crypts as potential stem cell niche with CK 14, p63, NGF, and ABCG2 as relevant markers for cells with regenerative capacity. The pseudostratified arrangement of the basal layer was a unique finding.

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“…Cluster 4, 9 and 10 were transcriptionally similar to each other ( Figure S1), we thus performed a differential gene expression and violin plot analyses, which showed high expression of KRT14 and TP63 in all three clusters; however KRT15, a LSCs and progenitor marker (Chen et al, 2010;Yoshida et al, 2006) was highly expressed in clusters 9 and 4 but not in cluster 10 ( Figure S5A-D). Through the differential gene expression analysis ( Figure S5A), we were able to identify a unique marker for cluster 10, CPVL, which was not expressed in the other epithelial clusters ( Figure S6A).…”

Section: Identification Of Epithelial Cell Populationsmentioning

confidence: 87%

A single cell atlas of human cornea that defines its development, limbal stem and progenitor cells and the interactions with the limbal niche

Collin

Zerti

Bojić

et al. 2020

Preprint

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To study the development and composition of human ocular surface, we performed single cell (sc) RNA-Seq at key embryonic, fetal and adult stages and generated the first atlas of the corneal cell types from development to adulthood. Our data indicate that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of proliferative epithelial progenitors, which predate the formation of limbal niche by a few weeks. Bioinformatic comparison of adult cell clusters identified GPHA2, a novel cell-surface marker for quiescent limbal stem cells (qLSCs), whose function is to maintain qLSCs self-renewal. Combining scRNA- and ATAC-Seq analysis, we identified multiple upstream regulators for qLSCs and transit amplifying (TA) cells and demonstrated a close interaction between the immune cells and epithelial stem and progenitor cells in the cornea. RNA-Seq analysis indicated loss of qLSCs and acquisition of proliferative limbal basal epithelial progenitor markers during ex vivo limbal epithelial cell expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of TA cells in the limbal epithelium as two key changes underlying the disease phenotype. Our scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining pathways/genes that can lead to improvement in ex vivo expansion and differentiation methods for cell based replacement therapies and better understanding and treatment of ocular surface disorders.

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Investigation of K14/K5 as a Stem Cell Marker in the Limbal Region of the Bovine Cornea

Cited by 25 publications

References 30 publications

CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability

CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability

Morphological and immunohistochemical characteristics of the equine corneal epithelium

A single cell atlas of human cornea that defines its development, limbal stem and progenitor cells and the interactions with the limbal niche

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