Differentially Expressed Genes Associated with Human Limbal Epithelial Phenotypes: New Molecules That Potentially Facilitate Selection of Stem Cell-Enriched Populations

Takács, Lili; Tóth, Enikő; Losonczy, Gergely; Szántó, Attila; Bähr-Ivacevic, Tomi; Beneš, Vladimír; Berta, András; Vereb, György

doi:10.1167/iovs.10-5242

Cited by 24 publications

(14 citation statements)

References 37 publications

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“…ß-catenin controlling the stability of Fzd, (also up-regulated in GFP + LRCs after 42 d chase) functions with the protein transcription factor 3 (Tcf3) 47 not yet reported in the corneal epithelium regulating cell cycle progression. Fzd7 and Tcf3, known as epidermal stem cell markers were up-regulated at 42 d chase onwards, but not at 28 d. Similar increased expression of the stem cell related genes interferon induced transmembrane protein 1 (Ifitm1, a marker significantly up-regulated in LSCs 48 ) and Keratin 15 (Krt15, an epithelial stem and progenitor marker 49,50 ) were also observed with Ifitm1 expression and Krt15 expression up-regulated at in GFP + LRCs.…”

Section: Discussionmentioning

confidence: 63%

Characterization of slow cycling corneal limbal epithelial cells identifies putative stem cell markers

Sartaj

Zhang

Park

et al. 2017

Sci Rep

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In order to identify reliable markers of corneal epithelial stem cells, we employed an inducible transgenic "pulse-chase" murine model (K5Tta × TRE-H2BGFP) to localize, purify, and characterize slow cycling cells in the cornea. The retention of GFP labeling in slowly dividing cells allowed for localization of these cells to the corneal limbus and their subsequent purification by FACS. Transcriptome analysis from slow cycling cells identified differentially expressed genes when comparing to GFP -faster-dividing cells. RNA-Seq data from corneal epithelium were compared to epidermal hair follicle stem cell RNA-Seq to identify genes representing common putative stem cell markers or determinants, which included Sox9, Fzd7, Actn1, Anxa3 and Krt17. Overlapping retention of GFP and immunohistochemical expression of Krt15, ΔNp63, Sox9, Actn1, Fzd7 and Krt17 were observed in our transgenic model. Our analysis presents an array of novel genes as putative corneal stem cell markers.Loss of the regenerative capacity of the ocular surface through the absence of corneal epithelial stem cells is a potentially blinding condition. Lack of definitive molecular markers to reproducibility locate, purify and expand corneal epithelial stem cells has hampered the ability to understand their biology and to use these cells for therapeutic transplantation.Stem cells from the cornea reside between the corneal periphery and the conjunctiva, known as the limbus. Limbal stem cells (LSCs) are clonogenic, regenerating new tissue in vivo and in vitro 1 exhibiting slow cycling phenotypic characteristics 2 . Classically, these cells have been characterized by the ability to retain tritiated thymidine or bromodeoxyurdine (BrdU) for long periods, yet have high proliferative potential 3-7 . More recently, a transgenic system that can genetically label slow cycling cells with GFP has been used to identify label retaining cells (LRCs) in skin 8,9 , sweat glands 10 , salivary glands 11 and the cornea 12 . In this model, one parental strain harbors the H2B-GFP transgene under the control of a tetracycline (doxycycline; dox) regulatory element (TRE), creating a tet-off system. The second strain expresses a transcription factor regulated by tetracycline (tTA) under the control of a cell-type-specific Keratin 5 (K5) promoter. Thus, K5 expressing cells will have nuclear GFP labeling in the "pulse" period, which is then turned off with dox administration, beginning the "chase" period.This approach allowed for the purification of cells and their subsequent molecular characterization of LRCs using next generation sequencing (NGS), yielding genes marking (and in some cases determining) "stemness". Thus, the molecular markers are identified without a pre-conceived notion as to which genes may be related to stemness. Comparison of these data from corneal epithelium with prior similar characterization of hair follicle stem cells (HFSCs) 13 were performed to identify common stem cell markers in these developmentally related tissues.

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Section: Discussionmentioning

confidence: 63%

Characterization of slow cycling corneal limbal epithelial cells identifies putative stem cell markers

Sartaj

Zhang

Park

et al. 2017

Sci Rep

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“…Subsequently, several groups conducted investigations using up-to-date experimental procedures such as microarray and deep RNA sequencing (Bath et al, 2013;Figueira et al, 2007;Kulkarni et al, 2010;Nakatsu et al, 2013;Takacs et al, 2011;Utheim et al, 2009). However, the molecular mechanism and gene expression profile of holoclonetype corneal epithelial stem cells remain entirely unknown.…”

Section: Gene Expression Profiling Of Holoclone-type Stem Cellsmentioning

confidence: 99%

Ocular surface reconstruction using stem cell and tissue engineering

Nakamura

Inatomi

Sotozono

et al. 2016

Progress in Retinal and Eye Research

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“…Reports in the literature [7,8,9] suggest that the basal epithelial cells of the limbal region in humans express several proteins, e.g. ATP-binding cassette subfamily G member 2 (ABCG2), cytokeratin (CK) 19, vimentin and α9 integrin, but were not especially positive for CK3/CK12, connexin 43, involucrin, some integrins (α2, α6, β4 subunits) and nestin compared with basal cells of differentiated corneal epithelium.…”

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confidence: 99%

“…At present, p63 (isoform ΔNp63α) and ABCG2 are considered the most useful cellular markers for the identification and isolation of progenitor cells in the limbal region. Recently, total RNA isolates of human limbal and central corneal epithelia were applied after transcription for hybridization on whole human genome expression microarrays [8] or quantitative real-time PCR [9]. A set of differentially expressed genes detected by both approaches was established, which may be useful for identifying putative corneal epithelial stem cells.…”

mentioning

confidence: 99%

“…Furthermore, several overexpressed genes related to development and differentiation (ITM2A, integral to membrane 2A), extracellular matrix components (SPON1, spondin-1), regulation of cell growth (IFITM1, interferon-induced transmembrane protein 1), melanin biosynthesis (DCT, dopachrome tautomerase) and Wnt pathway signaling transduction proteins (FZD7, frizzled 7; DKK4, dickkopf homolog 4) were also observed. Most were colocalized with other CCAAT enhancer binding protein-δ-positive putative resting LSCs [8]. …”

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confidence: 99%

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Progenitor Cells for Ocular Surface Regenerative Therapy

Casaroli‐Marano

Nieto‐Nicolau²,

Martínez-Conesa³

2012

Ophthalmic Res

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The integrity and normal function of the corneal epithelium are essential for maintaining the cornea’s transparency and vision. The existence of a cell population with progenitor characteristics in the limbus maintains a dynamic of constant epithelial repair and renewal. Currently, cell-based therapies for bio-replacement, such as cultured limbal epithelial transplantation and cultured oral mucosal epithelial transplantation, present very encouraging clinical results for treating limbal stem cell deficiencies. Another emerging therapeutic strategy consists of obtaining and implementing human progenitor cells of different origins using tissue engineering methods. The development of cell-based therapies using stem cells, such as human adult mesenchymal stromal cells, represents a significant breakthrough in the treatment of certain eye diseases and also offers a more rational, less invasive and more physiological approach to ocular surface regeneration.

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Differentially Expressed Genes Associated with Human Limbal Epithelial Phenotypes: New Molecules That Potentially Facilitate Selection of Stem Cell-Enriched Populations

Cited by 24 publications

References 37 publications

Characterization of slow cycling corneal limbal epithelial cells identifies putative stem cell markers

Characterization of slow cycling corneal limbal epithelial cells identifies putative stem cell markers

Ocular surface reconstruction using stem cell and tissue engineering

Progenitor Cells for Ocular Surface Regenerative Therapy

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