Identification of Notch-1 expression in the limbal basal epithelium

Ocular Surface Center, Doheny Eye Institute, Los Angeles, CA 90033, USA. <>
Molecular vision (Impact Factor: 1.99). 03/2007; 13:337-44.
Source: PubMed


To determine whether Notch-1, a ligand-activated transmembrane receptor known to maintain cells in an undifferentiated state, primarily progenitor cells in other systems, could be used as a stem cell marker in human limbal epithelium.
Human corneoscleral tissues obtained from the Doheny Eye & Tissue Transplant Bank were prepared for cross section and whole mount analysis. Tissue for whole mount was incubated in dispase; the epithelial sheet was removed and fixed in 4% paraformaldehyde. Sections and whole mount were stained with antibodies against Notch-1, Notch-2, beta-1 integrin, alpha-6, and the G2 subtype member of the ATP binding cassette transporter (ABCG2). Specificity of the Notch-1 antibody was determined by western blot analysis with Cos-7 cells transfected with Notch-1. Explant culture was performed and only primary cultures were used in this experiment.
Notch-1 was found to be expressed in the limbal basal region where stem cells reside. Notch-1 antigenicity was more pronounced in cell clusters, mainly in the palisades of Vogt. The central cornea was almost devoid of Notch-1. The intensity of Notch-1 staining in cultured cells from the limbal explants was high in only a few cells. The Notch-1 signal was diminished in dividing cells. Expression in cultured cells was more cytoplasmic; few cells showed additional nuclear staining. The Notch-1-stained whole mount showed only a few cells in the limbal region. A 300 kDa and a 110 kDa band confirmed the specificity of the antibody in Cos-7 cells transfected with Notch-1. Double staining for ABCG2 and Notch-1 showed some ABCG2-positive cells co-expressing Notch-1 in the limbal basal epithelium, indicating that Notch-1-expressing cells might be a unique subpopulation of cells with stem cell properties.
Immunofluorescence data shows that Notch-1 could be a possible marker for the stem cells in the limbal basal epithelium. Further studies and characterization of the Notch pathway in corneal development will provide valuable clues for the identification of stem cells.

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Available from: Shivaram Selvam, Apr 21, 2014
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    • "Recent evidence suggested Notch signaling is involved in maintaining the undifferentiated status of LSCs. Notch-1 protein diminished in dividing cells in culture [22] and in the proliferating limbal epithelium in response to corneal wounding [23]. Activated Notch signaling in cardiac myocytes has proved to be crucial for inhibiting TGFβ-mediated cardiac fibrosis induced by pressure overload [24]. "
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    ABSTRACT: Continuous culture of limbal epithelial stem cells (LSCs) slows down proliferation, which inevitably results in differentiation. Transforming growth factor-beta (TGFβ)-assisted epithelial-mesenchymal transition (EMT) is often found in the late stage of LSC culture. Thus, EMT is proposed to be part of the mechanism responsible for the loss of LSCs in culture. To explore the regulation mechanism of EMT, we investigated the early stage culture for factor(s) that may potentially prevent EMT. LSCs from the corneal limbus region of rabbits were isolated and expanded to confluence in culture (P0), and then serial passage of these LSCs (P1 to P3) was performed. EMT in LSCs was induced with TGFβ1, and the corresponding EMT signaling was confirmed with Smad2/3 phosphorylation. The expression of mesenchymal markers, including alpha-smooth muscle actin (α-SMA) and vimentin, was determined with western blot analysis. Proteins extracted from different passaged cells were also subjected to western blot analysis of TGFβ signaling components, including TGFβ1, TGFβ receptor I/II, and Smad2/3 as well as Smad7, the main negative regulator of TGFβ signaling. The mitogenic response was measured with the bromodeoxyuridine (BrdU) labeling index and real-time PCR using primers for Ki67. N-(N-[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester (DAPT), a gamma-secretase inhibitor, and Jagged-1 Notch ligand were used to block and activate Notch signaling, respectively, and their efficacy was evaluated by determining the expression of Hes1, a Notch signaling target. Mesenchymal marker induction and growth arrest were found in the TGFβ1-treated P1 cells, and the changes were less significant in the TGFβ1-treated P0 cells. Western blot analysis confirmed that the expressed levels of TGFβ signaling components, including TGFβ1, TGFβ receptor I/II, and Smad2/3, were relatively stable with passages. In contrast, the expression of Hes1 and Smad7 markedly decreased after the first passage, and with each passage, the levels diminished even further. Hes1 and Smad7 were expressed only in the limbal epithelium and not in the corneal epithelium. DAPT effectively blocked the expression of Hes1. DAPT also dose-dependently suppressed Smad7 expression in P0 cells, which was associated with the susceptibility of P0 cells to TGFβ1-induced Smad2/3 phosphorylation, EMT formation, and growth arrest. Reciprocally, Jagged-1 upregulated Smad7 expression in LSCs against TGFβ signaling. These findings indicate that Smad7 plays a crucial role in antagonizing EMT induced by TGFβ signaling and support our proposition that Smad7 is a Notch signaling target in LSCs, and may mediate the Notch function in preventing the occurrence of EMT.
    Molecular vision 04/2014; 20:522-534. · 1.99 Impact Factor
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    • "However, results from various groups suggest that the niche rules stem cell behavior by regulating the cell division pattern, in part through the active role of basement membrane components at the limbus. Recent results that strongly support that in the adult corneal epithelium asymmetric divisions may occur only at the limbus [124], together with evidence that restricts the expression of specific markers and the expression of cell proliferation and cell fate regulators such as ΔNp63α [145] and Notch1 [146] to stem cells, suggest that asymmetric cell division is part of the differentiation program in corneal epithelial cells [147]. Therefore, the basement membrane would provide limbal stem cells with information about their position and fate. "
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    ABSTRACT: Stem cells emerged as a concept during the second half of 19(th) century, first as a theoretical entity, but then became one of the most promising research fields in cell biology. This work describes the most important characteristics of adult stem cells, including the experimental criteria used to identify them, and discusses current knowledge that led to the proposal that stem cells existed in different parts of the eye, such as the retina, lens, conjunctiva, corneal stroma, Descemet's membrane, and the subject of this review: the corneal epithelium. Evidence includes results that support the presence of corneal epithelial stem cells at the limbus, as well as the major obstacles to isolating them as pure cell populations. Part of this review describes the variation in the basement membrane composition between the limbus and the central cornea, to show the importance of the corneal stem cell niche, its structure, and the participation of extracellular matrix (ECM) components in regulating corneal stem cell compartment. Results obtained by various laboratories suggest that the extracellular matrix plays a central role in regulating stem cell commitment, corneal differentiation, and participation in corneal wound healing, in addition to other environmental signals such as cytokines and growth factors. The niche could define cell division patterns in corneal stem cell populations, establishing whether stem cells divide asymmetrically or symmetrically. Characterization and understanding of the factors that regulate corneal epithelial stem cells should open up new paths for developing new therapies and strategies for accelerating and improving corneal wound healing.
    Molecular vision 07/2013; 19:1600-13. · 1.99 Impact Factor
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    • "Currently, slit-lamps are used for clinical examination of the limbal morphology, however, this technology does not allow for volumetric, high resolution imaging of structural details. Recently, cellular level resolution images of the human corneo-scleral limbus were obtained in vivo with commercial laser scanning confocal microscopes (LSCM), that showed detailed 2D en-face view of the Vogt palisades [11,12]. Similar studies were carried out to image in vivo nerves and nerve fiber bundles in the human cornea and limbus and provide thorough classification of the nerves in terms of size, shape, reflectivity and position in the cornea [13]. "
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    ABSTRACT: The limbus is the structurally rich transitional region of tissue between the cornea on one side, and the sclera and conjunctiva on the other. This zone, among other things, contains nerves passing to the cornea, blood and lymph vasculature for oxygen and nutrient delivery and for waste, CO(2) removal and drainage of the aqueous humour. In addition, the limbus contains stem cells responsible for the existence and healing of the corneal epithelium. Here we present 3D images of the healthy human limbus, acquired in vivo with a spectral domain optical coherence tomography system operating at 1060nm. Cross-sectional and volumetric images were acquired from temporal and nasal locations in the human limbus with ~3µm x 18µm (axial x lateral) resolution in biological tissue at the rate of 92,000 A-scans/s. The imaging enabled detailed mapping of the corneo-scleral tissue morphology, and visualization of structural details such as the Vogt palisades, the blood and lymph vasculature including the Schlemm's canal and the trabecular meshwork, as well as corneal nerve fiber bundles. Non-invasive, volumetric, high resolution imaging reveals fine details of the normal human limbal structure, and promises to provide invaluable information about its changes in health and disease as well as during and after corneal surgery.
    Biomedical Optics Express 07/2011; 2(7):1794-02. DOI:10.1364/BOE.2.001794 · 3.65 Impact Factor
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