Wnt/␤-Catenin Signaling Regulates Proliferation of Human Cornea Epithelial Stem/Progenitor Cells

PURPOSE. To investigate the expression and role of the Wnt signaling pathway in human limbal stem cells (LSCs). METHODS. Total RNA was isolated from the human limbus and central cornea. Limbal or cornea-specific transcripts were identified through quantitative real-time PCR. Protein expression of Wnt molecules was confirmed by immunohistochemistry on human ocular tissue. Activation of Wnt signaling using lithium chloride was achieved in vitro and its effects on LSC differentiation and proliferation were evaluated. RESULTS. Expression of Wnt2, Wnt6, Wnt11, Wnt16b, and four Wnt inhibitors were specific to the limbal region, whereas Wnt3, Wnt7a, Wnt7b, and Wnt10a were upregulated in the central cornea. Nuclear localization of ␤-catenin was observed in a very small subset of basal epithelial cells only at the limbus. Activation of Wnt/␤-catenin signaling increased the proliferation and colony-forming efficiency of primary human LSCs. The stem cell phenotype was maintained, as shown by higher expression levels of putative corneal epithelial stem cell markers, ATP-binding cassette family G2 and ⌬Np63␣, and low expression levels of mature cornea epithelial cell marker, cytokeratin 12. CONCLUSIONS. These findings demonstrate for the first time that Wnt signaling is present in the ocular surface epithelium and plays an important role in the regulation of LSC proliferation. Modulation of Wnt signaling could be of clinical application to increase the efficiency of ex vivo expansion of corneal epithelial stem/progenitor cells for transplantation. (Invest Ophthalmol Vis Sci. 2011;52:4734 -4741

Usability, inclusivity, and content evaluation of COVID-19 contact tracing apps in the United States.

We evaluated the usability of mobile COVID-19 contact tracing apps, especially for individuals with barriers to communication and limited digital literacy skills. We searched the Apple App Store, Google Play, peer-reviewed literature, and lay press to find contact tracing apps in the United States. We evaluated apps with a framework focused on user characteristics and user interface. Of the final 26 apps, 77% were on both iPhone and Android. 69% exceeded 9th grade readability, and 65% were available only in English. Only 12% had inclusive illustrations (different genders, skin tones, physical abilities). 92% alerted users of an exposure, 42% linked to a testing site, and 62% linked to a public health website within 3 clicks. Most apps alert users of COVID-19 exposure but require high English reading levels and are not fully inclusive of the U.S. population, which may limit their reach as public health tools

Preferential biological processes in the human limbus by differential gene profiling.

Corneal epithelial stem cells or limbal stem cells (LSCs) are responsible for the maintenance of the corneal epithelium in humans. The exact location of LSCs is still under debate, but the increasing need for identifying the biological processes in the limbus, where LSCs are located, is of great importance in the regulation of LSCs. In our current study we identified 146 preferentially expressed genes in the human limbus in direct comparison to that in the cornea and conjunctiva. The expression of newly identified limbal transcripts endomucin, fibromodulin, paired-like homeodomain 2 (PITX2) and axin-2 were validated using qRT-PCR. Further protein analysis on the newly identified limbal transcripts showed protein localization of PITX2 in the basal and suprabasal layer of the limbal epithelium and very low expression in the cornea and conjunctiva. Two other limbal transcripts, frizzled-7 and tenascin-C, were expressed in the basal epithelial layer of the limbus. Gene ontology and network analysis of the overexpressed limbal genes revealed cell-cell adhesion, Wnt and TGF-β/BMP signaling components among other developmental processes in the limbus. These results could aid in a better understanding of the regulatory elements in the LSC microenvironment

Wnt/β-Catenin Signaling Regulates Proliferation of Human Cornea Epithelial Stem/Progenitor Cells

This study identifies several Wnt signaling molecules specific to limbal epithelial stem cells. In addition, activation of this pathway in limbal epithelial stem cells induced cell proliferation but not differentiation in vitro

Preferential Biological Processes in the Human Limbus by Differential Gene Profiling

<div><p>Corneal epithelial stem cells or limbal stem cells (LSCs) are responsible for the maintenance of the corneal epithelium in humans. The exact location of LSCs is still under debate, but the increasing need for identifying the biological processes in the limbus, where LSCs are located, is of great importance in the regulation of LSCs. In our current study we identified 146 preferentially expressed genes in the human limbus in direct comparison to that in the cornea and conjunctiva. The expression of newly identified limbal transcripts endomucin, fibromodulin, paired-like homeodomain 2 (PITX2) and axin-2 were validated using qRT-PCR. Further protein analysis on the newly identified limbal transcripts showed protein localization of PITX2 in the basal and suprabasal layer of the limbal epithelium and very low expression in the cornea and conjunctiva. Two other limbal transcripts, frizzled-7 and tenascin-C, were expressed in the basal epithelial layer of the limbus. Gene ontology and network analysis of the overexpressed limbal genes revealed cell-cell adhesion, Wnt and TGF-β/BMP signaling components among other developmental processes in the limbus. These results could aid in a better understanding of the regulatory elements in the LSC microenvironment.</p></div

Protein expression of selected limbal transcripts in human ocular tissue.

<p>A) Minimal expression of PITX2 was observed in the cornea. B) Distinct PITX2 cytoplasmic expression was present in pockets within the basal layer and suprabasal layer of the limbal epithelium. Insert highlights both PITX2 cytoplasmic and nuclear localization in the basal epithelial cells. Thin arrows indicate cells with PITX2 nuclear localization. C) Minimal expression of PITX2 was also observed in the conjunctiva. D) Weak expression of FZD7 was detected in the basal layer of the cornea. E) Highly localized FZD7 expression was observed at the basal layer of the limbal epithelium. F) We observed low expression in the suprabasal and superficial layers of the conjunctiva for FZD7. G) Minimal expression of TNC was observed in the cornea, while distinct expression was present in the subepithelial stroma along the limbus (H). (I) We detected minor expression of TNC in the suprabasal and superficial layers of the conjunctiva. Thick arrows represent examples of superficial epithelial cells and arrowheads represent examples of basal epithelial cells in the limbus. Scale bar, 50 µm.</p

Gene ontology analysis of preferentially expressed signature limbal genes.

<p>Bar graph showing significance of enrichments terms from preferentially expressed genes in the limbal tissue.</p

Analysis of preferential transcription in the limbus compared to the cornea and conjunctiva.

<p>A) Venn diagram of 216 preferentially expressed limbal transcripts (including unknown and overlapping probes) between the limbal over corneal and conjunctival transcripts. B) Heat map of the 216 preferentially expressed limbal transcripts (including unknown and overlapping probes) between the limbal over corneal and conjunctival transcripts categorized by anatomical tissue. Red signifies overexpression and blue signifies underexpression.</p

Network map of preferentially expressed signature limbal genes.

<p>The IPA network map highlights the upregulation of genes from the TGF-β pathway and extracellular matrix processes. Fibronectin-1 is centrally connected to a number of upregulated limbal genes.</p