Some notes on my research on goblet cells. The next frontier for dry eye treatment. Thinking there must be a way to 3D print or
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Abstract
Goblet cells are terminally differentiated cells secreting mucins and anti-bacterial peptides that play an important role in maintaining the health of the cornea. In corneal stem cell deficiency, the progenitor cells giving rise to goblet cells on the cornea are presumed to arise from differentiation of cells that migrate onto the cornea from the neighboring conjunctiva. This occurs in response to the inability of corneal epithelial progenitor cells at the limbus to maintain an intact corneal epithelium. This study characterizes clusters of cells we refer to as compound niches at the limbal:corneal border in the unwounded mouse. Compound niches are identified by high expression of simple epithelial keratin 8 (K8) and 19 (K19). They contain variable numbers of cells in one of several differentiation states: slow-cycling corneal progenitor cells, proliferating cells, non-proliferating cells, and post-mitotic differentiated K12+Muc5ac+goblet cells. Expression of K12 differentiates these goblet cells from those in the conjunctival epithelium and suggests that corneal epithelial progenitor cells give rise to both corneal epithelial and goblet cells. After wounds that remove corneal epithelial cells near the limbus, compound niches migrate from the limbal:corneal border onto the cornea where K8+ cells proliferate and goblet cells increase in number. By contrast, no migration of goblet cells from the bulbar conjunctiva onto the cornea is observed. This study is the first description of compound niches and corneal goblet cells and demonstration of a role for these cells in the pathology typically associated with corneal stem cell deficiency.
Goblet cells can rapidly secrete mucins to form a mucus layer that protects against inhaled pathogens, toxins, and other foreign particles. Although this mucus layer normally plays a protective role, abnormal mucus production and clearance can contribute to respiratory disease pathologies (9, 10). Despite the importance of goblet cells in respiratory diseases, the origins of goblet cells and processes regulating their formation are poorly understood. The mucin, MUC5AC, is one of the major mucins found in airway secretions in asthma and COPD (7, 11, 12). A candidate implicated in the control of MUC5AC expression and goblet cell formation is the T helper (Th) type 2 cytokine, IL-13. Indeed, IL-13 gives rise to increased goblet cell numbers in both murine models of allergic asthma (13, 14) and in vitro models of the human respiratory epithelium (15). Moreover, IL-13 has been demonstrated to induce MUC5AC mucin expression in vivo and in vitro (15, 16).
Studies in the mouse have implicated Clara or ciliated cells as progenitors of goblet cells via a process of transdifferentiation (17, 18). Data supporting transdifferentiation of Clara cells as a mechanism of goblet cell formation include a study using an ovalbumin (OVA)-induced mouse model of goblet cell formation, where mucin expression was found in a subset of Clara cells in the proximal, but not distal, airways (17). In a separate study, an ultrastructural analysis of mouse lung after OVA challenge showed mucus-producing cells in the airways, with many characteristics of Clara cells (19). Additional indirect evidence of Clara cell transdifferentiation comes from an analysis of Clara and goblet cell numbers in the lung of a mouse OVA model, where a decrease in the numbers of Clara cells was accompanied by an increase in the number of goblet cells (20). In contrast, in a Sendai virus model, transdifferentiation of ciliated cells was implicated as a mechanism for the observed goblet cell metaplasia, involving IL-13 and EGFR pathways (18). One limitation of the studies to date examining the origins of goblet cells in the airways is that the mouse has been used as the model organism. Differences in human and mouse airway epithelial cell biology (21, 22) indicate the importance of performing studies to elucidate goblet cell progenitors in human as well as mouse model systems. In addition, a direct, lineage-tracing approach would help to clarify the confusion over mechanisms underlying mucus metaplasia.
Here, we have investigated the role of the ciliated cell as a progenitor of the goblet cell using a cell lineage–tagging approach and primary human bronchial epithelial cells (HBECs). A lentiviral cotransduction approach employing the FOXJ1 promoter was used to deliver constructs for permanent Cre/loxP-mediated enhanced green fluorescent protein (EGFP) tagging of ciliated cells. Subsequently, the fate of the EGFP-tagged ciliated cells after treatment with IL-13 to induce goblet cell formation was monitored by histology. By increasing our understanding of cell types that contribute to goblet cell formation in human model systems, new therapeutic approaches for targeting excessive mucus production in respiratory diseases may be identified.
The ocular surface is composed of conjunctival, limbal, and corneal epithelia which express distinct repertoires of keratins. While all three epithelial cell types express K5 and K14 keratins like the epidermis, the conjunctiva and limbal epithelia express “simple epithelial” keratins including K4, K8, K13, K18, and K19 1–5. Human corneal epithelial cells express the K3/K12 keratin pair 6. The mouse genome lacks an intact K3 gene but its corneal epithelium expresses K12, which is considered a marker of corneal epithelial cells 7, 8. Conjunctival and corneal epithelia have been reported to derive from distinct stem cell populations 9–15. Data from studies looking at expression of corneal specific proteins in addition to K12 within conjunctival and corneal epithelial cell lines largely confirm that the conjunctiva and corneal epithelium represent distinct cell lineages 16, 17.
The location of the limbal stem cells has been the topic of numerous studies. A Limbal Stem Cell Hypothesis (LSCH) was originally proposed 18, 19, has been expanded upon 14, 20–24, challenged 25, and reviewed extensively 26–34. In brief, the LSCH states that stem cells required for maintaining the corneal epithelium, called limbal stem cells (LSCs), reside within a niche in a subset of the limbal basal cells. In the human cornea, this niche takes the form of invaginations called limbal crypts 35. LSCs divide and give rise to progeny that migrate across the corneal surface converging at the center of the cornea. Similar to conjunctival and limbal epithelia, LSCs express simple epithelial keratins. Based on the LSCH, stem cell deficiency results from damage to the limbus which allows conjunctival epithelial and goblet cells to migrate onto the ocular surface. Clinically the LSCH is supported by the fact that limbal transplants are highly successful treatments for stem cell deficiency caused by chemical injury 36.
2. The overproduction of mucus is a key pathology associated with respiratory diseases, such as asthma and chronic obstructive pulmonary disease. These conditions are characterized by an increase in the number of mucus-producing goblet cells in the airways. We have studied the cellular origins of goblet cells using primary human bronchial epithelial cells (HBECs), which can be differentiated to form a stratified epithelium containing ciliated, basal and goblet cells. Treatment of differentiated HBEC cultures with the cytokine IL-13, an important mediator in asthma, increased the numbers of goblet cells and decreased the numbers of ciliated cells. To determine whether ciliated cells act as goblet cell progenitors, ciliated cells in HBEC cultures were hereditably labeled with enhanced green fluorescent protein (EGFP) using two lentiviral vectors, one which contained Cre recombinase under the control of a FOXJ1 promoter and a second Cytomegalovirus (CMV)–floxed-EGFP construct. The fate of the EGFP-labeled ciliated cells was tracked in HBEC cultures. Treatment with IL-13 reduced the numbers of EGFP-labeled ciliated cells compared with untreated cultures. In contrast, IL-13 treatment significantly increased the numbers of EGFP-labeled goblet cells. This study demonstrates that goblet cells formed in response to IL-13 treatment are in part or wholly derived from progenitors that express the ciliated cell marker, FOXJ1.
Goblet Cells Are Derived from a FOXJ1-Expressing Progenitor in a Human Airway Epithelium
Jonathan Turner 1, Jan Roger 1, Juliette Fitau 1, Delphine Combe 1, June Giddings 1, Gino Van Heeke 1, and Carol E. Jones 1
Comparative study of limbal stem cell deficiency diagnosis methods: detection of MUC5AC mRNA and goblet cells in corneal epithelium.
Garcia I1, Etxebarria J, Boto-de-Los-Bueis A, Díaz-Valle D, Rivas L, Martínez-Soroa I, Saenz N, López C, Del-Hierro-Zarzuelo A, Méndez R, Soria J, González N, Suárez T, Acera A.
Abstract
PURPOSE:
To evaluate a limbal stem cell deficiency (LSCD) diagnosis method based on the detection of the MUC5AC transcript by reverse transcription-polymerase chain reaction (RT-PCR) in comparison with the standard diagnostic method based on goblet cell detection by periodic acid-Schiff (PAS)-hematoxylin staining, using samples obtained from corneal epithelium impression cytology (IC).
DESIGN:
Transversal, comparative case series.
PARTICIPANTS:
We studied 59 eyes from 43 patients clinically diagnosed with LSCD.
METHODS:
Impression cytology was used to gather cells from corneal and conjunctival epithelium from the same eye. The presence of goblet cells in the cornea was determined by PAS-hematoxylin staining, whereas the presence of the MUC5AC transcript was detected by RT-PCR using a custom-designed primer pair.
MAIN OUTCOME MEASURES:
Goblet cells in the corneal epithelium were detected by light microscopy, and the MUC5AC transcript was detected as the corresponding PCR amplicon in agarose gels.
RESULTS:
Our study included 59 corneal samples, together with their respective conjunctival samples for RT-PCR assays. Of these, 47 samples were also available for comparative PAS-hematoxylin staining. The MUC5AC amplicon was detected in 56 of 59 (94.9%) corneal epithelium samples. In contrast, conventional IC staining detected goblet cells in only 17 of 47 (36.2%) samples; these were not found in 27 of 47 (57.4%) samples (negative results), and 3 of 47 (6.4%) showed inconclusive results.
CONCLUSIONS:
The detection of the MUC5AC transcript in corneal epithelium is a more sensitive method to diagnose LSCD than the conventional PAS-hematoxylin method, although a minimum RNA concentration of 1.2 ng/μl is required for negative results to be reliable. Moreover, RT-PCR is a highly specific and more objective technique. Overall, these findings indicate that molecular analysis facilitates a more precise clinical diagnosis of LSCD, thereby reducing the risk of surgical failure.
References:
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