Conjunctival Epithelial Stem Cells Research Articles

Design of functional decellularized matrix for conjunctival epithelial stem cell maintenance and ocular surface reconstruction

Conjunctival defects severely impair ocular surface homeostasis and vision.However, the existing conjunctival reconstruction methods are still unable to achieve satisfactory functional replacement of conjunctiva. Scaffold materials that provide a more stem-cell-friendly microenvironment are in urgent need of improvement. Herein we reported a functional conjunctival reconstruction scaffold constructed from decellularized matrix derived from rabbit subconjunctival fibroblasts (DM-SCF) loaded with conjunctival epithelial stem cells (CjESCs) for ocular surface repair. The DM-SCF had a thickness and mechanical strength close to that of normal conjunctiva and showed excellent biocompatibility. CjESCs inoculated on DM-SCF maintained good stem cell properties, which showed long-term maintenance of low differentiation and good proliferation activity.DM-SCF transplantation with CjESCs showed satisfactory results in rabbit models of a large conjunctival defect.The conjunctiva was reconstructed with abundant goblet cell expression and regular fiber arrangement. Protein mass spectrometry revealed the active extracellular matrix components of DM-SCF, regulating the activation of Wnt/β-catenin and Notch pathways for stem cell maintenance. Overall, our study developed a promising new strategy for ocular surface reconstruction by optimizing bioactive scaffold and providing functional stem cell niche, so as to achieve long-standing and superior functional repair.

Oral Mucosal Epithelial Transplantation and Limbal-Rigid Contact Lens: A Therapeutic Modality for the Treatment of Severe Ocular Surface Disorders.

Stevens-Johnson syndrome, ocular cicatricial pemphigoid, and severe thermal or chemical injury are considered severe ocular surface disorders (OSDs) because they affect the entire ocular surface, including corneal and conjunctival epithelial stem cells. In patients with severe OSDs, the long-term prognosis for limbal transplantation is poor, and the related corneal opacity and cicatrization lead to devastating visual impairment. To date, there is no standardized treatment to improve vision in cases with severe OSD. Investigating novel treatment methods for severe OSDs, our group began cultivated oral mucosal epithelial transplantation in 2002 and developed a limbal-supported rigid-type contact lens that can be applied as a nonsurgical treatment. When used in combination, these treatment methods make it possible to successfully restore vision in cases with severe OSDs.

Conjunctival reconstruction via enrichment of human conjunctival epithelial stem cells by p75 through the NGF-p75-SALL2 signaling axis.

Severe conjunctival diseases can cause significant conjunctival scarring, which seriously limits eye movement and affects patients' vision. Conjunctival reconstruction remains challenging due to the lack of efficient methods for stem cells enrichment. This study indicated that p75 positive conjunctival epithelial cells (CjECs) were mainly located in the basal layer of human conjunctival epithelium and showed an immature differentiation state in vivo. The p75 strongly positive (p75++) CjECs enriched by immuno‐magnetic beads exhibited high expression of stem cell markers and low expression of differentiated keratins. During continuous cell passage cultivation, p75++ CjECs showed the strongest proliferation potential and were able to reconstruct the conjunctiva in vivo with the most complete structure and function. Exogenous addition of NGF promoted the differentiation of CjECs by increasing nuclear localization of SALL2 in p75++ CjECs while proNGF played an opposite role. Altogether, p75++ CjECs present stem cell characteristics and exhibit the strongest proliferation potential so can be used as seed cells for conjunctival reconstruction, and NGF‐p75‐SALL2 signaling pathway was involved in regulating the differentiation of CjECs.

Research progress in conjunctival epithelial stem cells

Conjunctival epithelial stem cells are the common progenitor cells of both conjunctival epithelial cells and goblet cells, having the ability of self-renewal proliferation and differentiation. They play a central role in maintaining the homeostasis of ocular surface and damage repair. With the in-depth study of conjunctival epithelial stem cells, we can further understand their effective mechanism in regulating ocular surface homeostasis. Furthermore, conjunctival epithelial stem cells are good candidates as seed cells for reconstructing conjunctiva in tissue engineering. The research progress in conjunctival epithelial stem cells, such as their concept, features, location, identification, culture method in vitro, differentiation and clinical application were reviewed. Key words: Stem cell/cytology; Epithelial, conjunctiva; Goblet cells; Cell culture

An Update on Ocular Surface Epithelial Stem Cells: Cornea and Conjunctiva.

The human ocular surface (front surface of the eye) is formed by two different types of epithelia: the corneal epithelium centrally and the conjunctival epithelium that surrounds this. These two epithelia are maintained by different stem cell populations (limbal stem cells for the corneal epithelium and the conjunctival epithelial stem cells). In this review, we provide an update on our understanding of these epithelia and their stem cells systems, including embryology, new markers, and controversy around the location of these stem cells. We also provide an update on the translation of this understanding into clinical applications for the treatment of debilitating ocular surface diseases.

Progresses of in vitro culture and differentiation in conjunctival epithelial stem cells

Conjunctiva epithelial stem cells are bipotent cells giving rise to both conjunctival epithelial cells and goblet cells. They play a central role in ocular surface homeostasis and wound repair, and also have great capacities of self-renewal, proliferation and differentiation. As a result, conjunctival epithelial stem cells are main resources of seed cells for tissue engineering conjunctiva. To review the current research of conjunctival epithelia stem cells such as their features, distribution, ways to identify stem cells, in vitro culture and induced differentiation to goblet cells.

Potential localization of putative stem/progenitor cells in human bulbar conjunctival epithelium

Although the conjunctival fornix appears to contain the greatest proportion of stem cells, it is likely that pockets of conjunctival epithelial stem cells may also exist throughout the conjunctival epithelium. This study was to investigate the potential localization of putative stem/progenitor cells in the human bulbar conjunctival epithelium by evaluating 6 keratins and 13 molecules that have been previously proposed stem cell associated or differentiation markers. We found that cornea specific cytokeratin (CK) 3 was not expressed by the bulbar conjunctival epithelial cells. In contrast, CK4 and CK7 were expressed by the superficial cells of bulbar conjunctival epithelium. CK14 and CK15 were confined to the basal cell layer. CK19 was strongly expressed by all layers of the bulbar conjunctival epithelium. The expression patterns of molecular markers in the basal cells of human bulbar conjunctival epithelium were found to be similar to the corneal epithelium. Basal conjunctival epithelial cells strongly expressed stem cell associated markers, including ABCG2, p63, nerve growth factor (NGF) with its receptors tyrosine kinase receptor A (TrkA) and neurotrophin low-affinity receptor p75NTR, glial cell-derived neurotrophic factor (GDNF) with its receptor GDNF family receptor alpha 1 (GFRalpha-1), integrin beta1, alpha-enolase, and epidermal growth factor receptor (EGFR). The differentiation associated markers nestin, E-cadherin and involucrin were not expressed by these cells. These findings indicate that the basal cells of bulbar conjunctival epithelium shares a similar expression pattern of stem cell associated markers to the corneal epithelium, but has a unique pattern of differentiation associated cytokeratin expression.

Ocular Surface Stem Cells and Disease: Current Concepts and Clinical Applications

Corneal and conjunctival epithelial stem cells are responsible for the homeostasis and regeneration of the ocular surface epithelium. Corneal epithelial stem cells reside in the basal region of the limbus, while the conjunctival forniceal region appears to be the site that is enriched in conjunctival stem cells. Ocular surface disease arising from limbal stem cell deficiency is characterised by persistent epithelial defects, corneal vascularisation, chronic inflammation, scarring and conjunctivalisation, resulting in visual loss. Limbal stem cell transplantation replaces the corneal stem cell population in these eyes with the hope of restoring vision. More recently, the use of bioengineered ocular surface tissue-equivalents has had promising results, and may represent the future for replacement and regeneration of ocular tissues in various ocular disorders.

Epithelial stem cells: the eye provides a vision.

Epithelial stem cells play a central role in tissue homeostasis, wound repair, and carcinogenesis. Corneal epithelial stem cells have been demonstrated to reside in the limbal epithelium, while the fornical zone of the conjunctiva appears to be a predominant site of conjunctival epithelial stem cells. Stem cells of the corneal and conjunctival epithelia, as well as the hair follicle and interfollicular epidermis share important features: they are capable of self renewal; they are relatively quiescent (slow-cycling); they can be induced to proliferate; and they are multipotent. Its becoming apparent that a certain degree of flexibility exists between corneal and hair follicle keratinocytes.

Wistar Rat Palpebral Conjunctiva Contains More Slow-cycling Stem Cells That Have Larger Proliferative Capacity: Implication for Conjunctival Epithelial Homeostasis

Purpose: To determine the location of conjunctival epithelial stem cells. Methods: Wistar rats received daily injection of 5-bromo-2-deoxyuridine (BrdU) at a dose of 5 mg/100 g for 2 weeks followed by a 1-month BrdU-free period before death. After the rats were sacrificed, the orbital contents and eyelids were exenterated en bloc, fixed in buffer formalin, and embedded in paraffin. To compare the proliferative capacity of ocular epithelial cells, 1.0% phorbol myristate (TPA) in petrolatum was topically applied once daily to both eyes of Wistar rats for 12 days. After 6, 12, 18, and 24 hours and 2, 4, 6, 8, 10, and 12 days of TPA treatment, rats were administered BrdU intraperitoneally 7 hours before they were sacrificed. The ocular epithelium was fixed and processed for immunochemistry, and the labeling index (LI) of every epithelial zone was determined. Results: Slow-cycling cells, detected as label-retaining cells (LRCs), were found in bulbar, fornical, and palpebral epithelia and mucocutaneous junctions, as well as in limbal epithelia. The greatest numbers of LRCs were identified in palpebral epithelium. Under normal situations, in conjunctiva the LI was lowest in palpebral epithelium (2.1 ± 0.5) compared with bulbar (2.2 ± 0.5), fornical (2.3 ± 0.4) epithelia and mucocutaneous junction (3.4 ± 0.9), respectively. In cornea, the LI was lowest in limbal epithelium (1.8 ± 0.7) compared with central corneal epithelium (3.5 ± 0.6). Twenty-four hours after TPA treatment, an 8.2-fold increase in the palpebral epithelial basal cell labeling index was noted compared with 4.7-fold, 5.7-fold, and 3.8-fold increases in bulbar, fornical, and mucocutaneous junction epithelial basal cell labeling indices, and a sevenfold increase in the limbal basal cell labeling indices compared with a 2.1-fold increase in the corneal basal cell labeling index, respectively. Limbal and palpebral epithelia maintained a significantly greater proliferative response (5.5-to 6.3-fold increase, respectively) during chronic stimulation than corneal, bulbar, fornical epithelia, and mucocutaneous junction (0.6- to 2.3-fold increase, respectively). Conclusions: In Wistar rat conjunctiva, slow-cycling cells are primarily located in palpebral epithelium, which has greater proliferative capacity than other conjunctival epithelia. This finding means that, in the Wistar rat, the conjunctival epithelial stem cells are mainly located in palpebral epithelium. These data open new perspectives in ocular epithelial development and are relevant in conjunctival wound repair.

Expansion of Conjunctival Epithelial Progenitor Cells on Amniotic Membrane

Amniotic membrane (AM) reconstructed human conjunctival surfaces recover a goblet cell density higher than normal. Cultured rabbit conjunctival epithelial cells (RCE) on AM preferentially exhibit non-goblet epithelial differentiation. It was thus wondered if conjunctival progenitor cells that might have been preserved during ex vivo expansion on AM can still differentiate into conjunctival non-goblet epithelial and goblet cells under the influence of mesenchymal cells.Fourteen day old AM cultures of RCE were subcutaneously implanted in Balb/c athymic mice for 11 days and processed for PAS staining and immunostaining with monoclonal antibodies to conjunctival goblet cell mucin (MUC5AC, AM3), glycocalyx (AMEM2), cornea specific cytokeratins K3 (AE5) and K12 (AK2) and basal cell specific cytokeratin K14. Cell cycle kinetics were measured by BrdU labelling for 1 or 7 days. The 7 day labelled RCE were chased for 14 days in the same primary culture.After subcutaneous implantation, conjunctival non-goblet epithelial cells increased stratification and formed occasional cysts. The resultant epithelial phenotype was conjunctival with many PAS-positive, MUC5AC-positive, and AM3-positive goblet cells, AMEM2-positive suprabasal and superficial cells, and K14-positive basal cells, but was not corneal (negative to AE5 and AK2 staining). Twenty four hr BrdU labelling showed a labelling index of 42.5%. A higher labelling index or 69% was noted after continuous BrdU labelling for 7 days. A large number of label retaining basal cells with a labelling index of 84% were noted following 14 days of chase.Conjunctival epithelial progenitor cells for goblet and non-goblet cell differentiation are preserved by AM in vitro as evidenced by being able to differentiate into goblet cells in a permissive stromal environment, and being slow-cycling, and label retaining. This information is useful for future ex vivo expansion of conjunctival epithelial stem cells for conjunctival surface reconstruction.