Rat limbal niche cells can induce transdifferentiation of oral mucosal epithelial cells into corneal epithelial-like cells in vitro.
ABSTRACT: BACKGROUND:Cultivated oral mucosal epithelial cells (OMECs) are widely used in the treatment of limbal stem cell deficiency (LSCD) for their ocular reconstruction capability. As the most important component of the limbal microenvironment, limbal niche cells (LNCs) play a key role in the direction of stem cell differentiation. In this study, we investigated whether LNCs can induce the transdifferentiation of rat OMECs to corneal epithelial-like cells. METHODS:We isolated OMECs and LNCs from rats by dispase and collagenase, respectively, to establish a three-dimensional or Transwell coculturing system. NIH-3T3 cells and renewed LNCs were also used as feeder layers in the Transwell system to compare their ability to support the OMECs. The airlift method was used for the culture of OMECs to obtain a stratified epithelial sheet. Cocultured OMECs were characterized by reverse-transcription polymerase chain reaction, Western blotting, hematoxylin and eosin staining, and immunohistochemistry. RESULTS:The cocultured OMECs showed corneal epithelial-like morphology and expressed the corneal epithelial markers CK12 and Pax6 in most cocultured systems. Furthermore, we found that the expression level of CK12, Pax6, and proliferation marker Ki67 was upregulated when compared with that of other groups by renewing the LNCs in the Transwell system (p?
Project description:Limbal epithelial stem cells (LESCs) play important roles in corneal epithelial homeostasis and regeneration, and damage to the limbus will lead to limbal stem cell deficiency (LSCD), with conjunctivalization and even visual impairment. Cultured LESCs have been used for ocular surface reconstruction, and silk fibroin (SF) membranes have shown potential as a substrate for LESC cultivation. Both culture methods and the carriers of LESCs affect outcomes following LESC transplantation.Rabbit LESCs were cultured from tissue explant, single cell-suspension, and cell cluster culture methods. Ratios of p63? and/or ABCB5-positive LESCs, differentiated corneal epithelial cells (CK12 staining), and corneal tight junction formation (Claudin-1 staining) were examined to choose the most applicable LESC cultures. SF membranes were prepared and modified by 400-Da poly(ethylene glycol) (PEG). The characteristics of stem cells and normal corneal differentiation of LESCs cultured on PEG-modified SF membranes were further examined by immunofluorescence staining and flow cytometric analysis. LESCs cultured on PEG-modified SF membranes (LESC/SF grafts) and PEG-modified SF membranes (SF grafts) were transplanted onto rabbit corneas with total LSCD. New blood vessels, corneal epithelial defects, and cornea clarity were examined after transplantation. Furthermore, corneal epithelial thickness, stromal thickness, and the percentage area of CK12-positive corneal epithelium were quantified 4 months after transplantation.Tissue explant and single cell-suspension cultures harvested more p63? and/or ABCB5-positive LESCs, generated more CK12-positive corneal epithelial cells, and formed more corneal tight junctions than cell cluster cultures. Prepared PEG-modified SF membranes were transparent, flexible, and sturdy enough for surgical manipulation. LESCs cultured on PEG-modified SF membranes maintained characteristics of stem cells and normal corneal differentiation. LESC/SF grafts inhibited new blood vessels and rescued corneal epithelial defects in the rabbit total LSCD model. In addition, LESC/SF grafts repopulated the limbus and increased corneal epithelial thickness, stromal thickness, and the area percentage of CK12-positive corneal epithelium.LESCs from tissue explant and single cell-suspension cultures were more applicable corneal epithelial cells for ocular surface reconstruction. LESC/SF grafts repaired corneal epithelial defects and reversed LSCD, and PEG-modified SF membranes were suitable to be a carrier for LESC transplantation.
Project description:Homeostasis and function of limbal epithelial stem cells (LESCs) rely on the limbal niche, which, if dysfunctional, leads to limbal epithelial stem cell deficiency (LSCD) and impaired vision. Hence, recovery of niche function is a principal therapeutic goal in LSCD, but the molecular mechanisms of limbal niche homeostasis are still largely unknown. Here, we report that the neural crest transcription factor SOX10, which is expressed in neural crest-derived limbal niche cells (LNCs), is required for LNCs to promote survival of LESCs both in vivo and in vitro. In fact, using mice with a Sox10 mutation and in vitro coculture experiments, we show that SOX10 in LNCs stimulates the production of KIT ligand (KITL), which in turn activates in LESCs the KIT-AKT signalling pathway that protects the cells against activated CASPASE 3-associated cell death. These results suggest that SOX10 and the KITL/KIT-AKT pathway play key roles in limbal niche homeostasis and LESC survival. These findings provide molecular insights into limbal niche function and may point to rational approaches for therapeutic interventions in LSCD.
Project description:Background:Autologous cultivated oral mucosal epithelial transplantation (COMET) is an important treatment for limbal stem cell deficiency. However, peripheral corneal neovascularization after surgery hinders its application. This study aims to employ a culture system using allogenic limbal niche cells (LNCs) instead of mouse-derived 3T3 cells as a feeder layer that could relieve postoperative neovascularization. Methods:Rat oral mucosal epithelial cells (OMECs) were co-cultured with rat LNCs or 3T3 cells. Cultivated oral mucosal epithelial cells (COMECs) of different culture systems were identified by hematoxylin and eosin staining and immunocytochemistry. The expression levels of the angiogenesis-related factors were analyzed by RT-qPCR and western blotting/ELISA. Angiogenic potential was reconfirmed by cell viability and tube formation assays with human umbilical vein endothelial cells (HUVECs). Results:COMECs were obtained from both culture systems successfully. Immunocytochemistry showed approximately equal percentages of positive staining cells for p63α (p = 0.9177), ABCG2 (p = 0.526), Ki67 (p = 0.0987), and CK3 (p = 0.4000) in COMECs of different groups. RT-qPCR and western blotting/ELISA showed that COMECs of the LNC group expressed a significantly lower amount of basic fibroblast growth factor (bFGF) (p = 0.0038 for RT-qPCR, p = 0.0026 for western blotting) but more pigment epithelium-derived factor (PEDF) (p = 0.0172 for RT-qPCR, p = 0.0253 for western blotting) and soluble fms-like tyrosine kinase-1 (sFlt-1) (p < 0.0001 for RT-qPCR, p = 0.0064 for ELISA) than the COMECs of the 3T3 group. Furthermore, compared with COMECs of the 3T3 group, COMECs of the LNC group could reduce the viability (p = 0.0002) and tube formation (p = 0.0002) of HUVECs. Conclusions:LNCs could substitute 3T3 cells for expanding OMECs in vitro, and the COMECs obtained in this system are less likely to induce postsurgical neovascularization, which provides an alternative option for an ex vivo culture system and promotes the application of COMET.
Project description:Both BMP and Wnt signaling control stem cells in bulge/dermal papilla, intestinal crypt, and bone marrow. To explore their roles in the limbal niche, which govern corneal epithelial homeostasis, we established an in vitro model of sphere growth by reunion between single limbal epithelial progenitor cells (LEPCs) and aggregates of limbal niche cells (LNCs) in 3D Matrigel. Compared to LEPCs alone, spheres formed by LEPC+LNC exhibited higher clonal growth and less corneal epithelial differentiation. Furthermore, pSmad1/5/8 was in the nucleus of LEPCs, but not LNCs, and correlated with upregulation of BMP1, BMP3, BMP4, all three BMP receptors, and BMP target genes. Inactivation of BMP signaling in LNCs was correlated with upregulation of noggin preferentially expressed by LNCs. Additionally, ?-catenin was stabilized in the perinuclear cytoplasm in LEPCs and correlated with upregulation of Wnt7A and FZD5 preferentially expressed by LEPCs. Inactivation of Wnt signaling in LNCs was correlated with upregulation of DKK1/2 by LNCs. Addition of XAV939 that expectedly downregulated perinuclear ?-catenin in LEPCs led to significant reduction of epithelial clonal growth, but upregulated all three BMP receptors and downregulated LNC-derived noggin, resulting in activation of BMP signaling in LNCs. Addition of noggin that expectedly downregulated nuclear localization of pSmad1/5/8 in LEPCs led to nuclear localization of ?-catenin in larger LEPCs but membrane relocation of ?-catenin in smaller LEPCs and significant upregulation of DKK1/2. Hence, balancing acts between Wnt signaling and BMP signaling exist not only within LEPCs but also between LEPCs and LNCs to regulate clonal growth of LEPCs.
Project description:PURPOSE:To grade the severity of limbal stem cell deficiency (LSCD) based on the extent of clinical presentation and central corneal basal epithelial cell density (BCD). METHODS:This is a retrospective observational comparative study of 48 eyes of 35 patients with LSCD and 9 eyes of 7 normal subjects (controls). Confocal images of the central cornea were acquired. A clinical scoring system was created based on the extent of limbal and corneal surface involvement. LSCD was graded as mild, moderate, and severe stages based on the clinical scores. The degree of BCD reduction was given a score of 0 to 3. RESULTS:Compared with BCD in controls, BCD decreased by 23.0%, 40.4%, and 69.5% in the mild, moderate, and severe stages of LSCD classified by the clinical scoring system, respectively. The degree of BCD reduction was positively correlated with larger limbal and corneal surface involvement and when the central visual axis was affected (all P ? 0.0005). Mean corrected distance visual acuity logarithm of the minimum angle of resolution was 0.0 ± 0.0 in control eyes, 0.2 ± 0.5 in mild LSCD, 0.6 ± 0.4 in moderate LSCD, and 1.6 ± 1.1 in severe LSCD (P < 0.0001). There was a significant correlation between a higher clinical score and corrected distance visual acuity logarithm of the minimum angle of resolution (rho = 0.82; P < 0.0001) and a greater decrease in BCD (rho = -0.78; P < 0.0001). CONCLUSIONS:A clinical scoring system was developed to assess the extent of clinical presentation of LSCD. A classification system to grade the severity of LSCD can be established by combining the BCD score with the clinical score.
Project description:Limbal epithelial stem cell (LESC) deficiency (LSCD) leads to corneal abnormalities resulting in compromised vision and blindness. LSCD can be potentially treated by transplantation of appropriate cells, which should be easily expandable and bankable. Induced pluripotent stem cells (iPSCs) are a promising source of transplantable LESCs. The purpose of this study was to generate human iPSCs and direct them to limbal differentiation by maintaining them on natural substrata mimicking the native LESC niche, including feederless denuded human amniotic membrane (HAM) and de-epithelialized corneas. These iPSCs were generated with nonintegrating vectors from human primary limbal epithelial cells. This choice of parent cells was supposed to enhance limbal cell differentiation from iPSCs by partial retention of parental epigenetic signatures in iPSCs. When the gene methylation patterns were compared in iPSCs to parental LESCs using Illumina global methylation arrays, limbal-derived iPSCs had fewer unique methylation changes than fibroblast-derived iPSCs, suggesting retention of epigenetic memory during reprogramming. Limbal iPSCs cultured for 2 weeks on HAM developed markedly higher expression of putative LESC markers ABCG2, ?Np63?, keratins 14, 15, and 17, N-cadherin, and TrkA than did fibroblast iPSCs. On HAM culture, the methylation profiles of select limbal iPSC genes (including NTRK1, coding for TrkA protein) became closer to the parental cells, but fibroblast iPSCs remained closer to parental fibroblasts. On denuded air-lifted corneas, limbal iPSCs even upregulated differentiated corneal keratins 3 and 12. These data emphasize the importance of the natural niche and limbal tissue of origin in generating iPSCs as a LESC source with translational potential for LSCD treatment.
Project description:Purpose:The most common cause of severe limbal stem cell deficiency (LSCD) is chemical injury. We report a case of severe unilateral alkali injury complicated by total LSCD, treated with customized Simple limbal epithelial transplantation (SLET) combined with conjunctival-limbal autografting (CLAU). Observation:A 23-year-old female sustained a severe unilateral alkali chemical injury, resulting in total LSCD, treated with customized SLET combined with CLAU. We used two autologous limbal biopsies harvested from the fellow eye. One was used for CLAU and the second was split into multiple pieces, and was glued to bare stroma following resection of the corneal pannus. A stable ocular surface was achieved, and the donor eye remained healthy. Visual acuity improved from hand motion initially, to 20/20 over one year post-operatively. Conclusion and importance:This case report demonstrates the efficacy and safety of customized SLET with supplemental CLAU to treat total LSCD in severe ocular burns.
Project description:Tissue engineering has become an essential tool in the development of regenerative medicine. We have developed cell sheet-based techniques for use in regenerative medicine that have already been successfully used in clinical applications. Native corneal epithelium is produced from limbal stem cells located in the transition zone between the cornea and the bulbar conjunctiva. Limbal stem cell deficiency (LSCD) is a severe defect of the limbal stem cells leading to vision loss due to conjunctival epithelial invasion and neovascularization. Rabbit LSCD models were treated with transplantable autologous oral mucosal epithelial cell (OEC) sheets fabricated on temperature-responsive cell culture surfaces, after which, the ocular surfaces were clear and smooth with no observable defects. The central part of the reconstructed ocular surface was scraped and wounded, after which proliferating epithelial cells covered the scraped area within a few days. The ocular surfaces were clear and smooth even after repeated scrapings and consisted of only OECs or heterogeneously mixed with corneal epithelial cells. This study demonstrates that transplanted cell sheets containing oral mucosal epithelial stem cells could reconstruct the ocular surface to maintain cornea homeostasis; moreover, they provide an ideal microenvironment to support the proliferation of remaining native limbal stem cells.
Project description:We studied the reproducibility and stability of limbal stem cell deficiency (LSCD) in mice following controlled injuries to the corneal and limbal epithelia. In one method, corneal and limbal epithelia were entirely removed with a 0.5 mm metal burr. In the other, limbus to limbus epithelial removal with the burr was followed by thermal injury to the limbus. These two methods were compared with a previously published one. Unwounded corneas were used as control. The corneas were examined monthly for three months by slit lamp with fluorescein staining. Immunofluorescence staining for cytokeratin 12 and 8 on corneal wholemount and cross sections were performed to determine the phenotype of the epithelium. Mechanical shaving of the epithelium, with or without thermal injury, resulted in a reproducible state of LSCD marked by superficial neovascularization, reduce of keratin 12 expression and presence of goblet cells on the cornea. The phenotype was stable in 100% of the eyes up to at least three months. Thermal injury produced a more severe phenotype with more significant stromal opacification. These corneal injury models may be useful for studying the mechanisms leading to limbal stem cell deficiency.
Project description:Purpose:In this study, we evaluated the feasibility of recovering the corneal surface integrity in a patient suffering from unilateral LSCD through the transplantation of cultured autologous corneal epithelial cells. Methods:Human corneal epithelial cells (HCECs) were isolated from a limbal biopsy of the contralateral eye of a patient with unilateral LSCD and cultured in monolayer in the presence of an irradiated human fibroblasts feeder layer (iHFL). To produce a cultured autologous corneal epithelium (CACE), HCECs were seeded on a fibrin substrate and maintained in culture until confluence. The in vitro obtained CACE was then used to treat the affected eye of the patient. Two years later, a successful penetrating keratoplasty was performed. Results:Efficient restoration of the corneal epithelium was achieved following transplantation of CACE indicating probable re-colonization of the cornea by stem cells. Corneal transparency was restored after removing the scarred stroma by performing a penetrating keratoplasty. Conclusion:CACE produced in vitro was shown to restore a normal corneal surface capable of sustaining a viable and clear penetrating keratoplasty and reestablished a near normal vision in a unilateral LSCD patient.