Ex Vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement.
[Show abstract] [Hide abstract] ABSTRACT: Background aims. Autologous transplantation of ex vivo cultured cells the treatment of choice for patients with limbal stem cell deficiency. The most commonly used cell sources for transplantation limbal, conjunctival or oral mucosal tissue. Protocols vary for culturing each tissue type, and there are no comparative studies on transplantation outcomes using these different culture techniques.To overcome this limitation, we devised a simple protocol that can uniformly promote growth and differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage. Methods. Biopsies were cultured as explants on de-epithelialized human amniotic membrane in the presence of recombinant epidermal growth factor and insulin. Cultured cells were characterized using immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction for stem/progenitor markers (ABCG2 and P63α) and differentiation markers (CK3, CK12, CK4, CK13, CK15 and CONNEXIN 43). Fluorescence-activated cell sorter analysis was performed for ABCG2. Results. The results revealed that cells of all three biopsies differentiated into the corneal lineage. Positivity of CK3/12, CK4, CK12 and CONNEXIN 43 immunostaining and the relative mRNA expression of CK3, CK4, CK12, CK13, CK15 and CONNEXIN 43 could be detected in the cultured biopsies. Conclusions. Unlike tissue-specific protocols, our protocol can unequivocally promote differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage. This simple standardized protocol can be adapted for ocular surface reconstruction using stem cell transplantation.0Comments 1Citation
- "It has been reported that there is a direct correlation between corneal stem/progenitors and success of corneal surface transplantation . Using stem cell and differentiation markers, Shahdadfar et al. determined that a simple medium with serum was sufficient to culture limbal epithelial cells .The various modifications during the culture of limbal epithelial cells are listed in Table II. Our protocol differs in that it reduces the usage of several additional components , such as cholera toxin, hydrocortisone and apotransferrin .This makes it more affordable and adaptable. "
[Show abstract] [Hide abstract] ABSTRACT: Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.0Comments 0Citations
- "Loss of LESCs and/or function due to disease or injury can result in impaired corneal function, neovascularization, conjunctival ingrowth and ultimately loss of vision. LESC deficiency (LESCD) —partial or total, can be treated by restoring the limbal area using biopsies from the patient's healthy eye or transplanting LESCs harvested from autologous or cadaver donor tissue, then cultured and expanded ex vivo [5, 6]. Several groups including ours have isolated, cultured and characterized successfully LESCs– all of these studies describe novel methods for cultivating these cells on different biological and synthetic scaffolds in a medium containing or void of serum or other growth supplements[6– 9]. "
[Show abstract] [Hide abstract] ABSTRACT: Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal epithelium, and nanocarriers for corneal drug delivery are discussed. Attention is also paid to problems in wound healing understanding and treatment, such as lack of specific epithelial stem cell markers, reliable identification of stem cells, efficient prevention of haze and stromal scar formation, lack of data on wound regulating microRNAs in keratocytes and endothelial cells, as well as virtual lack of targeted systems for drug and gene delivery to select corneal cells. Copyright © 2015 Elsevier Ltd. All rights reserved.0Comments 6Citations
- "Recent efforts were focused on developing xenobiotic-free LESC cultures with no feeder cells or with human cells. Human autologous serum has been used to expand LESC on HAM (Shahdadfar et al., 2012) or on soft contact lenses (Bobba et al., 2015). The latter technique has been used in a clinical trial in 16 LSCD patients with reported success in 10 of 16 eyes (63%) at a median follow-up time of 2.5 years with the advantage of autologous transplantation (Bobba et al., 2015). "
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