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Limbal stem cells are responsible for the continuous renewal of the corneal epithelium. The destruction or dysfunction of these stem cells or their niche induces limbal stem cell deficiency (LSCD) leading to visual loss, chronic pain, and inflammation of the ocular surface. To restore the ocular surface in cases of bilateral LSCD, an extraocular source of stem cells is needed to avoid dependence on allogeneic limbal stem cells that are difficult to obtain, isolate, and culture. The aim of this work was to test the tolerance and the efficacy of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) to regenerate the ocular surface in two experimental models of LSCD that closely resemble different severity grades of the human pathology. hAT-MSCs transplanted to the ocular surface of the partial and total LSCD models developed in rabbits were well tolerated, migrated to inflamed tissues, reduced inflammation, and restrained the evolution of corneal neovascularization and corneal opacity. The expression profile of the corneal epithelial cell markers CK3 and E-cadherin, and the limbal epithelial cell markers CK15 and p63 was lost in the LSCD models, but was partially recovered after hAT-MSC transplantation. For the first time, we demonstrated that hAT-MSCs improve corneal and limbal epithelial phenotypes in animal LSCD models. These results support the potential use of hAT-MSCs as a novel treatment of ocular surface failure due to LSCD. hAT-MSCs represent an available, non-immunogenic source of stem cells that may provide therapeutic benefits in addition to reduce health care expenses. Stem Cells 2017;35:2160-2174.
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Córnea/fisiopatologia , Células-Tronco Mesenquimais/metabolismo , Nicho de Células-Tronco/fisiologia , Animais , Células Cultivadas , Humanos , CoelhosRESUMO
The transplantation of limbal epithelial stem cells (LESCs) cultured in vitro is a great advance in the treatment of patients suffering from LESC deficiency. However, the optimal technique for LESC isolation from a healthy limbal niche has not yet been established. Our aim was to determine which isolation method renders the highest recovery of functional LESCs from the human limbus. To achieve this purpose, we compared limbal primary cultures (LPCs) obtained from explants and cell suspensions on plastic culture plates. Cell morphology was observed by phase contrast and transmission electron microscopy. LESC, corneal epithelial cell, fibroblast, endothelial cell, melanocyte, and dendritic cell markers were analyzed by real time by reverse transcription polymerase chain reaction and/or immunofluorescence. In addition, colony forming efficiency (CFE) and the presence of holoclones, meroclones, and paraclones were studied. We observed that LPC cells obtained from both methods had cuboidal morphology, desmosomes, and prominent intermediate filaments. The expression of LESC markers (K14, K15, ABCG2, p63α) was similar or higher in LPCs established through cell suspensions, except the expression of p63α mRNA, and there were no significant differences in the expression of corneal epithelial markers (K3, K12). Endothelial cell (PECAM), melanocyte (MART-1), and dendritic cell (CD11c) proteins were not detected, while fibroblast-protein (S100A4) was detected in all LPCs. The CFE was significantly higher in LPCs from cell suspensions. Cells from confluent LPCs produced by explants generated only paraclones (100%), while the percentage of paraclones from LPCs established through cell suspensions was 90% and the remaining 10% were meroclones. In conclusion, LPCs established from cell suspensions have a cell population richer in functional LESCs than LPCs obtained from explants. These results suggest that in a clinical situation in which it is possible to choose between either of the isolation techniques from the donor limbal tissue, then the cell suspension is probably the best option as long as the cells are expanded following our culture conditions.
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Técnicas de Cultura de Células/métodos , Epitélio Corneano/ultraestrutura , Limbo da Córnea/ultraestrutura , Células-Tronco/ultraestrutura , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Separação Celular , Células Cultivadas , Epitélio Corneano/metabolismo , Feminino , Humanos , Limbo da Córnea/metabolismo , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Células-Tronco/metabolismo , Doadores de TecidosRESUMO
The aim of this work was to evaluate semi-synthetic biopolymers based on chitosan (CH) and gelatin (G) as potential in vitro carrier substrata for human limbal epithelial cells (hLECs). To that end, human corneal epithelial cells (HCE) were cultured onto different CH-G membranes. None of the polymers were cytotoxic and cell proliferation was higher when CH was functionalized with G. Expression levels of corneal epithelial markers (K3, K12, E-caherin, desmoplakin, and zonula occludens (ZO)-1) were better maintained in HCE cells grown on CH-G 20:80 membranes than other proportions. Consequently, CH-G 20:80 was chosen for the subsequent expansion of hLECs. Cells derived from limbal explants were successfully expanded on CH-G 20:80 membranes using a culture medium lacking components of non-human animal origin. The expression levels found for corneal (K3 and K12) and limbal epithelial stem cells (K15) specific markers were similar to or higher than those found in limbal cells grown onto the control substratum. Our results demonstrate that CH-G 20:80 membranes are suitable for the expansion and maintenance of stem cells derived from the limbal niche. These results strongly support the use of polymers as alternative substrata for the transplantation of cultivated limbal cells onto the ocular surface.
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Biopolímeros/química , Quitosana/química , Epitélio Corneano/citologia , Gelatina/química , Células-Tronco/citologia , Técnicas de Cultura de Células , Proliferação de Células , Sobrevivência Celular , Córnea/patologia , Meios de Cultura/química , Humanos , Teste de Materiais , Polímeros/química , Alicerces TeciduaisRESUMO
INTRODUCTION: The aim of this work is to evaluate the effect of mesenchymal stem cell transplantation (MSCT) and cultivated limbal epithelial transplantation (CLET) therapies on the limbus of patients suffering from limbal stem cell deficiency (LSCD). METHODS: A sub-analysis of a phase I-II randomized, controlled, and double-masked clinical trial was performed to assess the changes in the anatomical structures of the limbus. In vivo confocal microscopy (IVCM) analysis was carried out in LSCD eyes before and 12 months after allogeneic MSCT or CLET. Epithelial phenotype of the central cornea, as well as the presence of transition zones and palisades of Vogt in the limbus, were assessed using Wilcoxon test. RESULTS: Twenty-three LSCD (14 MSCT and nine CLET) eyes were included. The epithelial phenotype of the central cornea improved significantly (p < 0.001) from 15 (eight MSCT, seven CLET) and eight (six MSCT, two CLET) LSCD eyes showing conjunctival and mixed phenotypes, respectively, to eight (five MSCT, three CLET), five (two MSCT, three CLET), and ten (seven MSCT, three CLET) eyes showing conjunctival, mixed, and corneal phenotypes, respectively. Transition areas and palisades of Vogt were observed in at least one quadrant in nine (five MSCT, four CLET) and 16 (nine MSCT, seven CLET), and in four (two MSCT, two CLET) and six (three MSCT, three CLET) LSCD eyes before and after surgery, respectively. Changes in the transition zones and palisades were solely significant (p = 0.046) for the nasal and inferior quadrants, respectively. CONCLUSIONS: MSCT and CLET improved the central corneal epithelial phenotype despite only minor changes in the anatomical structures of the limbus, as detected by IVCM technology. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT01562002.
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BACKGROUND: An effective treatment for acute non-arteritic ischemic optic neuropathy (NA-AION) has not been known or proven yet. Previous studies have suggested a neuroprotective effect of allogeneic bone marrow-derived mesenchymal stem cells. This study aims to report the results of a clinical trial on patients with acute non-arteritic optic neuropathy (NA-AION) treated with an intravitreal injection of allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) (MSV®). METHODS: We conducted a prospective, non-randomized, clinical phase-II study (Eudra CT number 2016-003029-40; ClinicalTrials.gov Registry NCT03173638) that included 5 patients with acute unilateral NA-AION diagnosed within 2 weeks after symptom onset and who received an intravitreal injection of allogeneic BM-MSCs (0.05 ml; cell concentration: 1.5 × 106cells/mL). The patients underwent regular ophthalmological examinations and were followed for one year. RESULTS: In this trial, allogeneic BM-MSCs appeared to be safe as no patients developed signs of acute nor chronic intraocular inflammation or a significant change in intraocular pressure, although an epiretinal membrane was developed in one patient. A retrolental aggregate formed shortly after the injection spontaneously disappeared within a few weeks in another phakic patient, leaving a subcapsular cataract. Visual improvement was noted in 4 patients, and amplitudes of P100 on the visually evoked potentials recordings increased in three patients. The retinal nerve fiber layer and macular ganglion cell layer thicknesses significantly decreased during the follow-up. CONCLUSIONS: Besides the development of an epiretinal membrane in one patient, the intravitreal application of allogeneic BM-MSCs appeared to be intraocularly well tolerated. Consequently, not only NA-AION but also BM-MSCs deserve more clinical investigational resources and a larger randomized multicenter trial that would provide stronger evidence both about safety and the potential therapeutic efficacy of intravitreally injected allogeneic BM-MSCs in acute NA-AION. TRIAL REGISTRATION: Safety Assessment of Intravitreal Mesenchymal Stem Cells for Acute Non-Arteritic Anterior Ischemic Optic Neuropathy (NEUROSTEM). NCT03173638. Registered June 02, 2017 https://clinicaltrials.gov/ct2/show/NCT03173638 .
Assuntos
Membrana Epirretiniana , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Mesenquimais , Doenças do Nervo Óptico , Humanos , Inflamação , Estudos ProspectivosRESUMO
Optic nerve diseases include a wide variety of pathogenic conditions triggering injury or dysfunction of the optic nerves that lead to visual impairment or blindness in one or both eyes. Despite their pathogenic variety, most of them proceed through common mechanisms that allow them to investigate together. Nevertheless, roles of the cells, tissues, genes, growth factors, and proteins, and all underlying pathophysiological mechanisms need to be studied fully for better management of each optic nerve disease. This review presents a collection of information regarding ongoing and completed clinical trials (CT) of advanced therapies that deliver stem cell and gene therapy treatments as drugs to patients with optic nerve diseases as well as successes and failures achieved in treating these patients in the last few years. These drugs seem safe from creating neurotoxicity. It describes outcomes of a bibliographic search for stem cell therapy, gene therapy, and neuroprotection-based CT registered in the International ClinicalTrials.gov, the European EudraCT, and the Spanish REEC database, and related papers published in the PUBMED database by applying different search terminologies. This review overall informs the patients of optic neurodiseases that advanced therapies are progressing successfully in search of effective and safe treatments for them.
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Doenças do Nervo Óptico , Humanos , Nervo Óptico , Doenças do Nervo Óptico/prevenção & controle , Ensaios Clínicos como AssuntoRESUMO
Adipose-derived stem cells are a subtype of mesenchymal stem cell that offers the important advantage of being easily obtained (in an autologous manner) from low invasive procedures, rendering a high number of multipotent stem cells with the potential to differentiate into several cellular lineages, to show immunomodulatory properties, and to promote tissue regeneration by a paracrine action through the secretion of extracellular vesicles containing trophic factors. This secretome is currently being investigated as a potential source for a cell-free based regenerative therapy for human tissues, which would significantly reduce the involved costs, risks and law regulations, allowing for a broader application in real clinical practice. In the current article, we will review the existing preclinical and human clinical evidence regarding the use of such adipose-derived mesenchymal stem cells for the regeneration of the three main layers of the human cornea: the epithelium (derived from the surface ectoderm), the stroma (derived from the neural crest mesenchyme), and the endothelium (derived from the neural crest cells).
Assuntos
Células-Tronco Mesenquimais , Tecido Adiposo , Córnea , Humanos , Células-Tronco Multipotentes , Células-TroncoRESUMO
PURPOSE: Corneal epithelium is maintained by a population of stem cells (SCs) that have not been identified by specific molecular markers. The objective of this study was to find new putative markers for these SCs and to identify associated molecular pathways. METHODS: Real time PCR (rt-PCR) was performed in 24 human limbal and central corneal epithelial samples to evaluate the gene expression profile of known corneal epithelial SC-associated markers. A pool of those samples was further analyzed by a rt-PCR array (RT²-PCR-A) for 84 genes related to the identification, growth, maintenance, and differentiation of SCs. RESULTS: Cells from the corneal epithelium SC niche showed significant expression of ATP-binding cassette sub-family G member 2 (ABCG2) and cytokeratin (KRT)15, KRT14, and KRT5 genes. RT²-PCR-A results indicated an increased or decreased expression in 21 and 24 genes, respectively, in cells from the corneal SC niche compared to cells from the central corneal epithelium. Functional analysis by proprietary software found 4 different associated pathways and a novel network with the highest upregulated genes in the corneal SC niche. This led to the identification of specific molecules, chemokine (C-X-C motif) ligand 12 (CXCL12), islet-1 transcription factor LIM/homeodomain (ISL1), collagen-type II alpha 1 (COL2A), neural cell adhesion molecule 1 (NCAM1), aggrecan (ACAN), forkhead box A2 (FOXA2), Gap junction protein beta 1/connexin 32 (GJB1/Cnx32), and Msh homeobox 1 (MSX1), that could be used to recognize putative corneal epithelial SCs grown in culture and intended for transplantation. Other molecules, NCAM1 and GJB1/Cnx32, potentially could be used to positively purify them, and Par-6 partitioning defective 6 homolog alpha (PARD6A) to negatively purify them. CONCLUSIONS: Knowledge of these gene and molecular pathways has provided a better understanding of the signaling molecular pathways associated with progenitor-rich limbal epithelium. This knowledge potentially could give support to the design and development of innovative therapies with the potential to reverse corneal blindness arising from ocular surface failure.
Assuntos
Biomarcadores/metabolismo , Epitélio Corneano/metabolismo , Expressão Gênica , Redes Reguladoras de Genes , Limbo da Córnea/metabolismo , Transdução de Sinais/genética , Células-Tronco/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Autopsia , Diferenciação Celular/genética , Epitélio Corneano/citologia , Perfilação da Expressão Gênica , Humanos , Queratina-14/genética , Queratina-14/metabolismo , Queratina-15/genética , Queratina-15/metabolismo , Queratina-5/genética , Queratina-5/metabolismo , Limbo da Córnea/citologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Células-Tronco/citologiaRESUMO
Corneal failure is a highly prevalent cause of blindness. One special cause of corneal failure occurs due to malfunction or destruction of the limbal stem cell niche, upon which the superficial cornea depends for homeostatic maintenance and wound healing. Failure of the limbal niche is referred to as limbal stem cell deficiency. As the corneal epithelial stem cell niche is easily accessible, limbal stem cell-based therapy and regenerative medicine applied to the ocular surface are among the most highly advanced forms of this novel approach to disease therapy. However, the challenges are still great, including the development of cell-based products and understanding how they work in the patient's eye. Advances are being made at the molecular, cellular, and tissue levels to alter disease processes and to reduce or eliminate blindness. Efforts must be coordinated from the most basic research to the most clinically oriented projects so that cell-based therapies can become an integrated part of the therapeutic armamentarium to fight corneal blindness. We undoubtedly are progressing along the right path because cell-based therapy for eye diseases is one of the most successful examples of global regenerative medicine.
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Mesenchymal stem cells (MSCs) have unique and beneficial properties and are currently used to treat a broad variety of diseases. These properties include the potential for differentiation into other cell types, secretion of different trophic factors that promote a regenerative microenvironment, anti-inflammatory actions, selective migration to damaged tissues, and non-immunogenicity. MSCs are effective for the treatment of ocular surface diseases such as dry eye, corneal burns, and limbal stem cell deficiency (LSCD), both in experimental models and in humans. LSCD is a pathological condition in which damage occurs to the limbal epithelial stem cells, or their niche, that are responsible for the continuous regeneration of the corneal epithelium. If LSCD is extensive and/or severe, it usually causes corneal epithelial defects, ulceration, and conjunctival overgrowth of the cornea. These changes can result in neovascularization and corneal opacity, severe inflammation, pain, and visual loss. The effectiveness of MSCs to reduce corneal opacity, neovascularization, and inflammation has been widely studied in different experimental models of LSCD and in some clinical trials; however, the methodological disparity used in the different studies makes it hard to compare outcomes among them. In this regard, the MSC route of administration used to treat LSCD and other ocular surface diseases is an important factor. It should be efficient, minimally invasive, and safe. So far, intravenous and intraperitoneal injections, topical administration, and MSC transplantation using carrier substrata like amniotic membrane (AM), fibrin, or synthetic biopolymers have been the most commonly used administration routes in experimental models. However, systemic administration carries the risk of potential side effects and transplantation requires surgical procedures that could complicate the process. Alternatively, subconjunctival injection is a minimally invasive and straightforward technique frequently used in ophthalmology. It enables performance of local treatments using high cell doses. In this review, we provide an overview of the current status of MSC administration by subconjunctival injection, analyzing the convenience, safety, and efficacy for treatment of corneal failure due to LSCD in different experimental models. We also provide a summary of the clinical trials that have been completed, are in progress, or being planned.
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Doenças da Córnea , Epitélio Corneano , Limbo da Córnea , Células-Tronco Mesenquimais , Córnea , Doenças da Córnea/terapia , Humanos , Transplante de Células-Tronco , Células-TroncoRESUMO
Advanced therapy medicinal products (ATMPs) are a group of innovative and complex biological products for human use that comprises somatic cell therapy medicinal products, tissue engineered products, gene therapy medicinal products, and the so-called combined ATMPs that consist of one of the previous three categories combined with one or more medical devices. During the last few years, the development of ATMPs for the treatment of eye diseases has become a fast-growing field as it offers the potential to find novel therapeutic approaches for treating pathologies that today have no cure or are just subjected to symptomatic treatments. Therefore, it is important for all professionals working in this field to be familiar with the regulatory principles associated with these types of innovative products. In this review, we outline the legal framework that regulates the development of ATMPs in the European Union and other international jurisdictions, and the criteria that each type of ATMP must meet to be classified as such. To illustrate each legal definition, ATMPs that have already completed the research and development stages and that are currently used for the treatment of eye diseases are presented as examples.
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Cultured limbal epithelial stem cell transplantation is a clinical procedure used to regenerate the corneal epithelium in patients with limbal stem cell deficiency. The protocols used to expand limbal epithelial cells in vitro need to be optimized, since the scarcity of human ocular tissue donors is limiting the potential use of this procedure. Here, we describe a method to consecutively expand a single human limbal explant. With this method it is possible to obtain up to three limbal epithelial primary cultures from the same explant, thus increasing the efficiency of the in vitro cell culture.
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Técnicas de Cultura de Células/métodos , Doenças da Córnea/terapia , Epitélio Corneano/crescimento & desenvolvimento , Limbo da Córnea/crescimento & desenvolvimento , Doenças da Córnea/patologia , Epitélio Corneano/citologia , Epitélio Corneano/transplante , Humanos , Limbo da Córnea/citologia , Células-Tronco/citologiaRESUMO
Limbal epithelial stem cells (LESCs) are responsible for the renewal of corneal epithelium. Cultivated limbal epithelial transplantation is the current treatment of choice for restoring the loss or dysfunction of LESCs. To perform this procedure, a substratum is necessary for in vitro culturing of limbal epithelial cells and their subsequent transplantation onto the ocular surface. In this work, we evaluated poly-L/DL-lactic acid 70:30 (PLA) films functionalized with type IV collagen (col IV) as potential in vitro carrier substrata for LESCs. We first demonstrated that PLA-col IV films were biocompatible and suitable for the proliferation of human corneal epithelial cells. Subsequently, limbal epithelial cell suspensions, isolated from human limbal rings, were cultivated using culture medium that did not contain animal components. The cells adhered significantly faster to PLA-col IV films than to tissue culture plastic (TCP). The mRNA expression levels for the LESC specific markers, K15, P63α and ABCG2 were similar or greater (significantly in the case of K15) in limbal epithelial cells cultured on PLA-col IV films than limbal epithelial cells cultured on TCP. The percentage of cells expressing the corneal (K3, K12) and the LESC (P63α, ABCG2) specific markers was similar for both substrata. These results suggest that the PLA-col IV films promoted LESC attachment and helped to maintain their undifferentiated stem cell phenotype. Consequently, these substrata offer an alternative for the transplantation of limbal cells onto the ocular surface.
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Colágeno Tipo IV/química , Células Epiteliais/citologia , Epitélio Corneano/citologia , Poliésteres/química , Células-Tronco/citologia , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , HumanosRESUMO
Ocular stem cell transplantation derived from either autologous or allogeneic donor corneoscleral junction is a functional cell therapy to manage extensive and/or severe limbal stem cell deficiencies that lead to corneal epithelial failure. Mesenchymal stem cells have been properly tested in animal models of this ophthalmic pathology, but never in human eyes despite their potential advantages. We conducted a 6- to 12-month proof-of-concept, randomized, and double-masked pilot trial to test whether allogeneic bone marrow-derived mesenchymal stem cell transplantation (MSCT], nâ¯=â¯17) was as safe and as equally efficient as allogeneic cultivated limbal epithelial transplantation (CLET), (nâ¯=â¯11) to improve corneal epithelial damage due to limbal stem cell deficiency. Primary endpoints demanded combination of symptoms, signs, and the objective improvement of the epithelial phenotype in central cornea by in vivo confocal microscopy. This proof-of-concept trial showed that MSCT was as safe and efficacious as CLET. Global success at 6-12 months was 72.7%-77.8% for CLET cases and 76.5%-85.7% for MSCT cases (not significant differences). Central corneal epithelial phenotype improved in 71.4% and 66.7% of MSCT and CLET cases, respectively at 12 months (P = 1.000). There were no adverse events related to cell products. This trial suggests first evidence that MSCT facilitated improvement of a diseased corneal epithelium due to lack of its stem cells as efficiently as CLET. Consequently, not only CLET but also MSCT deserves more preclinical investigational resources before the favorable results of this proof-of-concept trial could be transformed into the larger numbers of the multicenter trials that would provide stronger evidence. (ClinicalTrials.gov number, NCT01562002.).
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Epitélio Corneano/citologia , Células-Tronco Mesenquimais/citologia , Adulto , Idoso , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudo de Prova de Conceito , Transplante de Células-TroncoRESUMO
PURPOSE: Transplantation of in vitro cultured limbal epithelial stem cells (LESCs) is a treatment widely used for LESC deficiency. However, the number of limbal tissue donors is limited, and protocols for LESC cultivation often include compounds and/or feeder layers that can induce side effects and/or increase the cost of the culture procedure. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same biopsy using a culture medium in which several potentially harmful compounds were replaced at the same time by biosafe supplements, allowing the LESC cultivation without feeder layers. MATERIALS AND METHODS: We established feeder layer-free LPCs with three culture media: (1) a modified supplemental hormonal epithelial medium, containing potential harmful components (cholera toxin, dimethylsulfoxide, and fetal bovine serum [FBS]), (2) IOBA-FBS, a medium with FBS but with no other harmful supplements, and (3) IOBA-HS, similar to IOBA-FBS but with human serum instead of FBS. Additionally, the same limbal explant was consecutively cultured with IOBA-HS producing three cultures. LPCs were characterized by real-time reverse transcription polymerase chain reaction and/or immunofluorescence. RESULTS: LPCs cultured with the three media under feeder layer-free conditions showed cuboidal cells and no significant differences in the percentage of positive cells for limbal (ABCG2, p63, and K14) and corneal (K3, K12) proteins. Except for ABCG2, the relative mRNA expression of the LESC markers was significantly higher when IOBA-FBS or IOBA-HS was used. LPC1 showed characteristics similar to LPC0, while LPC2 cell morphology became elongated and the expression of some LESC markers was diminished. CONCLUSION: IOBA-HS enables the culturing of up to two biosafe homologous LPCs from one limbal tissue under feeder layer-free conditions. The routine use of this culture medium could improve both the biosafety and the number of available LPCs for potential clinical transplantation, as well as decrease the expense of the culture procedure.
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Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Córnea/citologia , Meios de Cultivo Condicionados/farmacologia , Regulação da Expressão Gênica , Limbo da Córnea/citologia , Proteínas de Neoplasias/genética , Proteína A4 de Ligação a Cálcio da Família S100/genética , Transplante de Células-Tronco/métodos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/biossíntese , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Contagem de Células , Técnicas de Cultura de Células/métodos , Córnea/efeitos dos fármacos , Córnea/metabolismo , Perda de Células Endoteliais da Córnea/genética , Perda de Células Endoteliais da Córnea/patologia , Perda de Células Endoteliais da Córnea/terapia , Estudos de Viabilidade , Células Alimentadoras , Humanos , Microscopia de Fluorescência , Proteínas de Neoplasias/biossíntese , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína A4 de Ligação a Cálcio da Família S100/biossíntese , Doadores de TecidosRESUMO
PURPOSE: Corneal epithelium is maintained by limbal epithelial stem cells (LESCs), the loss of which can be catastrophic for corneal transparency. Effective therapies include the transplantation of cultivated LESCs, requiring optimization of in vitro cultivation protocols. Unfortunately, optimization studies are hampered by the limited number of ocular tissue donors. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same 1-2 mm(2) limbal explant (LE). METHODS: LEs were plated and maintained until outgrowth surrounded each, being removed at this point. LPCs were allowed to reach confluence (LPC0). The same removed LE was plated again, following the same procedure, obtaining LPC1. This procedure was repeated as often as possible up to six times. LPCs from each passage were analysed by real time reverse transcription-polymerase chain reaction and immunofluorescence-microscopy. RESULTS: LPCs from LPC0 to LPC2 presented a heterogeneous cell population, with cells positive for LESC markers K14, K15, ABCG2 and p63, differentiated corneal epithelial cell-specific markers K3 and K12, and for the fibroblast marker S100A4. These cells had an epithelial-like morphology. In LPC3-LPC4, elongated cell morphology appeared, and the presence of LESC markers decreased, while the presence of differentiated corneal epithelial-cell and fibroblast markers increased. CONCLUSION: One LE can be successfully cultivated up to three consecutive times while maintaining the LESC phenotype in the LPC cells. This protocol provides several homologous LPCs for basic research. Additionally, by using a cell-carrier, the resulting LPCs could serve reservoirs for potential autologous expanded LESC transplantations and/or for making correlations between laboratory and clinical outcomes.