RESUMO
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.
Assuntos
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
Human limbal epithelial stem cells (LESCs) are essential for the maintenance of the corneal epithelium of the ocular surface. LESCs are located within limbal crypts between the palisades of Vogt in the limbus; the interface between the peripheral cornea and conjunctiva. The limbal crypts have been proposed as a LESC niche owing to their support of epithelial cells, which can form holoclone colonies in vitro. Closely associated with the limbal crypts is a concentrated population of melanocytes. The anatomical location and close proximity to putative LESC suggests that melanocytes might play a role in maintenance of these stem cells in the niche. The aim of this study was to assess the ability of human limbal melanocytes (hLM) to support the expansion of human limbal epithelial cells (LECs) in vitro as an indicator of functional cell-cell interaction. After observing that hLM co-localize with clusters of compact epithelial cells in the native limbal crypts, hLM were isolated from crypt-rich cadaveric limbal biopsies and used as feeders for the culture of LECs. Interestingly, LECs grown on mitotically active hLM were able to generate large epithelial colonies that contained small and compact cells with morphological stem cell characteristics. Immunocytochemistry revealed that LECs expanded on hLM were positive for the expression of the putative stem cell markers CK15, Bmi-1 and p63α and negative for the marker of terminal cell differentiation CK3. LECs and hLM were finally co-cultured on RAFT (real architecture for 3D tissue) collagen tissue equivalents. In 3D co-cultures, hLM promoted multi-layering of the epithelial sheet in which basal cells were maintained in an undifferentiated state. Taken together, these observations suggest melanocytes could play an important role in the maintenance of LESCs in the native human limbal stem cell niche.
Assuntos
Epitélio Corneano/citologia , Limbo da Córnea/citologia , Melanócitos/fisiologia , Nicho de Células-Tronco/fisiologia , Células-Tronco/citologia , Biomarcadores , Comunicação Celular/fisiologia , Contagem de Células , Técnicas de Cocultura , Epitélio Corneano/metabolismo , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Limbo da Córnea/metabolismo , Microscopia Eletrônica de Transmissão , Células-Tronco/metabolismo , Doadores de Tecidos , Engenharia TecidualRESUMO
Background: The pathophysiological processes underlying the phenotypic spectrum of severe forms of epidermolysis bullosa (EB) are complex and poorly understood. Objective: To use burden mapping to explore relationships between primary pathomechanisms and secondary clinical manifestations in severe forms of EB (junctional and dystrophic EB [JEB/DEB]) and highlight strengths and weaknesses in evidence regarding the contribution of different pathways. Methods: Literature searches were performed to identify evidence regarding the pathophysiological and clinical aspects of JEB/DEB. Identified publications and clinical experience were used to construct burden maps to visually communicate plausible connections and their relative importance by subtype. Results: Our findings suggest that most of the clinical consequences of JEB/DEB may result from an abnormal state and/or faulty skin remodeling driven by a vicious cycle of delayed wound healing, predominantly mediated through inflammation. The quantity and quality of evidence varies by individual manifestations and disease subtype. Limitations: The burden maps are provisional hypotheses requiring further validation and are limited by the published evidence base and subjectivity in clinical opinion. Conclusions: Delayed wound healing appears to be a key driver of the burden of JEB/DEB. Further studies are warranted to understand the role of inflammatory mediators and accelerated wound healing in patient management.
RESUMO
The objective of this study was to determine whether corneal stromal cells (CSCs) from the limbal and central corneal stroma in dogs have multipotent mesenchymal stem/stromal cell (MSC) properties, and whether this cell population can be differentiated into keratocyte-like cells (KDCs). Normal, donated, mesocephalic dog corneas were used to isolate CSC in vitro. Immunohistochemistry demonstrated a distinct population of CD90 expressing cells in the anterior stroma throughout the limbal and central cornea. CSC could be cultured from both the limbal and central cornea and the culture kinetics showed a progenitor cell profile. The CSC expressed stem cell markers CD90, CD73, CD105, N-cadherin, and Pax6, while CD34 was negative. Limbal and central CSC differentiated into osteoblasts, chondrocytes, and adipocytes confirming their multipotency. Coculturing allogeneic peripheral blood mononuclear cells (PBMCs) with limbal CSCs did not affect baseline PBMC proliferation indicating a degree of innate immune privilege. Limbal CSC could be differentiated into KDCs that expressed Keratocan, Lumican, and ALDH1A3 and downregulated Pax6 and N-cadherin. In conclusion, canine CSCs have multipotent MSC properties similarly described in humans and could serve as a source of cells for cell therapy and studying corneal diseases.
Assuntos
Córnea/citologia , Células-Tronco Mesenquimais/citologia , Células Estromais/citologia , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Córnea/metabolismo , Cães , Feminino , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Células Estromais/metabolismoRESUMO
We report the repigmentation at the limbus in patients who underwent simple limbal epithelial transplant (SLET) for uniocular chemical injury. The first case is of an 8-year-old child who presented with grade 4 chemical injury, with limbal stem cell deficiency (LSCD) corresponding to 6 o' clock till 11 o' clock. He was managed by amniotic membrane graft in the acute stage and SLET after 6 months of the initial injury. The second case is of a 15-year-old female who presented with lime injury, which had resulted in 6 o' clock of limbal involvement (10 o' clock till 4 o' clock). The patient was managed on similar lines with amniotic membrane graft (AMG) in the acute phase and SLET after 6 months of injury. The ocular surface was stable in both the patients post-SLET. The effected limbus showed pigmentation at 8 months of follow-up which eventually became distinct and remained stable. We speculate that the pigmentation at limbus could be attributed to proliferation and movement of melanocytes from limbal biopsy in SLET. These may be capable of supporting the proliferation of limbal epithelial cells and modulation of corneal wound healing.
Assuntos
Queimaduras Químicas , Doenças da Córnea , Epitélio Corneano , Queimaduras Oculares , Limbo da Córnea , Adolescente , Queimaduras Químicas/diagnóstico , Queimaduras Químicas/cirurgia , Criança , Doenças da Córnea/diagnóstico , Doenças da Córnea/cirurgia , Células Epiteliais , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/diagnóstico , Queimaduras Oculares/cirurgia , Feminino , Humanos , Limbo da Córnea/cirurgia , Masculino , Pigmentação , Transplante de Células-Tronco/efeitos adversos , Transplante AutólogoRESUMO
Cultured limbal and oral epithelial cells have been successfully used to treat patients with limbal stem cell deficiency (LSCD). The most common culture method for these cell therapies utilizes amniotic membrane as a cell support and/or murine 3T3s as feeder fibroblasts. The aim of this study is to refine the production of autologous oral mucosal cell therapy for the treatment of LSCD. Real architecture for 3D tissue (RAFT) is used as an alternative cell culture support. In addition, oral mucosal cells (epithelial and fibroblast) are used as autologous alternatives to donor human limbal epithelial cells (HLE) and murine 3T3s. The following tissue equivalents are produced and characterized: first, for patients with bilateral LSCD, an oral mucosa tissue equivalent consisting of human oral mucosal epithelial cells on RAFT supported by human oral mucosal fibroblasts (HOMF). Second, for patients with unilateral LSCD, HLE on RAFT supported by HOMF. For both tissue equivalent types, features of the cornea are observed including a multi-layered epithelium with small cells with a stem cell like phenotype in the basal layer and squamous cells in the top layers, and p63α and PAX6 expression. These tissue equivalents may therefore be useful in the treatment of LSCD.
Assuntos
Córnea/metabolismo , Lesões da Córnea , Células Epiteliais , Fibroblastos , Mucosa Bucal , Células 3T3 , Animais , Lesões da Córnea/metabolismo , Lesões da Córnea/terapia , Células Epiteliais/metabolismo , Células Epiteliais/transplante , Fibroblastos/metabolismo , Fibroblastos/transplante , Humanos , Camundongos , Mucosa Bucal/metabolismo , Mucosa Bucal/transplante , Células-TroncoRESUMO
Epithelial stem cells of the ocular surface are essential for the maintenance of corneal transparency and therefore for vision. Human corneal/limbal epithelial stem cells (LESCs) are believed to reside in the limbus, the interface between the peripheral cornea and neighboring conjunctiva. A specific anatomical microenvironment called the niche regulates the proliferative and differentiation potential of LESCs and their daughter cells. This review covers multiple structural and functional aspects of the human limbal epithelial stem cell niche, including: anatomical features of the niche, composition of the local extracellular matrix, soluble factors and signaling pathways, interactions with surrounding stromal niche cells and melanocytes.
Assuntos
Nicho de Células-Tronco , Comunicação Celular , Células Epiteliais , Humanos , Limbo da Córnea , Células-TroncoRESUMO
Human limbal epithelial cells (HLE) and corneal stromal stem cells (CSSC) reside in close proximity in vivo in the corneal limbal stem cell niche. However, HLE are typically cultured in vitro without supporting niche cells. Here, we re-create the cell-cell juxtaposition of the native environment in vitro, to provide a tool for investigation of epithelial-stromal cell interactions and to optimize HLE culture conditions for potential therapeutic application. RAFT (Real Architecture For 3D Tissue) tissue equivalents (TEs) were used as a 3-dimensional substrate for co-culturing HLE and CSSC. Our results demonstrate that a monolayer of HLE that maintained expression of p63α, ABCB5, CK8 and CK15 (HLE markers), formed on the surface of RAFT TEs within 13 days of culture. CSSC remained in close proximity to HLE and maintained expression of mesenchymal stem cell markers. This simple technique has a short preparation time of only 15 days with the onset of HLE layering and differentiation observed. Furthermore, co-cultivation of HLE with another niche cell type (CSSC) directly on RAFT TEs, eliminates the requirement for animal-derived feeder cells. RAFT TEs may be useful for future therapeutic delivery of multiple cell types to restore the limbal niche following ocular surface injury or disease.
Assuntos
Células Epiteliais/citologia , Células-Tronco Mesenquimais/citologia , 5'-Nucleotidase/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Técnicas de Cocultura , Células Epiteliais/metabolismo , Epitélio Corneano/citologia , Humanos , Imuno-Histoquímica , Queratina-15/metabolismo , Queratina-8/metabolismo , Limbo da Córnea/citologia , Células-Tronco Mesenquimais/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Antígenos Thy-1/metabolismoRESUMO
The limbal epithelial stem cell niche provides a unique, physically protective environment in which limbal epithelial stem cells reside in close proximity with accessory cell types and their secreted factors. The use of advanced imaging techniques is described to visualize the niche in three dimensions in native human corneal tissue. In addition, a protocol is provided for the isolation and culture of three different cell types, including human limbal epithelial stem cells from the limbal niche of human donor tissue. Finally, the process of incorporating these cells within plastic compressed collagen constructs to form a tissue-engineered corneal limbus is described and how immunohistochemical techniques may be applied to characterize cell phenotype therein.
Assuntos
Epitélio Corneano/citologia , Imuno-Histoquímica/métodos , Limbo da Córnea/citologia , Nicho de Células-Tronco , Células-Tronco/citologia , Engenharia Tecidual/métodos , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Fibroblastos/citologia , Humanos , Microscopia Confocal/métodos , Microscopia Eletrônica de Varredura/métodosRESUMO
Limbal epithelial stem cells (LESCs) are essential to maintain the transparent ocular surface required for vision. Despite great advances in our understanding of ocular stem cell biology over the last decade, the exact location of the LESC niche remains unclear. In the present study we have used in vitro clonal analysis to confirm that limbal crypts provide a niche for the resident LESCs. We have used high-resolution imaging of the basal epithelial layer at the limbus to identify cells with a morphology consistent with stem cells that were only present within the basal layer of the limbal crypts. These cells are proximal to limbal stromal cells suggesting direct cell-to-cell interaction. Serial block-face scanning electron microscopy (SBFSEM) confirmed that the putative LESCs are indeed in direct contact with cells in the underlying stroma, a contact that is facilitated by focal basement membrane interruptions. Limbal mesenchymal cells previously identified in the human limbus collocate in the crypt-rich limbal stromal area in the vicinity of LESCs and may be involved in the cell-to-cell contact revealed by SBFSEM. We also observed a high population of melanocytes within the basal layer of the limbal crypts. From these observations we present a three dimensional reconstruction of the LESC niche in which the stem cell is closely associated and maintained by both dendritic pigmented limbal melanocytes and elongated limbal stromal cells.
Assuntos
Comunicação Celular , Células Epiteliais/citologia , Epitélio Corneano/citologia , Limbo da Córnea/citologia , Limbo da Córnea/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Biomarcadores/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Melanócitos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Nicho de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/ultraestrutura , Células Estromais/ultraestruturaRESUMO
Limbal epithelial stem cells are responsible for the maintenance of the human corneal epithelium and these cells reside in a specialised stem cell niche. They are located at the base of limbal crypts, in a physically protected microenvironment in close proximity to a variety of neighbouring niche cells. Design and recreation of elements of various stem cell niches have allowed researchers to simplify aspects of these complex microenvironments for further study in vitro. We have developed a method to rapidly and reproducibly create bioengineered limbal crypts (BLCs) in a collagen construct using a simple one-step method. Liquid is removed from collagen hydrogels using hydrophilic porous absorbers (HPAs) that have custom moulded micro-ridges on the base. The resulting topography on the surface of the thin collagen constructs resembles the dimensions of the stromal crypts of the human limbus. Human limbal epithelial cells seeded onto the surface of the constructs populate these BLCs and form numerous layers with a high proportion of the cells lining the crypts expressing putative stem cell marker, p63α. The HPAs are produced using a moulding process that is flexible and can be adapted depending on the requirements of the end user. Creation of defined topographical features using this process could be applicable to numerous tissue-engineering applications where varied 3-dimensional niche architectures are required.