Melanocytes as emerging key players in niche regulation of limbal epithelial stem cells.
TL;DR: Limbal melanocytes (LMel) represent essential components of the corneal epithelial stem cell niche and are known to protect limbal stem/progenitor cells (LEPCs) from UV damage by transfer of melanosomes.
Abstract: Purpose Limbal melanocytes (LMel) represent essential components of the corneal epithelial stem cell niche and are known to protect limbal epithelial stem/progenitor cells (LEPCs) from UV damage by transfer of melanosomes. Here, we explored additional functional roles for LMel in niche homeostasis, immune regulation and angiostasis. Methods Human corneoscleral tissues were morphologically analyzed in normal, inflammatory and wound healing conditions. The effects of LMel on LEPCs were analyzed in direct and indirect co-culture models using electron microscopy, immunocytochemistry, qRT-PCR, Western blotting and functional assays; limbal mesenchymal stromal cells and murine embryonic 3T3 fibroblasts served as controls. The immunophenotype of LMel was assessed by flow cytometry before and after interferon-γ stimulation, and their immunomodulatory properties were analyzed by mixed lymphocytes reaction, monocyte adhesion assays and cytometric bead arrays. Their angiostatic effects on human umbilical cord endothelial cells (HUVECs) were evaluated by proliferation, migration, and tube formation assays. Results LMel and LEPCs formed structural units in the human limbal stem cell niche in situ, which could be functionally replicated, including melanosome transfer, by co-cultivation in vitro. LMel supported LEPCs during clonal expansion and during epithelial wound healing by stimulating proliferation and migration, and suppressed their differentiation through direct contact and paracrine effects. Under inflammatory conditions, LMel were increased in numbers and upregulated expression of ICAM-1 and MHC II molecules (HLA-DR), but lacked expression of HLA-G, -DP, -DQ and costimulatory molecules CD80 and CD86. They were also found to be potent suppressors of alloreactive T- cell proliferation and cytokine secretion, which largely depended on direct cell-cell interaction. Moreover, the LMel secretome exerted angiostatic activity by inhibiting vascular endothelial cell proliferation and capillary network formation. Conclusion These findings suggest that LMel are not only professional melanin-producing cells, but exert various non-canonical functions in limbal niche homeostasis by regulating LEPC maintenance, immune responses, and angiostasis. Their potent regulatory, immunomodulatory and anti-angiogenic properties may have important implications for future regenerative cell therapies.
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22 Sep 2021
TL;DR: In this article, a review outlines the current understanding of the limbal niche, its pathology and the emerging approaches targeted at restoring the limbo-nomenclature and accelerating corneal regeneration.
Abstract: The protective function and transparency provided by the corneal epithelium are dependent on and maintained by the regenerative capacity of limbal epithelial stem cells (LESCs). These LESCs are supported by the limbal niche, a specialized microenvironment consisting of cellular and non-cellular components. Disruption of the limbal niche, primarily from injuries or inflammatory processes, can negatively impact the regenerative ability of LESCs. Limbal stem cell deficiency (LSCD) directly hampers the regenerative ability of the corneal epithelium and allows the conjunctival epithelium to invade the cornea, which results in severe visual impairment. Treatment involves restoring the LESC population and functionality; however, few clinically practiced therapies currently exist. This review outlines the current understanding of the limbal niche, its pathology and the emerging approaches targeted at restoring the limbal niche. Most emerging approaches are in developmental phases but show promise for treating LSCD and accelerating corneal regeneration. Specifically, we examine cell-based therapies, bio-active extracellular matrices and soluble factor therapies in considerable depth.
12 citations
TL;DR: In this paper, a decellularized human limbus (DHL) was used as a biomimetic scaffold for the transplantation of expanded limbal epithelial progenitor cells (LEPCs) on amniotic membrane or fibrin gel.
Abstract: The transplantation of ex vivo expanded limbal epithelial progenitor cells (LEPCs) on amniotic membrane or fibrin gel is an established therapeutic strategy to regenerate the damaged corneal surface in patients with limbal stem cell deficiency (LSCD), but the long-term success rate is restricted. A scaffold with niche-specific structure and extracellular matrix (ECM) composition might have the advantage to improve long-term clinical outcomes, in particular for patients with severe damage or complete loss of the limbal niche tissue structure. Therefore, we evaluated the decellularized human limbus (DHL) as a biomimetic scaffold for the transplantation of LEPCs. Corneoscleral tissue was decellularized by sodium deoxycholate and deoxyribonuclease I in the presence or absence of dextran. We evaluated the efficiency of decellularization and its effects on the ultrastructure and ECM composition of the human corneal limbus. The recellularization of these scaffolds was studied by plating cultured LEPCs and limbal melanocytes (LMs) or by allowing cells to migrate from the host tissue following a lamellar transplantation ex vivo. Our decellularization protocol rapidly and effectively removed cellular and nuclear material while preserving the native ECM composition. In vitro recellularization by LEPCs and LMs demonstrated the good biocompatibility of the DHL and intrastromal invasion of LEPCs. Ex vivo transplantation of DHL revealed complete epithelialization as well as melanocytic and stromal repopulation from the host tissue. Thus, the generated DHL scaffold could be a promising biological material as a carrier for the transplantation of LEPCs to treat LSCD.
9 citations
TL;DR: In this article , the authors examined the interactions between the ocular surface and its local draining lymphoid compartment, by encompassing the corneal epithelium and myeloid cells, conjunctival goblet cells, and regulatory T cells.
8 citations
TL;DR: A highly efficient isolation of pure LM, LMSC, and LEPC populations from a single preparation may allow for direct transcriptomic and proteomic profiling as well as functional studies on native unpassaged LNC, which can be considered as proper equivalents of LNC in vivo.
Abstract: The fate decision of limbal epithelial progenitor cells (LEPC) at the human corneal limbus is determined by the surrounding microenvironment with limbal niche cells (LNC) as one of its essential components. Research on freshly isolated LNC which mainly include limbal mesenchymal stromal cells (LMSC) and limbal melanocytes (LM) has been hampered by a lack of efficient protocols to isolate and purify these cells. We devised a protocol for rapid retrieval of pure LMSC, LM and LEPC populations by collagenase digestion of limbal tissue and subsequent fluorescence-activated cell sorting (FACS) using antibodies against CD90 and CD117. The sorted cells were characterized by immunophenotyping and functional assays. The effects of LMSC and LM on LEPC were studied in 3D co-cultures and LEPC differentiation status was assessed by immunohistochemistry. Enzymatic digestion and flow sorting yielded pure populations of LMSC (CD117−CD90+), LM (CD117+CD90−), and LEPC (CD117−CD90−). The LMSC exhibited self-renewal capacity (55.0 ± 4.6 population doublings), expressed mesenchymal stem cell markers (CD73, CD90, CD105, and CD44), and transdifferentiated to adipocytes, osteocytes, or chondrocytes. The LM exhibited self-renewal capacity and sustained melanin production. The sorted LEPC expressed epithelial progenitor markers (CK14, CK19, and CK15) and showed a colony-forming ability. Co-cultivation of LMSC and LM with LEPC resulted in a 4–5-layered stratified epithelium and supported the preservation of a LEPC phenotype, as reflected by increased p63+ and Ki67+ cells and decreased CK12+ cells compared with LEPC monocultures. A highly efficient isolation of pure LM, LMSC, and LEPC populations from a single preparation may allow for direct transcriptomic and proteomic profiling as well as functional studies on native unpassaged LNC, which can be considered as proper equivalents of LNC in vivo. The developed biomimetic 3D co-culture method could provide an experimental model for investigating the functional role of LNC in the limbal stem cell niche.
8 citations
TL;DR: It is demonstrated that P-cad is expressed by epithelial progenitor cells as well as melanocytes in the human limbal epithelial stem cell niche, and this findings led to further improvement of cell enrichment protocols to enhance the yield of LEPC.
Abstract: Interactions between limbal epithelial progenitor cells (LEPC) and surrounding niche cells, which include limbal mesenchymal stromal cells (LMSC) and melanocytes (LM), are essential for the maintenance of the limbal stem cell niche required for a transparent corneal surface. P-cadherin (P-cad) is a critical stem cell niche adhesion molecule at various epithelial stem cell niches; however, conflicting observations were reported on the presence of P-cad in the limbal region. To explore this issue, we assessed the location and phenotype of P-cad+ cells by confocal microscopy of human corneoscleral tissue. In subsequent fluorescence-activated cell sorting (FACS) experiments, we used antibodies against P-cad along with CD90 and CD117 for the enrichment of LEPC, LMSC and LM, respectively. The sorted cells were characterized by immunophenotyping and the repopulation of decellularized limbal scaffolds was evaluated. Our findings demonstrate that P-cad is expressed by epithelial progenitor cells as well as melanocytes in the human limbal epithelial stem cell niche. The modified flow sorting addressing P-cad as well as CD90 and CD117 yielded enriched LEPC (CD90−CD117−P-cad+) and pure populations of LMSC (CD90+CD117−P-cad−) and LM (CD90−CD117+P-cad+). The enriched LEPC showed the expression of epithelial progenitor markers and better colony-forming ability than their P-cad− counterparts. The cultured LEPC and LM exhibited P-cad expression at intercellular junctions and successfully repopulated decellularized limbal scaffolds. These data suggest that P-cad is a critical cell–cell adhesion molecule, connecting LEPC and LM, which may play an important role in the long-term maintenance of LEPC at the limbal stem cell niche; moreover, these findings led to further improvement of cell enrichment protocols to enhance the yield of LEPC.
5 citations
References
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TL;DR: The aim of this review is to critically discuss the immunogenicity and immunomodulatory properties of MSCs, both in vitro and in vivo, the possible underlying mechanisms, the potential clinical use of M SCs as modulators of immune responses in vivo and to indicate clinical safety concerns and recommendations for future research.
1,683 citations
TL;DR: Culture of limbal stem cells represent a source of cells for transplantation in the treatment of destruction of the human cornea due to burns and success--that is, the generation of normal epithelium on donor stroma--was associated with the percentage of p63-bright holoclone-forming stem cells in culture.
Abstract: limbus, causing limbal stem-cell deficiency. We investigated the long-term clinical results of cell therapy in patients with burn-related corneal destruction associated with limbal stem-cell deficiency, a highly disabling ocular disease. Methods We used autologous limbal stem cells cultivated on fibrin to treat 112 patients with corneal damage, most of whom had burn-dependent limbal stem-cell deficiency. Clinical results were assessed by means of Kaplan–Meier, Kruskal–Wallis, and univariate and multivariate logistic-regression analyses. We also assessed the clinical outcome according to the percentage of holoclone-forming stem cells, detected as cells that stain intensely (p63-bright cells) in the cultures. Results Permanent restoration of a transparent, renewing corneal epithelium was attained in 76.6% of eyes. The failures occurred within the first year. Restored eyes remained stable over time, with up to 10 years of follow-up (mean, 2.91±1.99; median, 1.93). In post hoc analyses, success — that is, the generation of normal epithelium on donor stroma — was associated with the percentage of p63-bright holoclone-forming stem cells in culture. Cultures in which p63-bright cells constituted more than 3% of the total number of clonogenic cells were associated with successful transplantation in 78% of patients. In contrast, cultures in which such cells made up 3% or less of the total number of cells were associated with successful transplantation in only 11% of patients. Graft failure was also associated with the type of initial ocular damage and postoperative complications. Conclusions Cultures of limbal stem cells represent a source of cells for transplantation in the treatment of destruction of the human cornea due to burns.
994 citations
TL;DR: Corneal epithelium, nonetheless, has considerable healing capacity, which is achieved primarily by migration of epithelial cells, and should correspond to higher demands on the generative capacity of the corneal basal cells compared with skin.
Abstract: THE human cornea is covered by a five-layered epithelium. Cells are continually shed from its surface and replaced by division of the basal cells, which has a mean generation time estimated to be about 4 days1. Because of the papillae in the skin, the relation between the area of the basal cell layer and the surface is about 20 : 1. Because it must be refractive, there can be no papillae on the cornea, and the relation between the basal cell layer and the surface is accordingly 1 : 1. This should correspond to higher demands on the generative capacity of the corneal basal cells compared with skin. The epidermal basal cells are in close contact with a well developed capillary network. There are no vessels in the cornea, and so it can be assumed that the supply of its epithelium is poorer. Corneal epithelium, nonetheless, has considerable healing capacity, which is achieved primarily by migration of epithelial cells.
617 citations
TL;DR: The results have generated several controversies, a few myths and a change in a major paradigm, and how they can be applied to the adult stem/progenitor cells from bone marrow, referred to as MSCs are reviewed.
572 citations
TL;DR: The results of this study suggest that the anti‐inflammatory and anti‐angiogenic action of MSC in the chemically burned corneas might be mediated in part through paracrine pathways involving soluble factors such as IL‐10, TGF‐β1, IL‐6 and TSP‐1.
Abstract: To investigate the anti-inflammatory and anti-angiogenic effects of mesenchymal stem cells (MSC) in the chemically burned corneas, we mechanically removed the corneal epithelium of rats after 100% alcohol instillation. The rats were then randomized into four groups: fresh media, conditioned media derived from the MSC culture (MSC-CM), MSC applied topically to the damaged corneas for 2 hours immediately after the injury or MSC-CM applied either once or 3 times per day for 3 consecutive days. Corneal surface was evaluated every week. After 3 weeks, the corneas were stained with the hematoxylin-eosin, and the expression of interleukin (IL)-2, interferon (IFN)-gamma, IL-6, IL-10, transforming growth factor (TGF)-beta1, thrombospondin-1 (TSP-1), matrix metalloproteinase-2 (MMP-2), and vascular endothelial growth factor (VEGF) were analyzed. CD4+ cells were assessed in the corneas. We found that both MSC and three-time applied MSC-CM (1) reduced corneal inflammation and neovascularization, (2) decreased IL-2 and IFN-gamma, although increased IL-10 and TGF-beta1 as well as IL-6, (3) reduced the infiltration of CD4+ cells, and (4) upregulated the expression of TSP-1, although downregulated that of MMP-2. Interestingly, whereas three-time application of MSC-CM was partially effective, transplantation of MSC achieved a better outcome in suppressing corneal inflammation. The results of this study suggest that the anti-inflammatory and anti-angiogenic action of MSC in the chemically burned corneas might be mediated in part through paracrine pathways involving soluble factors such as IL-10, TGF-beta1, IL-6 and TSP-1.
355 citations