Isolation and characterization of rabbit limbal niche cells.

Limbal niche cells (LNCs) are one of the most important supporting cells for corneal epithelial stem cells (CES), however, research on LNCs has been mostly limited to humans and rats previously. To expand the research work into the rabbit animal model, one of the most often used animals in stem cell study, this study was carried out for the in vitro isolation and identification of rabbit LNCs. Rabbit LNCs were isolated by collagenase A digestion method and single cells were obtained, the cells were then seeded on 5% Matrigel-coated plastic surface and cultured in modified embryonic stem cell medium (MESCM). Three biological replicates of the isolating and characterization were recorded from New Zealand White rabbits aged from 2.5 months to 5 months. LNC markers (VIM/CD90/CD105/SCF/PDGFRß) were analyzed using tyramide signal amplification (TSA) staining, immunohistochemical staining (IHC), western blotting (WB), and real-time reverse transcription polymerase chain reaction (qPCR). TSA staining suggested that VIM was highly expressed in rabbit limbus stroma, which was confirmed by WB, and P63α was expressed in the basal limbus epithelium. Pan-CK and CK12 were highly expressed in the central corneal epithelium but lightly expressed in the limbal epithelium. The WB result indicated that PDGFRß and VIM expressions in rabbit-LNCs P4 were higher than in P1 and P7. In addition, rabbit corneal epithelium highly expressed Paired Box 6 (PAX6) and Epidermal growth factor-like domain 6(EGFL6). For the three repeat experiments, the cell expansion activity of rabbit-LNC was highest at P4. Rabbit-LNCs were passaged from P0 to P7, and the number of cell doublings (NCD) of P4 for the three repeat experiments was 2.816, 2.737, and 2.849. qPCR showed that high mRNA expression levels of VIM, CD90, CD105, SCF, and PDGFRß in rabbit-LNCs P4. In conclusion, rabbit-LNCs could be successfully isolated by the collagenase A digestion method as used in human tissue. There were similar characteristics between rabbit and human LNCs (VIM+/CD90+/CD105+/SCF+/PAX6+/PDGFRß+).

Efficient Isolation and Expansion of Limbal Melanocytes for Tissue Engineering.

Limbal melanocytes (LMs) are found in the corneoscleral limbus basal epithelial layer and interact with neighboring limbal epithelial progenitor cells. The difficulty of isolating and cultivating LMs is due to the small fraction of LMs in the overall limbal population and the frequent contamination of primary cultures by other cell types. This has limited the research on freshly isolated LMs and the investigation of their biological significance in the maintenance of the limbal stem cell niche. Here, we describe an optimized protocol for the efficient isolation and expansion of LMs from cadaveric corneal limbal tissue using CD90 and CD117 as selective markers in fluorescence-activated cell sorting to obtain a pure population of LMs (CD90- CD117+) with self-renewal capacity and sustained melanin production. The isolation of pure LMs from a single preparation enables direct transcriptomic and proteomic analyses, as well as functional studies on freshly isolated LMs, which can be considered the proper counterparts of LMs in vivo and have potential applications in tissue engineering.

The Role of Sensory Innervation in Homeostatic and Injury-Induced Corneal Epithelial Renewal.

The cornea is the window through which we see the world. Corneal clarity is required for vision, and blindness occurs when the cornea becomes opaque. The cornea is covered by unique transparent epithelial cells that serve as an outermost cellular barrier bordering between the cornea and the external environment. Corneal sensory nerves protect the cornea from injury by triggering tearing and blink reflexes, and are also thought to regulate corneal epithelial renewal via unknown mechanism(s). When protective corneal sensory innervation is absent due to infection, trauma, intracranial tumors, surgery, or congenital causes, permanent blindness results from repetitive epithelial microtraumas and failure to heal. The condition is termed neurotrophic keratopathy (NK), with an incidence of 5:10,000 people worldwide. In this report, we review the currently available therapeutic solutions for NK and discuss the progress in our understanding of how the sensory nerves induce corneal epithelial renewal.

Characterization of Porcine Ocular Surface Epithelial Microenvironment.

The porcine ocular surface is used as a model of the human ocular surface; however, a detailed characterization of the porcine ocular surface has not been documented. This is due, in part, to the scarcity of antibodies produced specifically against the porcine ocular surface cell types or structures. We performed a histological and immunohistochemical investigation on frozen and formalin-fixed, paraffin-embedded ocular surface tissue from domestic pigs using a panel of 41 different antibodies related to epithelial progenitor/differentiation phenotypes, extracellular matrix and associated molecules, and various niche cell types. Our observations suggested that the Bowman's layer is not evident in the cornea; the deep invaginations of the limbal epithelium in the limbal zone are analogous to the limbal interpalisade crypts of human limbal tissue; and the presence of goblet cells in the bulbar conjunctiva. Immunohistochemistry analysis revealed that the epithelial progenitor markers cytokeratin (CK)15, CK14, p63α, and P-cadherin were expressed in both the limbal and conjunctival basal epithelium, whereas the basal cells of the limbal and conjunctival epithelium did not stain for CK3, CK12, E-cadherin, and CK13. Antibodies detecting marker proteins related to the extracellular matrix (collagen IV, Tenascin-C), cell-matrix adhesion (ß-dystroglycan, integrin α3 and α6), mesenchymal cells (vimentin, CD90, CD44), neurons (neurofilament), immune cells (HLA-ABC; HLA-DR, CD1, CD4, CD14), vasculature (von Willebrand factor), and melanocytes (SRY-homeobox-10, human melanoma black-45, Tyrosinase) on the normal human ocular surface demonstrated similar immunoreactivity on the normal porcine ocular surface. Only a few antibodies (directed against N-cadherin, fibronectin, agrin, laminin α3 and α5, melan-A) appeared unreactive on porcine tissues. Our findings characterize the main immunohistochemical properties of the porcine ocular surface and provide a morphological and immunohistochemical basis useful to research using porcine models. Furthermore, the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.

Influence of Organ Culture on the Characteristics of the Human Limbal Stem Cell Niche.

Organ culture storage techniques for corneoscleral limbal (CSL) tissue have improved the quality of corneas for transplantation and allow for longer storage times. Cultured limbal tissue has been used for stem cell transplantation to treat limbal stem cell deficiency (LSCD) as well as for research purposes to assess homeostasis mechanisms in the limbal stem cell niche. However, the effects of organ culture storage conditions on the quality of limbal niche components are less well described. Therefore, in this study, the morphological and immunohistochemical characteristics of organ-cultured limbal tissue are investigated and compared to fresh limbal tissues by means of light and electron microscopy. Organ-cultured limbal tissues showed signs of deterioration, such as edema, less pronounced basement membranes, and loss of the most superficial layers of the epithelium. In comparison to the fresh limbal epithelium, organ-cultured limbal epithelium showed signs of ongoing proliferative activity (more Ki-67+ cells) and exhibited an altered limbal epithelial phenotype with a loss of N-cadherin and desmoglein expression as well as a lack of precise staining patterns for cytokeratin ((CK)14, CK17/19, CK15). The analyzed extracellular matrix composition was mainly intact (collagen IV, fibronectin, laminin chains) except for Tenascin-C, whose expression was increased in organ-cultured limbal tissue. Nonetheless, the expression patterns of cell-matrix adhesion proteins varied in organ-cultured limbal tissue compared to fresh limbal tissue. A decrease in the number of melanocytes (Melan-A+ cells) and Langerhans cells (HLA-DR+, CD1a+, CD18+) was observed in the organ-cultured limbal tissue. The organ culture-induced alterations of the limbal epithelial stem cell niche might hamper its use in the treatment of LSCD as well as in research studies. In contrast, reduced numbers of donor-derived Langerhans cells seem associated with better clinical outcomes. However, there is a need to consider the preferential use of fresh CSL for limbal transplants and to look at ways of improving the limbal stem cell properties of stored CSL tissue.

HC-HA/PTX3 from amniotic membrane reverts senescent limbal niche cells to Pax6+ neural crest progenitors to support limbal epithelial progenitors.

Quiescence and self-renewal of human corneal epithelial progenitor/stem cells (LEPC) are regulated by the limbal niche, presumably through close interaction with limbal (stromal) niche cells (LNC). Paired box homeotic gene 6 (Pax6), a conserved transcription factor essential for eye development, is essential for proper differentiation of limbal and corneal epithelial stem cells. Pax6 haploinsufficiency causes limbal stem cell deficiency, which leads to subsequent corneal blindness. We previously reported that serial passage of nuclear Pax6+ LNC resulted in the gradual loss of nuclear Pax6+ and neural crest progenitor status, the latter of which was reverted upon recovery of Pax6. These findings suggest Pax6 plays a pivotal role in supporting the self-renewal of LEPC in limbal niche. Herein, we show that HC-HA/PTX3, a unique matrix purified from amniotic membrane (AM) and consists of heavy chain 1of inter-α-trypsin inhibitor covalently linked to hyaluronic acid and complexed with pentraxin 3, is capable of reverting senescent LNC to nuclear Pax6+ neural crest progenitors that support self-renewal of LEPC. Such reversion is causally linked to early cell aggregation mediated by activation of C-X-C chemokine receptor type 4 (CXCR4)-mediated signaling followed by activation of bone morphogenetic protein (BMP) signaling. Furthermore, CXCR4-mediated signaling, but not BMP signaling, controls recovery of the nuclear Pax6+ neural crest progenitors. These findings not only explain why AM helps in vivo and ex vivo expansion of human LEPC, but they also illuminate the potential role of HC-HA/PTX3 as a surrogate matrix niche that complements stem cell-based therapies in regenerative medicine.

Efficient Isolation and Functional Characterization of Niche Cells from Human Corneal Limbus.

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.

KIT ligand produced by limbal niche cells under control of SOX10 maintains limbal epithelial stem cell survival by activating the KIT/AKT signalling pathway.

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.

A validated porcine corneal organ culture model to study the limbal response to corneal epithelial injury.

Limbal epithelial stem cells are required for the maintenance and repair of the corneal epithelial surface. The difficulty in obtaining human corneal tissue for research purposes means that animal models for studying the corneal and limbal epithelium are extremely useful. Porcine corneal tissue represents an attractive experimental model, however, functional analysis of the limbal epithelial cell population is needed to validate the use of this tissue. Single cell clonal analysis revealed that holoclone-generating cells were enriched in the limbus as compared with the central cornea (38.3% vs 8.3%) and that label-retaining cells were also enriched in the limbus and compared with the central cornea (44.7 ± 6.4 vs 4.7 ± 1.5). Furthermore, it was demonstrated that in a 3D-printed organ culture system, porcine tissue was capable of maintaining and healing the corneal epithelium. Ki67 staining of corneal sections revealed that in response to central epithelial wounding, a greater proportion of progenitors in the basal limbal epithelium enter an actively dividing state. The authors present a comprehensively validated model system for studying the interactions between limbal niche factors and limbal epithelial stem cell fate.

Limbal niche cells can reduce the angiogenic potential of cultivated oral mucosal epithelial cells.

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.

Differentiation potential of limbal fibroblasts and bone marrow mesenchymal stem cells to corneal epithelial cells.

The cornea is covered by a stratified epithelium that is renewed by stem cells located in the peripheral region of the cornea known as the limbus. This stroma of the limbus contains stromal keratocytes that, when expanded in culture, are termed limbal fibroblasts (LFs). It is thought that LFs exhibit similar characteristics to bone marrow mesenchymal stem cells (BM MSCs) and help maintain the epithelial stem cell phenotype in the limbal region. In this study, we aimed at reprogramming stage-specific embryonic antigen-4 (SSEA4+) LFs and BM MSCs into corneal epithelial lineage using a three-dimensional culture system and embryonic stem cell medium. After enrichment, SSEA4+ cells showed a higher level of stem cell marker expression such as Sox2, Oct4, Nanog, Rex1, ABCG2, and TRA-1-60, and colony-forming efficiency than did SSEA4- cells. SSEA4+, as compared to SSEA4- cells, had a greater propensity to form spheres that, in turn, were induced into ectodermal lineage and further differentiated into functional corneal epithelium. Results show that LFs were similar to BM MSCs in marker profiles, and together with the differences noted between SSEA4+ and SSEA4- cells, point to LFs' being tissue-specific MSCs. However, LFs showed a greater potential for differentiation into corneal epithelium, indicating the potential importance of tissue-specific adult progenitors in their reprogramming capacity into cells of interest. This study opens a new avenue for investigating the molecular mechanism involved in maintaining a limbal stem cell niche and thus a potentially important clinical application to treat corneal epithelial stem cell loss.

Basic Fibroblast Growth Factor Supports the Function of Limbal Niche Cells via the Wnt/ß-Catenin Pathway.

Purpose: To test the effects and underlying mechanisms of basic fibroblast growth factor (bFGF) on the limbal niche cell (LNC) function ex vivo. Methods: By using different concentrations of bFGF (0, 4, 8, 12, and 16 ng/mL) and fibroblast growth factor receptor (FGFR) inhibitors, the effects of bFGF on LNC proliferation, expression of stem cell markers, and transcription levels of the ß-catenin were investigated. Single-cell RNA sequencing (scRNA-seq) was used to analyze the action and mechanisms of FGFR subtypes and the Wnt/ß-catenin pathway during LNC culture. An mature corneal epithelial cell (MCEC)/LNC three-dimensional model was constructed to verify whether bFGF activates the Wnt/ß-catenin pathway in LNC by inhibiting FGFR or ß-catenin targets. Results: scRNA-seq showed that FGFR1 is the main receptor in LNC, along with the molecules in the Wnt pathway, including WNT2, FZD7, LRP5, LRP6, and ß-catenin. The 12 ng/mL bFGF treatment group showed higher LNC proliferation rate and transcription levels of OCT4, SOX2, NANOG, and ß-catenin than any other groups (P < 0.001). In the MCEC/LNC co-culture model, MCEC/LNC treated with 12 ng/mL bFGF promoted the aggregation of the spheres than other groups, associated with increased transcription levels of P63α, WNT2, ß-catenin, and a decreased transcription level of CK12 (P < 0.001). Wnt/ß-catenin inhibitor LF3 treatment reversed the abovementioned effect of bFGF. Conclusions: bFGF could maintain and promote the stemness of LNC via the FGFR1/Wnt2/FZD7/LRP6 axis in a concentration-dependent manner.

PAX6 Expression Patterns in the Adult Human Limbal Stem Cell Niche.

Paired box 6 (PAX6), a nuclear transcription factor, determines the fate of limbal epithelial progenitor cells (LEPC) and maintains epithelial cell identity. However, the expression of PAX6 in limbal niche cells, primarily mesenchymal stromal cells (LMSC), and melanocytes is scarce and not entirely clear. To distinctly assess the PAX6 expression in limbal niche cells, fresh and organ-cultured human corneoscleral tissues were stained immunohistochemically. Furthermore, the expression of PAX6 in cultured limbal cells was investigated. Immunostaining revealed the presence of PAX6-negative cells which were positive for vimentin and the melanocyte markers Melan-A and human melanoma black-45 in the basal layer of the limbal epithelium. PAX6 staining was not observed in the limbal stroma. Moreover, the expression of PAX6 was observed by Western blot in cultured LEPC but not in cultured LMSC or LM. These data indicate a restriction of PAX6 expression to limbal epithelial cells at the limbal stem cell niche. These observations warrant further studies for the presence of other PAX isoforms in the limbal stem cell niche.

Concise Review: Bioengineering of Limbal Stem Cell Niche.

The corneal epithelium is composed of nonkeratinized stratified squamous cells and has a significant turnover rate. Limbal integrity is vital to maintain the clarity and avascularity of the cornea as well as regeneration of the corneal epithelium. Limbal epithelial stem cells (LESCs) are located in the basal epithelial layer of the limbus and preserve this homeostasis. Proper functioning of LESCs is dependent on a specific microenvironment, known as the limbal stem cell niche (LSCN). This structure is made up of various cells, an extracellular matrix (ECM), and signaling molecules. Different etiologies may damage the LSCN, leading to limbal stem cell deficiency (LSCD), which is characterized by conjunctivalization of the cornea. In this review, we first summarize the basics of the LSCN and then focus on current and emerging bioengineering strategies for LSCN restoration to combat LSCD.

Isolation and ex vivo Expansion of Limbal Mesenchymal Stromal Cells.

Limbal mesenchymal stromal cells (LMSC), a cellular component of the limbal stem cell niche, have the capability of determining the fate of limbal epithelial progenitor cells (LEPC), which are responsible for the homeostasis of corneal epithelium. However, the isolation of these LMSC has proven to be difficult due to the small fraction of LMSC in the total limbal population, and primary cultures are always hampered by contamination with other cell types. We recently published the efficient isolation and functional characterization of LMSC from the human corneal limbus using CD90 as a selective marker. We observed that flow sorting yielded a pure population of LMSC with superior self-renewal capacity and transdifferentiation potential, and supported the maintenance of the LEPC phenotype. Here, we describe an optimized protocol for the isolation of LMSC from cadaveric corneal limbal tissue by combined collagenase digestion and flow sorting with expansion of LMSC on plastic. Graphical abstract.

Consecutive dosing of UVB irradiation induces loss of ABCB5 expression and activation of EMT and fibrosis proteins in limbal epithelial cells similar to pterygium epithelium.

Pterygium pathogenesis is often attributed to a population of altered limbal stem cells, which initiate corneal invasion and drive the hyperproliferation and fibrosis associated with the disease. These cells are thought to undergo epithelial to mesenchymal transition (EMT) and to contribute to subepithelial stromal fibrosis. In this study, the presence of the novel limbal stem cell marker ABCB5 in clusters of basal epithelial pterygium cells co-expressing with P63α and P40 is reported. ABCB5-positive pterygium cells also express EMT-associated fibrosis markers including vimentin and α-SMA while their ß-catenin expression is reduced. By using a novel in vitro model of two-dose UV-induced EMT activation on limbal epithelial cells, we could observe the dysregulation of EMT-related proteins including an increase of vimentin and α-SMA as well as downregulation of ß-catenin in epithelial cells correlating to downregulation of ABCB5. The sequential irradiation of limbal fibroblasts also induced an increase in vimentin and α-SMA. Taken together, these data demonstrate for the first time the expression of ABCB5 in pterygium stem cell activity and EMT-related events while the involvement of limbal stem cells in pterygium pathogenesis is exhibited via sequential irradiation of limbal epithelial cells. The later in vitro approach can be used to further study the involvement of limbal epithelium UV-induced EMT in pterygium pathogenesis and help identify novel treatments against pterygium growth and recurrence.

Limbal Bio-Engineered Tissue Employing 3D Nanofiber-Aerogel Scaffold to Facilitate LSCs Growth and Migration.

Constrained by the existing scaffold inability to mimic limbal niche, limbal bio-engineered tissue constructed in vitro is challenging to be widely used in clinical practice. Here, a 3D nanofiber-aerogel scaffold is fabricated by employing thermal cross-linking electrospinned film polycaprolactone (PCL) and gelatin (GEL) as the precursor. Benefiting from the cross-linked (160 °C, vacuum) structure, the homogenized and lyophilized 3D nanofiber-aerogel scaffold with preferable mechanical strength is capable of refraining the volume collapse in humid vitro. Intriguingly, compared with traditional electrospinning scaffolds, the authors' 3D nanofiber-aerogel scaffolds possess enhanced water absorption (1100-1300%), controllable aperture (50-100 µm), and excellent biocompatibility (optical density value, 0.953 ± 0.021). The well-matched aperture and nanostructure of the scaffolds with cells enable the construction of limbal bio-engineered tissue. It is foreseen that the proposed general method can be extended to various aerogels, providing new opportunities for the development of novel limbal bio-engineered tissue.

Transcriptomic Landscape and Functional Characterization of Human Induced Pluripotent Stem Cell-Derived Limbal Epithelial Progenitor Cells.

Limbal stem cell deficiency (LSCD) is a complex, multifactorial disease affecting limbal epithelial progenitor cells (LEPC), which are essential for maintaining corneal stability and transparency. Human induced pluripotent stem cell-derived (hiPSC-) LEPC are a promising cell source for the treatment of LSCD. However, their similarity to native tissue-derived (T-) LEPC and their functional characterization has not been studied in detail. Here, we show that hiPSC-LEPC and T-LEPC have rather similar gene expression patterns, colony-forming ability, wound-healing capacity, and melanosome uptake. In addition, hiPSC-LEPC exhibited lower immunogenicity and reduced the proliferation of peripheral blood mononuclear cells compared with T-LEPC. Similarly, the hiPSC-LEPC secretome reduced the proliferation of vascular endothelial cells more than the T-LEPC secretome. Moreover, hiPSC-LEPC successfully repopulated decellularized human corneolimbal (DHC/L) scaffolds with multilayered epithelium, while basal deposition of fibrillary material was observed. These findings suggest that hiPSC-LEPC exhibited functional properties close to native LEPC and that hiPSC-LEPC-DHC/L scaffolds might be feasible for transplantation in patients suffering from LSCD in the future. Although hiPSC-LEPC-based stem cell therapy is promising, the current study also revealed new challenges, such as abnormal extracellular matrix deposition, that need to be overcome before hiPSC-LEPC-based stem cell therapies are viable.

P-Cadherin Is Expressed by Epithelial Progenitor Cells and Melanocytes in the Human Corneal Limbus.

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.

Melanocytes as emerging key players in niche regulation of limbal epithelial stem cells.

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.