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Cellular Stiffness as a Novel Stemness Marker in the Corneal Limbus.

ABSTRACT: Healthy eyes contain a population of limbal stem cells (LSCs) that continuously renew the corneal epithelium. However, each year, 1 million Americans are afflicted with severely reduced visual acuity caused by corneal damage or disease, including LSC deficiency (LSCD). Recent advances in corneal transplant technology promise to repair the cornea by implanting healthy LSCs to encourage regeneration; however, success is limited to transplanted tissues that contain a sufficiently high percentage of LSCs. Attempts to screen limbal tissues for suitable implants using molecular stemness markers are confounded by the poorly understood signature of the LSC phenotype. For cells derived from the corneal limbus, we show that the performance of cell stiffness as a stemness indicator is on par with the performance of ΔNP63α, a common molecular marker. In combination with recent methods for sorting cells on a biophysical basis, the biomechanical stemness markers presented here may enable the rapid purification of LSCs from a heterogeneous population of corneal cells, thus potentially enabling clinicians and researchers to generate corneal transplants with sufficiently high fractions of LSCs, regardless of the LSC percentage in the donor tissue.

SUBMITTER: Bongiorno T

PROVIDER: S-EPMC5072985 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Isolation and enrichment of melanocytes from human corneal limbus using CD117 (c-Kit) as selection marker.

Project description:Limbal melanocytes (LM) are located in the basal epithelial layer of the corneoscleral limbus and interact with adjacent limbal epithelial progenitor cells. The exploration of their biological role in the maintenance of the limbal stem cell niche has been limited by the difficulty of LM isolation and cultivation. Here, we report on a facile protocol for the efficient isolation and enrichment of pure populations of human LMs by fluorescence-activated cell sorting (FACS) using antibodies raised against the cell surface marker CD117 (c-Kit). The enriched LMs retain self-renewal capacity and sustained melanin production, and are suitable to study the potential of LMs in stem cell-based corneal tissue engineering.

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Substrate Stiffness Modulates Stemness and Differentiation of Rabbit Corneal Endothelium Through the Paxillin-YAP Pathway.

Project description:PurposeTo explore the role of substrate stiffness and the mechanism beneath corneal endothelial cells' (CECs') stemness maintenance and differentiation.MethodsCECs were divided into central zone (8 mm trephined boundary) and peripheral zone (8 mm trephined edge with attached limbal). Two zones were analyzed by hematoxylin-eosin staining and scanning electron microscopy for anatomic structure. The elastic modulus of Descemet's membrane (DM) was analyzed by atomic force microscopy. Compressed type I collagen gels with different stiffness were constructed as an in vitro model system to test the role of stiffness on phenotype using cultured rabbit CECs. Cell morphology, expression and intracellular distribution of Yes-associated protein (YAP), differentiation (ZO-1, Na+/K+-ATPase), stemness (FOXD3, CD34, Sox2, Oct3/4), and endothelial-mesenchymal transition (EnMT) markers were analyzed by immunofluorescence, quantitative RT-PCR, and Western blot.ResultsThe results showed that the peripheral area of rabbit and human DM is softer than the central area ex vivo. Using the biomimetic extracellular matrix collagen gels in vitro model, we then demonstrated that soft substrate weakens the differentiation and EnMT in the culture of CECs. It was further proved by the inhibitor experiment that soft substrate enhances stemness maintenance via inhibition of paxillin-YAP signaling, which was activated on a stiff substrate.ConclusionsOur findings confirm that substrate stiffness modulates the stemness maintenance and differentiation of CECs and suggest a potential strategy for CEC-based corneal tissue engineering.

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Changes in the corneal thickness and limbus after 1 year of scleral contact lens use.

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

Project description: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.

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Protection of Corneal Limbus from Riboflavin Prevents Epithelial Stem Cell Loss after Collagen Cross-Linking.

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Effect of central corneal curvature on corneal material stiffness parameter acquired by dynamic corneal responses.

Project description:The stress-strain index (SSI) is a measure of corneal material stiffness, which is obtained using the Corvis ST algorithm based on dynamic corneal response parameters. The reduced SSI corresponds to the longer axial length (AL). In a previous study, we found SSI increases as the corneal curvature flattens, whereas a flatter corneal curvature indicates a longer AL (emmetropia or myopia). Therefore, in this cross-sectional study, we aimed to address these contradictory findings. First, we characterized the features of SSI, curvature radius of the anterior corneal surface (CR), and AL and analyzed their correlation with advanced myopia. Next, we compared the relationship between AL and SSI after adjusting for the effect of CR. We found a significant positive correlation between SSI and CR, which contradicts the developmental law of axial myopia. Furthermore, after accounting for the effect of CR, we observed a stronger correlation between SSI and AL than that in the unadjusted model. In conclusion, CR is an independent influencing factor for SSI in addition to AL, which masked the decrease in SSI caused by prolonged AL in axial myopia.

| S-EPMC10619688 | biostudies-literature

Genome-wide analysis suggests a differential microRNA signature associated with normal and diabetic human corneal limbus.

Project description:Small non-coding RNAs, in particular microRNAs (miRNAs), regulate fine-tuning of gene expression and can impact a wide range of biological processes. However, their roles in normal and diseased limbal epithelial stem cells (LESC) remain unknown. Using deep sequencing analysis, we investigated miRNA expression profiles in central and limbal regions of normal and diabetic human corneas. We identified differentially expressed miRNAs in limbus vs. central cornea in normal and diabetic (DM) corneas including both type 1 (T1DM/IDDM) and type 2 (T2DM/NIDDM) diabetes. Some miRNAs such as miR-10b that was upregulated in limbus vs. central cornea and in diabetic vs. normal limbus also showed significant increase in T1DM vs. T2DM limbus. Overexpression of miR-10b increased Ki-67 staining in human organ-cultured corneas and proliferation rate in cultured corneal epithelial cells. MiR-10b transfected human organ-cultured corneas showed downregulation of PAX6 and DKK1 and upregulation of keratin 17 protein expression levels. In summary, we report for the first time differential miRNA signatures of T1DM and T2DM corneal limbus harboring LESC and show that miR-10b could be involved in the LESC maintenance and/or their early differentiation. Furthermore, miR-10b upregulation may be an important mechanism of corneal diabetic alterations especially in the T1DM patients.

| S-EPMC5471258 | biostudies-literature

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