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Nature Communications Apr 2020In today's clinics, a cell-resolution view of the cornea can be achieved only with a confocal microscope (IVCM) in contact with the eye. Here, we present a common-path...
In today's clinics, a cell-resolution view of the cornea can be achieved only with a confocal microscope (IVCM) in contact with the eye. Here, we present a common-path full-field/spectral-domain OCT microscope (FF/SD OCT), which enables cell-detail imaging of the entire ocular surface in humans (central and peripheral cornea, limbus, sclera, tear film) without contact and in real-time. Real-time performance is achieved through rapid axial eye tracking and simultaneous defocusing correction. Images contain cells and nerves, which can be quantified over a millimetric field-of-view, beyond the capability of IVCM and conventional OCT. In the limbus, palisades of Vogt, vessels, and blood flow can be resolved with high contrast without contrast agent injection. The fast imaging speed of 275 frames/s (0.6 billion pixels/s) allows direct monitoring of blood flow dynamics, enabling creation of high-resolution velocity maps. Tear flow velocity and evaporation time can be measured without fluorescein administration.
Topics: Adult; Angiography; Biomedical Engineering; Blood Flow Velocity; Cornea; Equipment Design; Female; Humans; Limbus Corneae; Male; Microscopy; Optical Imaging; Software; Tomography, Optical Coherence; Young Adult
PubMed: 32313067
DOI: 10.1038/s41467-020-15792-x -
Frontiers in Bioscience (Scholar... Nov 2022Energy storage, transmission and dissipation are important considerations of normal mechanical homeostasis. In this paper we present a new technique termed vibrational...
BACKGROUND
Energy storage, transmission and dissipation are important considerations of normal mechanical homeostasis. In this paper we present a new technique termed vibrational optical coherence tomography (VOCT) to study the anterior anatomic structures of the pig eye to better understand how energy applied to the cornea is dissipated without delamination occurring.
METHODS
VOCT uses infrared light and an applied sinusoidal audible sound wave to image and measure the resonant frequency and modulus of individual macromolecular components of tissue non-invasively. We have measured the resonant frequencies and calculated the moduli of tissues in the anterior portion of the pig eye using VOCT.
RESULTS
While both pig and human eyes have similar resonant frequencies, they do differ in the peak amplitudes near the frequencies of 80, 120, 150 and 250 Hz. It is known that the stroma of pig cornea is much thicker than that of human corneas and these differences may explain the normalized peak height differences. The similarity of the resonant frequency peaks near 80, 120, 150 and 250 Hz of cornea, sclera and limbus suggest that the anatomically described layers in these tissues are connected into a single biomechanical unit that can store external mechanical energy and then transmit it for dissipation. Since the energy stored and dissipated is proportional to the modulus and the ability of the tissue to deform under stress, energy storage in these tissues is related to the stiffness.
CONCLUSIONS
It is concluded that stored energy is transmitted to the posterior segment of the eye for dissipation through the attachment with the sclera. This mechanism of energy dissipation may protect the cornea from changes in shape, curvature, and refractive power. However, ultimately, energy dissipation through thinning of the sclera may cause globe elongation observed in subjects with myopia and glaucoma.
Topics: Humans; Animals; Swine; Sclera; Elastic Modulus; Limbus Corneae; Cornea; Myopia
PubMed: 36575840
DOI: 10.31083/j.fbs1404030 -
International Journal of Molecular... Nov 2021Limbal epithelial stem/progenitor cells (LSCs) reside in a niche that contains finely tuned balances of various signaling pathways including Wnt, Notch, BMP, Shh, YAP,... (Review)
Review
Limbal epithelial stem/progenitor cells (LSCs) reside in a niche that contains finely tuned balances of various signaling pathways including Wnt, Notch, BMP, Shh, YAP, and TGFβ. The activation or inhibition of these pathways is frequently dependent on the interactions of LSCs with various niche cell types and extracellular substrates. In addition to receiving molecular signals from growth factors, cytokines, and other soluble molecules, LSCs also respond to their surrounding physical structure via mechanotransduction, interaction with the ECM, and interactions with other cell types. Damage to LSCs or their niche leads to limbal stem cell deficiency (LSCD). The field of LSCD treatment would greatly benefit from an understanding of the molecular regulation of LSCs in vitro and in vivo. This review synthesizes current literature around the niche factors and signaling pathways that influence LSC function. Future development of LSCD therapies should consider all these niche factors to achieve improved long-term restoration of the LSC population.
Topics: Animals; Epithelium, Corneal; Eye; Humans; Limbus Corneae; Mechanotransduction, Cellular; Stem Cell Niche; Stem Cells
PubMed: 34769405
DOI: 10.3390/ijms222111975 -
American Journal of Ophthalmology Jul 2022Taking into consideration prior human experience with treating limbal stem cell deficiency (LSCD) with cultivated limbal epithelial cells (CLEC) from other countries, we... (Review)
Review
PURPOSE
Taking into consideration prior human experience with treating limbal stem cell deficiency (LSCD) with cultivated limbal epithelial cells (CLEC) from other countries, we have set a goal to optimize and standardize the techniques of CLEC preparation (called CALEC by our group) for the clinical trial in the United States.
METHODS
We performed an extensive literature review of all human trials, case series, and reports involving autologous cultivated limbal epithelial cell transplantation. Allogeneic cultivated limbal epithelial cell transplantations were reported only when combined with autologous studies. We also searched prior animal data aiding in detailing regulatory toxicology requirements.
RESULTS
Between 1997 and 2020, the analysis of human trials revealed 21 studies on autologous grafts, and 13 studies analyzing both autologous grafts and allogeneic grafts. Of a total of 34 studies, 6 studies used good manufacturing process (GMP) facilities, and 11 studies had no animal-derived products or murine feeder layers, whereas only 1 study had both. Overall, the treatment with autologous CLEC grafts was 68.9% successful. In total there were 6 preclinical studies using rabbits, serving as surrogate studies to assess the safety and toxicity of cultivated limbal epithelial cells for human trials. Based on prior human experience, we further optimized the manufacturing conditions with GMP-grade and serum and animal-free reagents, and developed cell characterization assays for the CALEC product release.
CONCLUSIONS
These data were used to develop a novel and consistent manufacturing process using only qualified and validated reagents for performing the first clinical trial on CALEC transplantation to treat LSCD in the United States.
Topics: Animals; Cell Transplantation; Cells, Cultured; Corneal Diseases; Epithelial Cells; Epithelium, Corneal; Humans; Limbus Corneae; Mice; Rabbits; Scleral Diseases; Stem Cell Transplantation; Transplantation, Autologous
PubMed: 35314191
DOI: 10.1016/j.ajo.2022.03.015 -
Investigative Ophthalmology & Visual... Apr 2023Corneal sensory nerves protect the cornea from injury. They are also thought to stimulate limbal stem cells (LSCs) to produce transparent epithelial cells constantly,...
PURPOSE
Corneal sensory nerves protect the cornea from injury. They are also thought to stimulate limbal stem cells (LSCs) to produce transparent epithelial cells constantly, enabling vision. In other organs, Schwann cells (SCs) associated with tissue-innervating axon terminals mediate tissue regeneration. This study defines the critical role of the corneal axon-ensheathing SCs in homeostatic and regenerative corneal epithelial cell renewal.
METHODS
SC localization in the cornea was determined by in situ hybridization and immunohistochemistry with SC markers. In vivo SC visualization and/or ablation were performed in mice with inducible corneal SC-specific expression of tdTomato and/or Diphtheria toxin, respectively. The relative locations of SCs and LSCs were observed with immunohistochemical analysis of harvested genetically SC-prelabeled mouse corneas with LSC-specific antibodies. The correlation between cornea-innervating axons and the appearance of SCs was ascertained using corneal denervation in rats. To determine the limbal niche cellular composition and gene expression changes associated with innervation-dependent epithelial renewal, single-cell RNA sequencing (scRNA-seq) of dissociated healthy, de-epithelized, and denervated cornea limbi was performed.
RESULTS
We observed limbal enrichment of corneal axon-associated myelinating and non-myelinating SCs. Induced local genetic ablation of SCs, although leaving corneal sensory innervation intact, markedly inhibited corneal epithelial renewal. scRNA-seq analysis (1) highlighted the transcriptional heterogenicity of cells populating the limbal niche, and (2) identified transcriptional changes associated with corneal innervation and during wound healing that model potential regulatory paracrine interactions between SCs and LSCs.
CONCLUSIONS
Limbal SCs are required for innervation-dependent corneal epithelial renewal.
Topics: Animals; Mice; Rats; Cornea; Epithelial Cells; Epithelium, Corneal; Limbus Corneae; Schwann Cells; Stem Cells
PubMed: 37036418
DOI: 10.1167/iovs.64.4.7 -
Cells Oct 2023Epithelial and stromal/mesenchymal limbal stem cells contribute to corneal homeostasis and cell renewal. Extracellular vesicles (EVs), including exosomes (Exos), can be...
Epithelial and stromal/mesenchymal limbal stem cells contribute to corneal homeostasis and cell renewal. Extracellular vesicles (EVs), including exosomes (Exos), can be paracrine mediators of intercellular communication. Previously, we described cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos in non-diabetic (N) and diabetic (DM) limbal epithelial cells (LECs). Presently, we quantify the miRNA and proteome profiles of human LEC-derived Exos and their regulatory roles in N- and DM-LSC. We revealed some miRNA and protein differences in DM vs. N-LEC-derived Exos' cargos, including proteins involved in Exo biogenesis and packaging that may affect Exo production and ultimately cellular crosstalk and corneal function. Treatment by N-Exos, but not by DM-Exos, enhanced wound healing in cultured N-LSCs and increased proliferation rates in N and DM LSCs vs. corresponding untreated (control) cells. N-Exos-treated LSCs reduced the keratocyte markers ALDH3A1 and lumican and increased the MSC markers CD73, CD90, and CD105 vs. control LSCs. These being opposite to the changes quantified in wounded LSCs. Overall, N-LEC Exos have a more pronounced effect on LSC wound healing, proliferation, and stem cell marker expression than DM-LEC Exos. This suggests that regulatory miRNA and protein cargo differences in DM- vs. N-LEC-derived Exos could contribute to the disease state.
Topics: Humans; MicroRNAs; Exosomes; Limbus Corneae; Cornea; Diabetes Mellitus; Epithelial Cells; Stromal Cells; Cell Communication
PubMed: 37947602
DOI: 10.3390/cells12212524 -
Indian Journal of Ophthalmology Jul 2023To study and compare the demographic and clinical profile of acute ocular burns (AOB) in children and adults.
PURPOSE
To study and compare the demographic and clinical profile of acute ocular burns (AOB) in children and adults.
METHODS
This retrospective case series included 271 children (338 eyes) and 1300 adults (1809 eyes) who presented to two tertiary eye care centers within one month of sustaining AOB. Data regarding demographics, causative agents, severity of injury, visual acuity (VA), and treatment were collected and analyzed.
RESULTS
Males were more commonly affected particularly among adults (81% versus 64%, P < 0.00001). Among children, 79% sustained domestic injuries, whereas 59% of adults had work-place injuries (P < 0.0001). Most cases were due to alkali (38%) and acids (22%). Edible lime (chuna, 32%), superglue (14%), and firecrackers (12%) in children, and chuna (7%), insecticides, lye, superglue (6% each), toilet cleaner (4%) and battery acid (3%) in adults, were the main causative agents. The percentage of cases with Dua grade IV-VI was greater in children (16% versus 9%; P = 0.0001). Amniotic membrane grafting and/or tarsorrhaphy were needed in 36% and 14% of affected eyes in children and adults, respectively (P < 0.00001). The median presenting VA was logMAR 0.5 in children and logMAR 0.3 in adults (P = 0.0001), which improved significantly with treatment in both groups (P < 0.0001), but the final VA in eyes with Dua grade IV-VI burns was poorer in children (logMAR 1.3 versus logMAR 0.8, P = 0.04).
CONCLUSION
The findings clearly delineate the at-risk groups, causative agents, clinical severity, and treatment outcomes of AOB. Increased awareness and data-driven targeted preventive strategies are needed to reduce the avoidable ocular morbidity in AOB.
Topics: Male; Child; Adult; Humans; Eye Burns; Retrospective Studies; Burns, Chemical; Corneal Transplantation; Acids; Demography; Limbus Corneae; Corneal Diseases
PubMed: 37417107
DOI: 10.4103/IJO.IJO_3330_22 -
PLoS Biology Oct 2023The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells (LSCs), while the nontransparent epidermis relies on...
The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells (LSCs), while the nontransparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human LSCs from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors (TFs) that define specific cell fates and established their regulatory hierarchy. Single-cell RNA-seq (scRNA-seq) analyses of the cornea and the epidermis confirmed these shared and cell type-specific TFs. Notably, the shared and LSC-specific TFs can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the LSC fate and its association with corneal opacity.
Topics: Humans; Limbus Corneae; Cornea; Epithelium, Corneal; Transcription Factors; Cell Differentiation; Corneal Opacity
PubMed: 37856539
DOI: 10.1371/journal.pbio.3002336 -
Stem Cell Research & Therapy Jul 2020Destruction or dysfunction of limbal epithelial stem cells (LESCs) leads to unilateral or bilateral limbal stem cell deficiency (LSCD). Fifteen years have passed since... (Review)
Review
Destruction or dysfunction of limbal epithelial stem cells (LESCs) leads to unilateral or bilateral limbal stem cell deficiency (LSCD). Fifteen years have passed since the first transplantation of ex vivo cultivated oral mucosal epithelial cells (COMET) in humans in 2004, which represents the first use of a cultured non-limbal autologous cell type to treat bilateral LSCD. This review summarizes clinical outcomes from COMET studies published from 2004 to 2019 and reviews results with emphasis on the culture methods by which grafted cell sheets were prepared.
Topics: Cell Transplantation; Cells, Cultured; Corneal Diseases; Epithelial Cells; Epithelium, Corneal; Humans; Limbus Corneae; Stem Cell Transplantation; Transplantation, Autologous
PubMed: 32693830
DOI: 10.1186/s13287-020-01783-8 -
Experimental Eye Research Oct 2020Homeostasis of the corneal epithelium is ultimately maintained by stem cells that reside in a specialized microenvironment within the corneal limbus termed palisades of... (Review)
Review
Homeostasis of the corneal epithelium is ultimately maintained by stem cells that reside in a specialized microenvironment within the corneal limbus termed palisades of Vogt. This limbal niche nourishes, protects, and regulates quiescence, self-renewal, and fate decision of limbal epithelial stem/progenitor cells (LEPCs) toward corneal epithelial differentiation. This review focuses on our current understanding of the mechanism by which limbal (stromal) niche cells (LNCs) regulate the aforementioned functions of LEPCs. Based on our discovery and characterization of a unique extracellular matrix termed HC-HA/PTX3 (Heavy chain (HC1)-hyaluronan (HA)/pentraxin 3 (PTX3) complex, "-" denotes covalent linkage; "/" denotes non-covalent binding) in the birth tissue, i.e., amniotic membrane and umbilical cord, we put forth a new paradigm that HC-HA/PTX3 serves as a surrogate matrix niche by maintaining the in vivo nuclear Pax6+ neural crest progenitor phenotype to support quiescence and self-renewal but prevent corneal fate decision of LEPCs. This new paradigm helps explain how limbal stem cell deficiency (LSCD) develops in aniridia due to Pax6-haplotype deficiency and further explains why transplantation of HC-HA/PTX3-containing amniotic membrane prevents LSCD in acute chemical burns and Stevens Johnson syndrome, augments the success of autologous LEPCs transplantation in patients suffering from partial or total LSCD, and assists ex vivo expansion (engineering) of a graft containing LEPCs. We thus envisage that this new paradigm based on regenerative matrix HC-HA/PTX3 as a surrogate niche can set a new standard for regenerative medicine in and beyond ophthalmology.
Topics: C-Reactive Protein; Cell Differentiation; Cells, Cultured; Corneal Diseases; Humans; Limbus Corneae; Serum Amyloid P-Component; Stem Cell Niche
PubMed: 32795525
DOI: 10.1016/j.exer.2020.108181