Differentiation Of Corneal Epithelial Cells Research Articles

Elucidating the mechanism of corneal epithelial cell repair: unraveling the impact of growth factors.

The repair mechanism for corneal epithelial cell injuries encompasses migration, proliferation, and differentiation of corneal epithelial cells, and extracellular matrix remodeling of the stromal structural integrity. Furthermore, it involves the consequential impact of corneal limbal stem cells (LSCs). In recent years, as our comprehension of the mediating mechanisms underlying corneal epithelial injury repair has advanced, it has become increasingly apparent that growth factors play a pivotal role in this intricate process. These growth factors actively contribute to the restoration of corneal epithelial injuries by orchestrating responses and facilitating specific interactions at targeted sites. This article systematically summarizes the role of growth factors in corneal epithelial cell injury repair by searching relevant literature in recent years, and explores the limitations of current literature search, providing a certain scientific basis for subsequent basic research and clinical applications.

Topical Insulin for Ocular Surface Disease.

Background: Insulin and insulin-like growth factor (IGF)-1 receptors are present in ocular tissues such as corneal epithelium, keratocytes, and conjunctival cells. Insulin plays a crucial role in the growth, differentiation, and proliferation of corneal epithelial cells, as well as in wound healing processes in various tissues. Purpose: This review explores the potential role of topical insulin in the treatment of ocular surface diseases. Specifically, it examines its impact on corneal nerve regeneration, sub-basal plexus corneal nerves, and its application in conditions like corneal epithelial defects, dry eye disease, and diabetic keratopathy. Methods: The review analyzes studies conducted over the past decade that have investigated the use of topical insulin in ocular surface diseases. It focuses on indications, drug preparation methods, side effects, efficacy outcomes, and variations in insulin concentrations and dosages used. Results: While off-label use of topical insulin has shown promising results in refractory corneal epithelial defects, its efficacy in dry eye disease is yet to be demonstrated. Variations in concentrations, dilutions, and dosing guidelines have been reported. However, limited data on ocular penetration, ocular toxicity, and systemic side effects pose challenges to its widespread utility. Conclusion: This review synthesizes findings from ocular investigations on topical insulin to assess its potential applicability in treating ocular surface and corneal diseases. By highlighting indications, preparation methods, side effects, and efficacy outcomes, it aims to provide insights into the current status and future prospects of using topical insulin in ophthalmic practice.

Examination of Subbasal Nerve Plexus and Central Corneal Stromal Microstructure in Subjects With Congenital Aniridia, Using in Vivo Confocal Laser Scanning Microscopy

Purpose During life up to 70% of aniridia subjects develop aniridia-associated keratopathy (AAK). AAK is characterized by limbal stem cell insufficiency, impaired corneal epithelial cell differentiation and abnormal cell adhesion, which leads to centripetal spreading vascularization, conjunctivalization, and thickening of the cornea. Our aim was to examine the subbasal nerve plexus and central corneal stromal microstructure in subjects with congenital aniridia, using in vivo confocal laser scanning microscopy CLSM. Methods 31 eyes of 18 patients (55.6% males, mean age: 25.22 ± 16.35 years) with congenital aniridia and 46 eyes of 29 healthy subjects (41.4% males, mean age 30 ± 14.82 years) were examined using the Rostock Cornea Module of Heidelberg Retina Tomograph-III. At the subbasal nerve plexus, corneal nerve fiber density (CNFD), corneal nerve fiber length (CNFL), corneal total branch density (CTBD), and corneal nerve fiber width (CNFW) were analyzed using ACCMetrics software. Keratocyte density in the anterior, middle and posterior stroma was assessed manually. Results The CNFD (2.02 ± 4.08 vs 13.99 ± 6.34/mm2), CNFL (5.78 ± 2.68 vs 10.56 ± 2.82 mm/mm2) and CTBD (15.08 ± 15.62 vs 27.44 ± 15.05/mm2) were significantly lower in congenital aniridia subjects than in controls (p < 0.001 for all). CNFW was significantly higher in aniridia subjects than in controls (0.03 ± 0.004 vs 0.02 ± 0.003 mm/mm2) (p = 0.003). Keratocyte density was significantly lower in all stromal layers of aniridia subjects than in controls (p < 0.001 for all). Stromal alterations included confluent keratocytes, keratocytes with long extensions and hyperreflective dots between keratocytes in aniridia. Conclusions Decrease in CNFD, CNFL, and CTBD, as well as increase in CNFW well refer to the congenital aniridia-associated neuropathy. The decreased keratocyte density and the stromal alterations may be related to an increased cell death in congenital aniridia, nevertheless, stromal changes in different stages of AAK have to be further analyzed in detail.

The single-cell transcriptomic atlas and RORA-mediated 3D epigenomic remodeling in driving corneal epithelial differentiation

Proper differentiation of corneal epithelial cells (CECs) from limbal stem/progenitor cells (LSCs) is required for maintenance of ocular homeostasis and clear vision. Here, using a single-cell transcriptomic atlas, we delineate the comprehensive and refined molecular regulatory dynamics during human CEC development and differentiation. We find that RORA is a CEC-specific molecular switch that initiates and drives LSCs to differentiate into mature CECs by activating PITX1. RORA dictates CEC differentiation by establishing CEC-specific enhancers and chromatin interactions between CEC gene promoters and distal regulatory elements. Conversely, RORA silences LSC-specific promoters and disrupts promoter-anchored chromatin loops to turn off LSC genes. Collectively, our work provides detailed and comprehensive insights into the transcriptional dynamics and RORA-mediated epigenetic remodeling underlying human corneal epithelial differentiation.

The sub‐basal nerve plexus and central corneal stromal microstructure in subjects with congenital aniridia, using in vivo confocal microscopy

Purpose: Congenital aniridia is a rare disease, with a global prevalence of 1 in 40 000 to 1 in 100 000. During life up to 70% of aniridia sufferers develop aniridia‐associated keratopathy (AAK) with limbal stem cell insufficiency, impaired corneal epithelial cell differentiation, abnormal cell adhesion and wound healing. AAK is characterized by centripetal spreading vascularization, conjunctivalization, and thickening of the cornea. We examined the sub‐basal nerve plexus and central corneal stromal microstructure in subjects with congenital aniridia, using in vivo confocal laser scanning microscopy (IVCM).Methods: 31 eyes of 18 patients (55.6% males, mean age: 25.22 ± 16.35 years) with congenital aniridia and 47 eyes of 29 healthy subjects (41.4% males, mean age 30 ± 14.82 years) were examined using the Rostock Cornea Module of Heidelberg Retina Tomograph‐III. At the sub‐basal nerve plexus, corneal nerve fibre density (CNFD), corneal nerve fibre length (CNFL), corneal total branch density (CTBD) and corneal nerve fibre width (CNFW) were analysed using ACCMetrics software. Keratocyte density in the anterior, middle and posterior stroma was assessed manually.Results: The CNFD (2.02 ± 4.08 vs. 13.99 ± 6.34/mm2), CNFL (5.78 ± 2.68 vs. 10.56 ± 2.82 mm/mm2) and CTBD (15.08 ± 15.62 vs. 27.44 ± 15.05/mm2) were significantly lower in congenital aniridia subjects, than in controls (p &lt; 0.001 for all). CNFW was significantly higher in aniridia subjects, than in controls (0.03 ± 0.004 vs. 0.02 ± 0.003 mm/mm2) (p &lt; 0.003). Keratocyte density was significantly lower in all stromal layers of congenital aniridia subjects, than in controls (p &lt; 0.001 for all). Stromal alterations included confluent keratocytes, keratocytes with long extensions and hyperreflective dots between keratocytes in congenital aniridia.Conclusions: CNFD, CNFL and CTBD decrease and CNFW increase well refer to the congenital aniridia associated neuropathy. The decreased keratocyte density contradicts the inflammatory hypothesis of congenital aniridia. Corneal stromal changes in different stages of AAK have to be further analysed in detail0.

Lithium chloride promotes diabetic corneal epithelial wound healing by activating the Wnt/β‑catenin signaling pathway.

Corneal epithelial abnormality is a common manifestation of diabetic keratopathy and leads to delayed epithelial wound healing. The Wnt/β-catenin signaling pathway participates in the development, differentiation and stratification of corneal epithelial cells. The present study compared the expression of Wnt/β-catenin signaling pathway related factors, including Wnt7a, β-catenin, cyclin D1 and phosphorylated (p-) glycogen synthase kinase 3 β (Gsk3b) between normal and diabetic mouse corneas, by reverse transcription-quantitative PCR, western blotting and immunofluorescence staining. It was found that the expression of the Wnt/β-catenin signaling pathway related factors was downregulated in diabetic corneas. Upon corneal epithelium scraping, the wound healing rate was significantly increased in diabetic mice after topical treatment with lithium chloride. After further investigation, significantly upregulated levels of Wnt7a, β-catenin, cyclin D1 and p-Gsk3b were found in the diabetic group 24 h after treatment, accompanied by β-catenin nuclear translocation observed by immunofluorescence staining. These results suggest that active Wnt/β-catenin pathway can promote diabetic corneal epithelial wound healing.

Downregulation of p38 MAPK Signaling Pathway Ameliorates Tissue-Engineered Corneal Epithelium.

Tissue-engineered corneal epithelium transplantation is effective treatment for severe limbal stem cell deficiency (LSCD), while epithelial terminal differentiation, tans-differentiation, and insufficient stem cell during construction affect the quality of tissue-engineered corneal epithelium. In this study, we applied SB203580 in the culture medium to downregulate the p38 mitogen-activated protein kinase (MAPK) signaling pathway during construction of tissue-engineered corneal epithelium. With application of SB203580, tissue-engineered corneal epithelium showed enhanced strength and condensed structure. The expression of progenitor cell markers ATP-binding cassette sub-family G member 2, tumor protein p63, keratin 14, and Wnt family member 7A was increased, differentiation markers keratin 12, paired box 6, keratin 10, and keratin 13 and trans-differentiation markers actin alpha 2, smooth muscle and snail family transcriptional repressor 1 was decreased, while cell junction markers claudin 1 and cadherin 1 was increased in the tissue-engineered corneal epithelium. The Wnt/catenin beta 1 signaling pathway was upregulated in the epithelium after p38 MAPK inhibition. Transplantation of tissue-engineered corneal epithelium treated with SB203580 to rabbit LSCD model showed faster wound healing and improved epithelial quality. We conclude that downregulation of p38 MAPK signaling pathway helps maintain the stemness and prevent terminal differentiation and abnormal differentiation of corneal epithelial cells during epithelium construction process, and thus can improve the quality of tissue-engineered corneal epithelium. Impact statement Downregulation of p38 MAPK signaling pathway helps maintain the self-renewal of limbal stem cells and prevents terminal differentiation and abnormal differentiation of corneal epithelial cells. Small molecules modulating p38 MAPK signaling pathway ameliorate tissue-engineered corneal epithelium.

Interleukin-13 increases the stemness of limbal epithelial stem cells cultures.

This study aimed to determine the effect of interleukin-13 (IL13) on the stemness, differentiation, proliferation, clonogenicity, and morphology of cultured limbal epithelial cells (LECs). Human limbal explants were used to culture LECs up to the second passage (P0-P2) with or without IL13 (IL13+ and IL13-, respectively). Cells were analyzed by qPCR (for the expression of ΔNp63α, BMI-1, keratin (K) 3, K7, K12, K14, K17, mucin 4, and MKI67) and immunofluorescence staining for p63α. The clonogenic ability was determined by colony-forming assay (CFA), and their metabolic activity was measured by WST-1 assay. The results of the CFA showed a significantly increased clonogenic ability in P1 and P2 cultures when LECs were cultured with IL13. In addition, the expression of putative stem cell markers (ΔNp63α, K14, and K17) was significantly higher in all IL13+ cultures compared to IL13-. Similarly, immunofluorescence analysis showed a significantly higher percentage of p63α positive cells in P2 cultures with IL13 than without it. LECs cultures without IL13 lost their cuboidal morphology with a high nucleocytoplasmic ratio after P1. The use of IL13 also led to significantly higher proliferation in P2, which can be reflected by a higher ability to reach confluence in P2 cultures. On the other hand, IL13 had no effect on corneal epithelial cell differentiation (K3 and K12 expression), and the expression of the conjunctival marker K7 significantly increased in all IL13+ cultures compared to the respective cell culture without IL13. This study showed that IL13 enhanced the stemness of LECs by increasing the clonogenicity and the expression of putative stem cell markers of LECs while maintaining their stem cell morphology. We established IL13 as a culture supplement for LESCs, which increases their stemness potential in culture, even after the second passage, and may lead to the greater success of LESCs transplantation in patients with LSCD.

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.

The O-GlcNAc modification promotes terminal differentiation of human corneal epithelial cells.

Dynamic modification of nuclear and cytoplasmic proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) plays an important role in orchestrating the transcriptional activity of eukaryotic cells. Here, we report that the O-GlcNAc modification contributes to maintaining ocular surface epithelial homeostasis by promoting mucin biosynthesis and barrier function. We found that induction of human corneal epithelial cell differentiation stimulated the global transfer of O-GlcNAc to both nuclear and cytosolic proteins. Inflammatory conditions, on the other hand, were associated with a reduction in the expression of O-GlcNAc transferase at the ocular surface epithelia. Loss- and gain-of-function studies using small interfering RNA targeting O-GlcNAc transferase, or Thiamet G, a selective inhibitor of O-GlcNAc hydrolase, respectively, revealed that the presence of O-GlcNAc was necessary to promote glycocalyx barrier function. Moreover, we found that Thiamet G triggered a correlative increase in both surface expression of MUC16 and apical epithelial cell area while reducing paracellular permeability. Collectively, these results identify intracellular protein O-glycosylation as a novel pathway responsible for promoting the terminal differentiation of human corneal epithelial cells.

Temporal Regulation of Notch Signaling and Its Influence on the Differentiation of Ex Vivo Cultured Limbal Epithelial Cells

ABSTRACTPurpose: Notch signaling plays a vital role in the differentiation and proliferation of corneal epithelial cells from limbal stem cells. The temporal regulation of Notch signaling during this differentiation remains unknown. Hence, we investigated the importance of temporal activation/blockage of Notch signaling during corneal differentiation.Methods: Human limbal epithelial cultures were established with and without Notch activators (rec-Human Jagged1 Fc chimera) and pharmacological blockers (LY-411575). The modulation of Notch signaling was done at different time points during cell differentiation, which were collected on Day 14 for further analysis of differentiation, proliferation, maturation and apoptosis using RT-qPCR and immunofluorescence staining.Results: The activation of Notch signaling at Day 8 resulted in the highest number of mature corneal epithelial cells (p = .008) and pro-apoptosis marker BAX (p = .0001) with no increase in the number of corneal progenitors, and proliferation marker Ki67 compared to untreated controls. Cultures grown in the presence of Notch signaling blockers showed a significantly higher number of corneal progenitors (p = .0001) and proliferation marker Ki67 (p = .02) but lower corneal epithelial marker CK3/CK12 (p = .0007) and no difference in the pro-apoptotic marker BAX compared to untreated controls.Conclusion: During the differentiation of limbal epithelial cells to the corneal epithelial cell type, Day 8 seems to be a crucial window to modulate Notch signaling for a customized outcome.

The Effect of Micro- and Nanoscale Surface Topographies on Silk on Human Corneal Limbal Epithelial Cell Differentiation

We previously reported that micro- and nano-scale topographic pitch created on silk films mimic features of the corneal basement membrane by providing biophysical cues to direct corneal epithelial cell adherence and migration. However, the effect of these topographical features on corneal limbal epithelial cell differentiation has not been explored. We hypothesize in the current study that various topographical pitch created on silk may affect corneal epithelial stem cell differentiation and alter the expression of genes involved in cell differentiation and self-renewal. We patterned silk films with different topographic pitch via soft lithography and observed human corneal limbal epithelial cell behavior. Colony forming assay demonstrated increased colony forming efficiency on patterned silk films. Cells cultured on nanoscale patterned silk films also expressed lower levels of putative keratocyte differentiation markers and higher levels of putative limbal stem cell markers. RNA-Seq analysis further implicated the involvement of pathways related to stem cell differentiation and self-renewal, including Notch, ERK/MAPK and Wnt/β-catenin signaling. We conclude that patterned silk film substrates can be used as scaffolds and provide biophysical cues to corneal limbal stem cells that may maintain corneal epithelial stem cells at a less differentiated state.

Role of Corneal Stromal Cells on Epithelial Cell Function during Wound Healing.

Following injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs) and participate in wound repair. Simultaneously, ongoing bi-directional communications between corneal stromal-epithelial cells also play a vital role in mediating the process of wound healing. Factors produced by stromal cells are known to induce proliferation, differentiation, and motility of corneal epithelial cells, which are also subsequently the main processes that occur during wound healing. In this context, the present study aims to investigate the effect of SFs conditioned medium (SFCM) on corneal epithelial cell function along with substance P (SP). Antibody microarrays were employed to profile differentially expressed cell surface markers and cytokines in the presence of SFCM and SP. Antibody microarray data revealed enhanced expression of the ITGB1 in corneal epithelial cells following stimulation with SP whereas SFCM induced abundant expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. All these proteins have either direct or indirect roles in epithelial cell growth, movement and adhesion related signaling cascades during tissue regeneration. We also observed activation of MAPK signaling pathway along with increased expression of focal adhesion kinase (FAK), paxillin, vimentin, β-catenin and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT) regulating transcription factors Slug and ZEB1 expression were enhanced in the presence of SFCM. SP enriched the expression of integrin subunits α4, α5, αV, β1 and β3 whereas SFCM increased α4, α5, αV, β1 and β5 integrin subunits. We also observed increased expression of Serpin E1 following SP and SFCM treatment. Wound healing scratch assay revealed enhanced migration of epithelial cells following the addition of SFCM. Taken together, we conclude that SFCM-mediated sustained activation of ZEB1, Slug in combination with upregulated migration-associated integrins and ERK (Extracellular signal-regulated kinase)-FAK-paxillin axis, may lead to induce type 2 EMT-like changes during corneal epithelial wound healing.

Characterization of enhancers and the role of the transcription factor KLF7 in regulating corneal epithelial differentiation

During tissue development, transcription factors bind regulatory DNA regions called enhancers, often located at great distances from the genes they regulate, to control gene expression. The enhancer landscape during embryonic stem cell differentiation has been well characterized. By contrast, little is known about the shared and unique enhancer regulatory mechanisms in different ectodermally derived epithelial cells. Here we use ChIP sequencing (ChIP-seq) to identify domains enriched for the histone marks histone H3 lysine 4 trimethylation, histone H3 lysine 4 monomethylation, and histone H3 lysine 27 acetylation (H3K4me3, H3K4me1, and H3K27ac) and define, for the first time, the super enhancers and typical enhancers active in primary human corneal epithelial cells. We show that regulatory regions are often shared between cell types of the ectodermal lineage and that corneal epithelial super enhancers are already marked as potential regulatory domains in embryonic stem cells. Kruppel-like factor (KLF) motifs were enriched in corneal epithelial enhancers, consistent with the important roles of KLF4 and KLF5 in promoting corneal epithelial differentiation. We now show that the Kruppel family member KLF7 promotes the corneal progenitor cell state; on many genes, KLF7 antagonized the corneal differentiation-promoting KLF4. Furthermore, we found that two SNPs linked previously to corneal diseases, astigmatism, and Stevens-Johnson syndrome fall within corneal epithelial enhancers and alter their activity by disrupting transcription factor motifs that overlap these SNPs. Taken together, our work defines regulatory enhancers in corneal epithelial cells, highlights global gene-regulatory relationships shared among different epithelial cells, identifies a role for KLF7 as a KLF4 antagonist in corneal epithelial cell differentiation, and explains how two SNPs may contribute to corneal diseases.

Template Curvature Influences Cell Alignment to Create Improved Human Corneal Tissue Equivalents.

To accurately create corneal stromal equivalents with native-like structure and composition, a new biofunctionalized, curved template is developed that allows the precise orientation of cells and of their extracellular matrix. This template is the first demonstration that curvature alone is sufficient to induce the alignment of human corneal stromal cells, which in turn are able to biofabricate stromal tissue equivalents with cornea-like shape and composition. Specifically, tissues self-released from curved templates show a highly organized nanostructure, comprised of aligned collagen fibrils, significantly higher expression of corneal stroma-characteristic markers keratocan, lumican, decorin, ALDH3, and CHST6 (p = 0.012, 0.033, 0.029, 0.003, and 0.02, respectively), as well as significantly higher elastic modulus (p = 0.0001) compared with their planar counterparts. Moreover, curved tissues are shown to support the growth, stratification, and differentiation of human corneal epithelial cells in vitro, while maintaining their structural integrity and shape without any supporting carriers, scaffolds, or crosslinking agents. Together, these results demonstrate that corneal stromal cells can align and create highly organized, purposeful tissues by the influence of substrate curvature alone, and without the need of additional topographical cues. These findings can be important to further understand the mechanisms of corneal biosynthesis both in vitro and in vivo.

Multi-layered silk film coculture system for human corneal epithelial and stromal stem cells.

With insufficient options to meet the clinical demand for cornea transplants, one emerging area of emphasis is on cornea tissue engineering. In the present study, the goal was to combine the corneal stroma and epithelium into one coculture system, to monitor both human corneal stromal stem cell (hCSSC) and human corneal epithelial cell (hCE) growth and differentiation into keratocytes and differentiated epithelium in these three-dimensional tissue systems in vitro. Coculture conditions were first optimized, including the medium, air-liquid interface culture, and surface topography and chemistry of biomaterial scaffold films based on silk protein. The silk was used as scaffolding for both stromal and epithelial tissue layers because it is cell compatible, can be surface patterned, and is optically clear. Next, the effects of proliferating and differentiating hCEs and hCSSCs were studied in this in vitro system, including the effects on cell proliferation, matrix formation by immunochemistry, and gene expression by quantitative reverse transcription-polymerase chain reaction. The incorporation of both cell types into the coculture system demonstrated more complete differentiation and growth for both cell types compared to the corneal stromal cells and corneal epithelial cells alone. Silk films for corneal epithelial culture were optimized to combine a 4.0-μm-scale surface pattern with bulk-loaded collagen type IV. Differentiation of each cell type was in evidence based on increased expression of corneal stroma and epithelial proteins and transcript levels after 6weeks in coculture on the optimized silk scaffolds.

Effect of microgravity on the mesenchymal stem cell characteristics of limbal fibroblasts

BackgroundMesenchymal stem cells (MSCs) are important for regenerative medicine. Limbal fibroblasts (LFs), present in the corneal limbus, have been shown to possess MSC characteristics, and can differentiate into other cell types. The current study sought to investigate the effect of microgravity on the proliferation and differentiation of LFs, and identify culture conditions to obtain a high proportion of LFs possessing MSC characteristics. MethodsA rotary cell culture system was used to generate microgravity. Cellular proliferation and MSC marker (CD14, CD45, CD90, CD105, and SSEA4) expression were evaluated by WST-1 test and flow cytometry, respectively. Differentiation of LFs into adipocytes, osteocytes, and chondrocytes was examined. The effects of LF-conditioned medium on limbal stem cell differentiation were assessed. ResultsThe cellular proliferation rates under microgravity were significantly lower than those under normal gravity (0.44 vs. 0.18 at 24 h, and 0.70 vs. 0.44 at 48 h, both P ≤ 0.004). Higher proportions of cells expressed CD90 (95.33% vs 81.69%), CD105 (95.32% vs 87.96%), and SSEA4 (68.26% vs 26.13%) under microgravity than under normal gravity. The differentiation potential of LFs was more prominent under microgravity. The LF-conditioned medium attenuated the differentiation of limbal corneal epithelial stem cells. ConclusionUnder microgravity, LFs showed a higher proportion of MSC characteristics and were easily induced into different linage cells. Culture in a microgravity environment may allow harvesting a greater number of MSC-like LFs for stem cell therapy in ocular surface reconstruction.

CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation.

BackgroundThe purpose of the study is to elicit the epigenetic mechanism involving CCCTC binding factor (CTCF)-mediated chromatin remodeling that regulates PAX6 gene interaction with differentiation-associated genes to control corneal epithelial differentiation.MethodsCell cycle progression and specific keratin expressions were measured to monitor changes of differentiation-induced primary human limbal stem/progenitor (HLS/P), human corneal epithelial (HCE) and human telomerase-immortalized corneal epithelial (HTCE) cells. PAX6-interactive and differentiation-associated genes in chromatin remodeling mediated by the epigenetic factor CTCF were detected by circular chromosome conformation capture (4C) and ChIP (Chromatin immunoprecipitation)-on-chip approaches, and verified by FISH (Fluorescent in situ hybridization). Furthermore, CTCF activities were altered by CTCF-shRNA to study the effect of CTCF on mediating interaction of Pax6 and differentiation-associated genes in corneal epithelial cell fate.ResultsOur results demonstrated that differentiation-induced human corneal epithelial cells expressed typical corneal epithelial characteristics including morphological changes, increased keratin12 expression and G0/G1 accumulations. Expressions of CTCF and PAX6 were suppressed and elevated following the process of differentiation, respectively. During corneal epithelial cell differentiation, differentiation-induced RCN1 and ADAM17 were found interacting with PAX6 in the process of CTCF-mediated chromatin remodeling detected by 4C and verified by ChIP-on-chip and FISH. Diminished CTCF mRNA with CTCF-shRNA in HTCE cells weakened the interaction of PAX6 gene in controlling RCN1/ADAM17 and enhanced early onset of the genes in cell differentiation.ConclusionOur results explain how epigenetic factor CTCF-mediated chromatin remodeling regulates interactions between eye-specific PAX6 and those genes that are induced/associated with cell differentiation to modulate corneal epithelial cell-specific differentiation.

Spatiotemporally Regulated Ablation of Klf4 in Adult Mouse Corneal Epithelial Cells Results in Altered Epithelial Cell Identity and Disrupted Homeostasis.

In previous studies, conditional disruption of Klf4 in the developing mouse ocular surface from embryonic day 10 resulted in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells, revealing the importance of Klf4 in ocular surface maturation. Here, we use spatiotemporally regulated ablation of Klf4 to investigate its functions in maintenance of adult corneal epithelial homeostasis. Expression of Cre was induced in ternary transgenic (Klf4(LoxP/LoxP)/Krt12(rtTA/rtTA)/Tet-O-Cre) mouse corneal epithelium by doxycycline administered through intraperitoneal injections and drinking water, to generate corneal epithelium-specific deletion of Klf4 (Klf4(Δ/ΔCE)). Corneal epithelial barrier function was tested by fluorescein staining. Expression of selected Klf4-target genes was determined by quantitative PCR (QPCR), immunoblotting, and immunofluorescent staining. Klf4 was efficiently ablated within 5 days of doxycycline administration in adult Klf4(Δ/ΔCE) corneal epithelium. The Klf4(Δ/ΔCE) corneal epithelial barrier function was disrupted, and the basal cells were swollen and rounded after 15 days of doxycycline treatment. Increased numbers of cell layers and Ki67-positive proliferating cells suggested deregulated Klf4(Δ/ΔCE) corneal epithelial homeostasis. Expression of tight junction proteins ZO-1 and occludin, desmosomal Dsg and Dsp, basement membrane laminin-332, and corneal epithelial-specific keratin-12 was decreased, while that of matrix metalloproteinase Mmp9 and noncorneal keratin-17 increased, suggesting altered Klf4(Δ/ΔCE) corneal epithelial cell identity. Ablation of Klf4 in the adult mouse corneas resulted in the absence of characteristic corneal epithelial cell differentiation, disrupted barrier function, and squamous metaplasia, revealing that Klf4 is essential for maintenance of the adult corneal epithelial cell identity and homeostasis.

Stromal–epithelial interaction study: The effect of corneal epithelial cells on growth factor expression in stromal cells using organotypic culture model

Interactions between stromal and epithelial cells play important roles in the development, homeostasis, and pathological conditions of the cornea. Soluble cytokines are critical factors in stromal–epithelial interactions, and growth factors secreted from corneal stromal cells contribute to the regulation of proliferation and differentiation of corneal epithelial cells (CECs). However, the manner in which the expression of growth factors is regulated in stromal cells has not been completely determined. To study stromal–epithelial cell interactions, we used an organotypic culture model. Human or rabbit CECs (HCECs or RCECs) were cultured on amniotic membranes placed on human corneal fibroblasts (HCFs) embedded in a collagen gel. The properties of the organotypic culture were examined by hematoxylin–eosin staining and immunofluorescence. In the organotypic culture, HCECs or RCECs were stratified into two–three layers after five days and five–seven layers after nine days. However, stratification was not observed when the HCECs were seeded on a collagen gel without fibroblasts. K3/K12 were expressed on day 9. The HCF-embedded collagen gels were collected on days 3, 5, or 9 after seeding the RCECs, and mRNA expression of growth factors FGF7, HGF, NGF, EGF, TGF-α, SCF, TGF-β1, TGF-β2, and TGF-β3 were quantified by real-time PCR. mRNA expression of the growth factors in HCFs cultured with RCECs were compared with those cultured without RCECs, as well as in monolayer cultures. mRNA expression of TGF-α was markedly increased in HCFs cultured with RCECs. However, mRNA expression of the TGF-β family was suppressed in HCFs cultured with RCECs. Principal component analysis revealed that mRNA expression of the growth factors in HCFs were generally similar when they were cultured with RCECs. In organotypic cultures, the morphological changes in the CECs and the expression patterns of the growth factors in the stromal cells clearly demonstrated stromal–epithelial cell interactions, and the results suggest that stromal cells and epithelial cells may act in concert in the cornea.